Multi-well electroporation devices and methods

Abstract

The present disclosure describes systems, devices, and methods for high-throughput electroporation of a donor cell. The systems can include a perforated membrane having one or more layers, each comprising a plurality of channels disposed through each layer. The systems can also include spacer arrays in contact with a perforated membrane to create a plurality of cell electroporation chambers for high-throughput cell electroporation.

Description

MULTI- WELL ELECTROPORATION DEVICES AND METHODSCROSS-REFERENCE

[0001] This application claims the benefit ofU.S. Provisional Application No. 63 / 591,116, filed on October 17, 2023, and U.S. Provisional Application No. 63 / 624,237, filed January 23, 2024, which are incorporated herein by reference in their entirety.BACKGROUND

[0002] Cell electroporation is primarily used to introduce biomolecules, like DNA, RNA, or drugs, proteins, or other charged biomolecules into cells by temporarily disrupting the cell membrane with an electric field, which allowing for efficient delivery of nucleic acids and / or other biomolecules that would normally not be membrane permeable. Cell electroporation is commonly used in for gene delivery and for drug delivery to treat diseases. Tools are developed to allow for faster, more efficient, and scalable electroporation methods. Additionally, high- throughput electroporation platforms capable of multiplexed electroporation can increase the efficiency of screening and production of therapeutics. While existing high-throughput electroporation platforms afford the ability to deliver many nucleic acids or biomolecules of interest to large and diverse populations of cells, the electroporation conditions imposed upon cells influence key aspects such as cell viability, magnitude of extracellular vesicle secretion, and uniformity of nucleic acid expression. Currently available cell electroporation systems typically employ electrical stimulation that disrupts the entire cell membrane. For example, developing a means to focally electroporate cells may increase electroporation efficiency, increase cell health, and expedite the development of therapeutics.SUMMARY

[0003] Described herein are systems, devices, and apparatuses and uses or methods thereof that offer high-yield and high-throughput delivery of nucleic acids and other biomolecules into cells. This disclosure presents a solution to the problems of current methods of increasing the efficiency and bandwidth of cell electroporation: culturing a cell on a perforated membrane having an array of channels such that the cell is in contact with the openings of the channels. The channels are from about 500 nm to about 20 pm to minimize the surface area of the cell membrane disrupted during application of an electric field to the cells via the openings of the channels within the perforated membrane. The systems provided by the instant disclosure enable multiplex high-throughput electroporation to deliver one or many nucleic acids or otherbiomolecules to one or many cell populations in contact with the channels of the perforated membrane.

[0004] In one aspect, the present disclosure provides a perforated membrane, comprising: a first layer comprising a plurality of first channels disposed through the first layer; and a second layer in contact with the first layer, the second layer comprising a plurality of second channels disposed through the second layer; wherein: a first average thickness of the first layer is different from a second average thickness of the second layer; a first channel of the plurality of first channels is in fluid communication of a second channel of the plurality of second channels; a first average diameter of the first channel is different from a second average diameter of the second channel.

[0005] In another aspect, this disclosure provides a method of making a perforated membrane from photoresist materials, comprising: (a) spin coating a first layer of a first photoresist material onto a substrate; (b) soft baking the first layer obtained in (a); (c) exposing the soft baked first layer obtained in (b) to a first UV radiation using a first photomask; (d) spin coating a second layer of a second photoresist material onto the first layer after (c); (e) soft baking the second layer obtained in (d); (f) exposing the soft baked second layer obtained in (e) to another UV radiation using a second photomask; (g) conducting a post exposure bake; and (g) developing both the first layer and the second layer after (g), thereby making a perforated membrane comprising two layers.

[0006] Also provided herein is a system for high-throughput cell electroporation comprising: (a) a perforated membrane, wherein the perforated membrane comprises: (i) a first layer comprising a plurality of first channels disposed through the first layer; and (ii) a second layer in contact with the first layer, the second layer comprising a plurality of second channels disposed through the second layer, wherein: a first average thickness of the first layer is different from a second average thickness of the second layer; a first channel of the plurality of first channels is in fluid communication of a second channel of the plurality of second channels; a first average diameter of the first channel is different from a second average diameter of the second channel; and at least one donor cell in contact with the perforated membrane.

[0007] Another aspect of the instant disclosure describes a system for high-throughput cell electroporation comprising: (a) a perforated membrane, wherein the perforated membrane comprises channels disposed through the perforated membrane; (b) a first array spacer; (c) a second array spacer; and (d) at least one donor cell, wherein the first array spacer, the second array spacer, and the at least one donor cell are in contact with the perforated membrane.

[0008] This disclosure further provides a method of producing and collecting extracellular vesicles comprising: (a) providing an electroporation device comprising multiple wells, suchdevice comprising a perforated membrane situated between two spacer sheets, wherein: the spacer sheets comprise complementary arrays of holes defining cross-sectional area of the wells; the wells comprise a portion of the perforated membrane; and the portion of the perforated membrane separates a cell culture chamber from an electroporation buffer chamber such that pores within the perforated membrane fluidically couple the cell culture chamber with the electroporation buffer chamber; (b) introducing donors cells into the cell culture chamber; introducing a polynucleotide, DNA, RNA, vector, or plasmid into the electroporation buffer chamber; (d) applying an electric field, a current, or a voltage through the electroporation device, thereby electroporating the donor cells; and (e) collecting extracellular vesicles (EVs) produced by the donors cells.

[0009] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure.Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0012] FIG. 1 depicts a workflow schematic of the fabrication process of a dual -layer perforated membrane in accordance with some embodiments of the inventions described herein. Non- limiting examples of materials for fabricating the dual-layer perforated membrane can be SU-8 epoxy -based photoresist.

[0013] FIG. 2 depicts a scanning electron micrograph with a perspective view of a dual-layer perforated membrane with a patterned array of “bottle” -shaped channels (e.g., channels having a small-diameter portion and a large-diameter portion) in accordance with some embodiments of the inventions described herein. A non-limiting example of a dual-layer perforated membranewith a patterned SU-8 film showing double-layer microstructures. The thin layer of the duallayer perforated membrane is made of SU-8 TF 6002 photoresist with channels (i.e., pores) having a diameter of approximately 1 pm and a depth / length of approximately 2 pm. The thick layer is composed of SU-8 3010 photoresist with a patterned array of channels (i.e. , pores) having a diameter of approximately 4 pm large pores, whereby one opening of the large channels is aligned with the small 1 -pm-diameter pores. The total thickness of the SU-8 film is ~10 pm.

[0014] FIG. 3 depicts a scanning electron micrograph with a side view of a “bottle” -shaped channel (e.g., nanochannel or microchannel) within the dual-layer perforated membrane shown in FIG. 2

[0015] FIG. 4A depicts a scanning electron micrograph with a perspective view of a dual -layer perforated membrane in accordance with some embodiments of the inventions described herein. As non-limiting example, of the SU-8 dual-layer structure is made of a thin layer composed of SU-8 2002 photoresist and a thick layer made of SU-8 2005 photoresist.

[0016] FIG. 4B depicts of a scanning electron micrograph with a side view of the dual -layer perforated membrane shown in FIG. 4A.

[0017] FIG. 5 depicts quantification of dystrophin (DMD) mRNA copies per electroporated donor cell produced from cell nanoelectroporated (CNP) human skin fibroblast cells. A total of 24,000 cells were positioned on a 0.9 cm x 0.9 cm surface area of either a dual -layer perforated membrane (SU-8 double-layer surfaces; SU-8) or a silicon perforated membrane (Si-CNP; Si). Electroporation conditions: 100V, 4.5 mm electrode -to-electrode distance, ten 10-ms pulses with 0.1 -second interval. EVs were collected in the culture medium for 24 hours after cell nanoelectroporation.

[0018] FIG. 6 depicts a workflow schematic of fabrication process of a polymer cell electroporation surface by Sacrificial Template Imprinting (STI) technology in accordance with some embodiments of the inventions described herein.

[0019] FIG. 7A depicts an optical image of a PVA-based sacrificial template mold (male mold), as shown in FIG. 6, and in accordance with some embodiments of the inventions described herein. FIG. 7B depicts an optical image of a PDMS cell electroporation / nanoelectroporation (CEP / CNP) surface prepared by the sacrificial template imprinting process shown in FIG. 6 and provided herein.

[0020] FIG. 8 depicts a schematic of a high-throughput single-array electroporation device (e.g., cell electroporation / nanoelectroporation (CEP / CNP) device) in accordance with one embodiment of the inventions described herein. Inset: Side view spanning a single cell culture well of the high-throughput single-array electroporation device. Donor cells are adhered to the top surface of a Si CNP surface with channel openings (e.g., nanochannel openings) and in fluidicconnection with the electroporation buffer solution (e.g., buffer containing DNA plasmids of interest) via the channels of the Si CNP surface. An electric pulse is applied by an electric pulse generator across the cathode and anode structures of the high-throughput single-array cell electroporation device to mediate electroporation of the donor cells with the genetic cargo (e.g., DNA plasmids) in the electroporation buffer.

[0021] FIG. 9A depicts a schematic diagram of a spacer array of the high-throughput singlearray electroporation device in accordance with some embodiments of the inventions described herein. The spacer array creates isolated cell culture wells and / or isolated electroporation media chambers for high-throughput electroporation (e.g., high-throughput cell nanoelectrorporation (HIT-CNP)). FIG. 9B depicts an image of the high-throughput single-array electroporation device with the spacer array in accordance with some embodiments of the inventions described herein.

[0022] FIG. 10 A depicts a scanning electron micrograph (SEM) of a patterned array of channels in a perforated electroporation membrane made of silicon (Si CNP surface).

[0023] FIG. 10B depicts a scanning electron micrograph (SEM) of non-patterned channels in a tracked etched perforated electroporation membrane (TEM).

[0024] FIG. 11A depicts a schematic of a high-throughput double-array electroporation device (e.g., cell nanoelectroporation (CNP) device) with a perforated electroporation membrane (e.g., track etched random positioning of channels or Silicon (Si) patterned array of channels) sandwiched between two PDMS spacer arrays in accordance with one embodiment of the inventions described herein. Inset: Side view spanning a double-layers high-throughput electroporation device. Donor cells are added to individual cell culture wells defined by the holes within the polymer spacer array and adhered to the top surface and channel openings (e.g., nanochannel openings) of a track etched membrane, patterned perforated silicon membrane, or polymer dual-layer perforated membrane. Here, the sandwiched arrays form individual cell culture wells, each of which are in fluidic connection with an individual electroporation reagent wells (e.g., buffer containing DNA plasmids of interest) via the channels of the perforated membrane. Any perforated membrane is compatible with this double-array layer-based cell electroporation device configuration. Each cell culture well can seed different types of cells from neighboring cell culture wells, and each electroporation reagent well can be loaded with different types, concentrations, or cocktails of transfection reagents (e.g., DNA plasmids, RNAs, or small molecules). An electric pulse is applied by an electric pulse generator across the cathode and anode structures of the high-throughput single-array electroporation device to mediate electroporation of the donor cells with the genetic cargo (e.g., DNA plasmids) in the electroporation buffer.

[0025] FIG. 1 IB depicts a schematic of the completed double-well array chamber layer-based cell electroporation system for high-throughput cell electroporation with the cathode coil array inserted into each of the electroporation reagent wells.

[0026] FIG. 12 depicts a schematic of the workflow for use of a double-array cell electroporation system for high-throughput cell electroporation (e.g., cell electroporation / nanoelectroporation (CEP / CNP)) in accordance with some embodiments of the inventions described herein. A perforated electroporation membrane is sandwiched between two spacer arrays to create complementary arrays of isolated cell culture wells and electroporation reagent chambers. In this configuration each individual cell culture well is fluidically coupled to one of the electroporation reagent chambers. The donor cells are adhered to the surface of the perforated electroporation membrane within one or more of the isolated cell culture wells. Upon adhering to the perforated membrane, the donor cells form contacts with the openings of the channels (e.g., nanochannels). The double-array cell electroporation system is then flipped upside down, the electroporation reagent wells are loaded with electroporation buffer containing the genetic cargo of interest (e.g, DNA plasmids, RNAs, etc.), and the cathode coil array (FIG. 13) is then inserted into the electroporation reagent wells to complete the electrical circuit for high-throughput cell electroporation / nanoelectroporation (CEP / CNP).

[0027] FIG. 13 depicts a photograph of a 5 x 5 cathode coil configured for use with the automated cell electroporation electric pulse generation system of FIG. 15A - FIG. 15D.

[0028] FIG. 14 depicts quantification of qPCR results of COL1 Al mRNA production in extracellular vesicles (EVs) as the enriched genetic material from the same number of donor cells (e.g., nHDF cells) by cell nanoelectroporation using a silicon perforated electroporation membrane with patterned channels (CNP) or a track etched perforated electroporation membrane with non-patterned channels (TEP). All data are presented as mean ± SD; CTR: control of nonelectroporated cells (i.e., cells that were not subjected to electrical stimulation).

[0029] FIG. 15A depicts an automated cell electroporation electric pulse generation system configured to generate sequential electric pulses for high-throughput single-array and doublearray electroporation devices with multiple cell culture well designs. The electric pulse generation system comprises an electric pulse generator with adjustable voltage capabilities, a shock pulse converter, and a shock box FIG. 15B depicts a schematic diagram of the electric pulse generator of the automated cell electroporation electric pulse generation system shown in FIG. 15A and in accordance with some embodiments of the inventions described herein. FIG. 15C depicts a photograph of the shock pulse converter of the automated cell electroporation electric pulse generation system shown in FIG. 15A and in accordance with some embodiments of the inventions described herein. The shock pulse generator sends electrical output to eachelectrode sequentially via a 25 -pin connector. FIG. 15D depicts a photograph of the shock box of the automated cell electroporation electric pulse generation system shown in FIG. 15A and in accordance with some embodiments of the inventions described herein . The shock box is configured to initiate high-throughput cell electroporation (e.g., cell electroporation / nanoelectroporation (CEP / CNP)) in individual cell culture wells.DETAILED DESCRIPTION OF THE INVENTIONOverview

[0030] Described herein are systems, devices, and apparatuses and uses or methods thereof that offer high-yield and high-throughput delivery of biomolecules into cells. In some cases, the systems, devices, and apparatuses enable intracellular delivery of nucleic acids, or other biomolecules, into high quantities of cells. In some cases, the systems, devices, and apparatuses enable non-endocytic delivery of nucleic acids, or other biomolecules, into high quantities of cells. In some cases, the systems, devices, and apparatuses enable multiplexed high-throughput electroporation to at least one type of cell or at least two different types of cells. In some cases, the systems, devices, and apparatuses enable multiplexed high-throughput electroporation to deliver at least one nucleic acid or other biomolecule into at least one type of cell or at least two different types of cells. In some cases, the systems, devices, and apparatuses enable multiplexed high-throughput electroporation to deliver at least two nucleic acids or other biomolecules into at least one type of cell or at least two different types of cells. In some cases, the systems, devices, and apparatuses enable non-endocytic delivery of at least one nucleic acid or other biomolecule into at least one type of cell or at least two different types of cells. In some cases, the systems, devices, and apparatuses enable non-endocytic delivery of at least two nucleic acids or other biomolecules into at least one type of cell or at least two different types of cells. In some cases, the systems, devices, and apparatuses allow for rapid cell electroporation, high expression of at least one nuclei acid or other biomolecule, and fast post-transfection cell collection. In many cases, the systems, devices, apparatuses, and methods provided herein can achieve uniform and precise delivery of various transfection reagents from small oligodeoxynucleotides to large plasmid DNAs and nanoparticles into at least one or more than one type of cell. In some cases, the delivery of at least one, or more than one, nucleic acids or other biomolecules to the at least one or more than one type of cells comprises positioning cells against pores (e.g., nano- or micro-sized pores) of a perforated membrane, whereby the cells are electroporated or nanoelectroporated (CEP / CNP) upon applying an electric field across the perforated membrane to deliver the at least one, or more than one, transfection reagents or other biomolecules to the cells.I. Perforated Membranes

[0031] In one aspect, the present disclosure provides a perforated membrane, comprising: a first layer comprising a plurality of first channels disposed through the first layer; and a second layer in contact with the first layer, the second layer comprising a plurality of second channels disposed through the second layer; wherein: a first average thickness of the first layer is different from a second average thickness of the second layer; a first channel of the plurality of first channels is in fluid communication of a second channel of the plurality of second channels; a first average diameter of the first channel is different from a second average diameter of the second channel.

[0032] In one aspect, this disclosure provides a perforated membrane with unique features. Generally, the perforated membrane is a flattened structure having a first surface and a second surface that is opposite the first surface. The first surface and the second surface are generally substantively parallel to one another, and the planes of the first surface and the second surface are generally perpendicular to the height of the perforated membrane. In some cases, the perforated membrane is a rigid perforated membrane. Alternatively, in some cases, the perforated membrane is a flexible perforated membrane. Generally, the perforated membrane comprises a plurality of channels that allows passage of a substance from the first surface to the second surface of the perforated membrane (i.e. through the perforated membrane). For example, the plurality of channels can allow passage of a liquid substance (e.g., cell culture medium, an electroporation buffer) or biomolecules (e.g., nucleic acids, DNAs, DNA plasmids, RNAs, proteins, or smallmolecule drugs) from the first surface to the second surface of the perforated membrane (i.e. through the perforated membrane). Individual channels of the plurality of channels can comprise a cylindrical shape, a cuboid shape, or any other three-dimensional shape that allows passage of a substance, voltage, or electrical current from the first surface to the second surface of the perforated membrane.

[0033] In some embodiments, the perforated membrane is configured to pass an electrical voltage of at least about 1 millivolt (mV). In some cases, the perforated membrane is configured to pass an electrical voltage of no more than about 500 volts (V). The perforated membrane can also be configured to pass an electrical voltage from about 1 mV to about 300 V or from about 1 V to about 200 V. In some cases, a voltage is applied to the channels of the perforated membrane are capable of passing a voltage through the perforated membrane. In some cases, the voltage is at least about 1 volt (V) to about 300 V. In some cases, the voltage is at least about 1 volt (V) to about 400 V. In some cases, the voltage is at least about 1 volt (V) to about 500 V. In some cases, the voltage is at least about 1 volt (V) to about 100 V. In some cases, the voltage is at least about 1 volt (V) to about 200 V.

[0034] In some embodiments, the perforated membrane is configured to pass an electrical current of at least about 0.001 Amperes (Amp). In some cases, the perforated membrane is configured to pass an electrical current of no more than about 20 Amperes (Amp). The perforated membrane can also be configured to pass an electrical current from about 0.01 Amp to about 15 Amp or from about 0.01 Amp to about 10 Amp. some cases, the channels of the perforated membrane are capable of passing an electrical current through the perforated membrane or an electrical current is applied to the device. In some cases, the channels can pass an electrical current of at least about 0.01 amperes (Amp) to about 10 Amp. In some cases, the current is at least about 0.01 amperes (Amp) to about 0. 1 Amp. In some cases, the current is at least about 0.01 amperes (Amp) to about 0.5 Amp. In some cases, the channels can pass an electrical current of at least about 0.01 amperes (Amp) to about 1 Amp. In some cases, the current is at least about 0.01 amperes (Amp) to about 2 Amp. In some cases, the current is f at least about 0.01 amperes (Amp) to about 3 Amp. In some cases, the current is at least about 0.01 amperes (Amp) to about 4 Amp. In some cases, the channels canpass an electrical current of at least about 0.01 amperes (Amp) to about 5 Amp. In some cases, the current is an electrical current of at least about 0.01 amperes (Amp) to about 6 Amp. In some cases, the channels can pass an electrical current of at least about 0.01 amperes (Amp) to about 7 Amp. In some cases, the current is at least about 0.01 amperes (Amp) to about 8 Amp. In some cases, the current is of at least about 0.01 amperes (Amp) to about 9 Amp. In some cases, the current is at least about 0.01 amperes (Amp) to about 10 Amp. In some cases, the channels can pass an electrical current of at least about 0.01 Amp, 0.05 Amp, 0.1 Amp, 0.2 Amp, 0.3 Amp, 0.4 Amp, 0.5 Amp, 0.6 Amp, 0.7 Amp, 0.8 Amp, 0.9 Amp, or at least about 1 .0 Amp.

[0035] Accordingly, the individual channels of the plurality of channels comprise a diameter. In some cases, the channels have a diameter of at least about 100 nanometers (nm), 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1,000 nm, 1,100 nm, 1,200 nm, 1,300 nm, 1,400 nm, l,500 nm, 1,600 nm, 1,700 nm, 1,800 nm, 1,900 nm, 2,000 nm. In some cases, the channels have a diameter of at least about 100 nanometers (nm)to about 2,000 nm. In some cases, the channels have a diameter of at least about 200 nanometers (nm) to about 2,000 nm. In some cases, the channels have a diameter of at least about 200 nanometers (nm) to about 1 ,000 nm. In some cases, the channels have a diameter of at least about 200 nanometers (nm) to about 500 nm. In some cases, the channels have a diameter of at least about 200 nanometers (nm) to about 1,500 nm. In some cases, the channels have a diameter of at least about 1,000 nanometers (nm) to about 2,000 nm. In some cases, the channels have variable diameters throughout the perforated membrane. In some cases, the channels have consistent diameters throughout the perforated membrane. In some cases, the channels are nanochannels comprising a diameter of from about 100 nm to about 2,000 nm. In some cases, the channels comprise a diameter of no more thanabout 20 pm, no more than about 15 pm, about 10 pm, no more than about 8 pm, no more than about 5 pm, no more than about 4 pm, no more than about 3 pm, more than about 2 pm, or no more than about 1.5 pm.

[0036] In some cases, the channels comprise a diameter of from about 0.3 pm to about 50 pm, from about 0.3 pm to about 20 pm, from about 0.4 pm to about 10 pm, from about 0.4 pm to about 5 pm, from about 0.4 pm to about 2 pm, or preferably from about 0.4 pm to about 1.5 pm. In some cases, the channels are microchannels comprising a diameter from about 0.8 pm to about 10 pm, from about 0.8 pm to about 5 pm, or from about 1 pm to about 5 pm. In some cases, the individual channels of the plurality of channels comprise a diameter that is substantially constant throughout the length of the individual channels of the plurality of channels, spanning the distance between the first surface and the second surface of the perforated membrane. It is also conceived that the individual channels of the plurality of channels can have complex three-dimensional shapes. In some cases, the individual channels of the plurality of channels comprise a plurality of segments having different diameters, such that a diameter of a first segment is different than a diameter of a second segment.

[0037] In some cases, the plurality of channels of the perforated membrane comprises a nonpatterned arrangement or non-patterned distribution of the channels throughout the perforated membrane. In some cases, the plurality of channels of the perforated membrane comprise s a patterned distribution of the channels throughout the perforated membrane. In some cases, the plurality of channels of the perforated membrane comprises more than one patterned distribution of channels throughout the perforated membrane, whereby a first cross-sectional portion of the perforated membrane has a first distance between channels along the length and / or width of the perforated membrane and a second cross-sectional portion of the perforated membrane that has a second distance between channels along the length and / or width of the perforated membrane, wherein the second distance between channels of the second cross-sectional portion of the perforated membrane is different than the first distance between channels of the first cross- sectional portion of the perforated membrane.

[0038] The plurality of channels can be distributed throughout the perforated membrane in either homogeneous or heterogenous densities (i.e. number of individual channels per unit surface area of the perforated membrane). In some cases, the plurality of channels of the perforated membrane comprises a channel density of at least about 1 x 105channels per cm2. In some cases, the plurality of channels of the perforated membrane comprises a channel density of at most about 1 x 108channels per cm2. In some cases, the plurality of channels of the perforated membrane comprises a channel density from about 1 x 105channels per cm2to about 1 x 108channels percm2. In some cases, the plurality of channels of the perforated membrane comprises a channel density from about 5 * 105channels per cm2to about 5 * 106channels per cm2.

[0039] The perforated membrane has a thickness (i.e. height or distance along z-axis). In some cases, the thickness of the perforated membrane is at least about 10 pm and provides easy handling using tweezers during cell electroporation (e.g., cell electroporation / nanoelectroporation (CEP / CNP)) operations. In some cases, the perforated membrane has a thickness of at least about 5 micrometers (pm), at least about 10 pm, at least about 20 pm, at least about 30 pm, at least about 40 pm, or at least about 50 pm. In some cases, the thickness of the perforated membrane is from about 5 pm to about 50 pm. In some cases, the thickness of the perforated membrane is from about 5 pm to about 40 pm. In some cases, the thickness of the perforated membrane is from about 5 pm to about 30 pm. In some cases, the thickness of the perforated membrane is from about 5 pm to about 20 pm. In some cases, the thickness of the perforated membrane is from about 5 pm to about 10 pm.

[0040] The perforated membrane has size dimensions defined by a width and a length of the first surface distances (i.e. distances along the x-axis andy-axis of the first surface). In some cases, the first surface of the perforated membrane has a length of at least about 2 cm. In some cases, the first surface of the perforated membrane has a length of at no more than about 100 cm. In some cases, the first surface of the perforated membrane has a length of from about 2 cm to about 30 cm or from about 5 cm to about 15 cm. In some cases, the first surface of the perforated membrane has a length of about 7 cm. In some cases, the first surface of the perforated membrane has a width of at least about 2 cm. In some cases, the first surface of the perforated membrane has a width of at no more than about 100 cm. In some cases, the first surface of the perforated membrane has a width of from about 2 cm to about 30 cm or from about 5 cm to about 15 cm. In some cases, the first surface of the perforated membrane has a width of about 7 cm.

[0041] In some cases, distances along the x-axis andy-axis of the second surface of the perforated membrane are substantially identical to the distances along the x-axis and y-axis of the first surface.

[0042] In some cases, the perforated membrane comprising comprises a single layer, such that the perforated membrane is made of a single material or a single chemical composition. In some cases, the perforated membrane comprising comprises a single layer, such that the perforated membrane is made of a homogeneous concentration of the single material or single chemical composition. In some case, the perforated membrane comprises at least one layer, such that the perforated membrane is made of one or more layers, whereby each layer is a distinct cross- sectional segment along the z-axis (i.e. height) of the perforated membrane that has distinct features from an adjacent layer or a second layer along the z-axis of the perforated membrane. Adistinct feature of a first layer of the perforated membrane from a second layer of the perforated membrane can include, for example, a different diameter of channels, a different thickness (i.e. height or distance alone z-axis), or a different material or chemical composition.

[0043] The perforated membrane described herein can comprise a polymer material, a silicon material, or an Aluminum Oxide material. More preferably, the perforated membrane comprises a polymer material. In some cases, the perforated membrane comprises polycarbonate, polyester, or polyimide.

[0044] Accordingly, the perforated membrane is configured to electroporate a donor cell with a transfection reagent, including transfection reagents such as a DNA, a RNA, a nucleic acid, a synthetic nucleic acid molecule, other biomolecule, or a small molecule.First layer

[0045] In some cases, the perforated membrane comprises a first layer, wherein the first layer comprises a plurality of first channels disposed through the first layer.

[0046] The first layer of the perforated membrane has a first average thickness (i.e. height or distance along the z-axis). In some cases, the first average thickness of the first layer of the perforated membrane is at least about 1 pm. In some cases, the first average thickness of the first layer of the perforated membrane is from about 1 pm to about 5,000 pm. In some cases, the first average thickness of the first layer of the perforated membrane is from about 1 pm to about 1 ,000 pm. In some cases, the first average thickness of the first layer of the perforated membrane is from about 1 pm to about 200 pm. In some cases, the first average thickness of the first layer of the perforated membrane is from about 1 pm to about 100 pm, 1 pm to about 50 pm, 1 pm to about 40 pm, 1 pm to about 30 pm, 1 pm to about 20 pm, 1 pm to about 10 pm, or 1 pm to about 5 pm. In some cases, the first average thickness of the first layer of the perforated membrane is about 20 pm, about 15 pm, about 12 pm, about 10 pm, about 8 pm, about 6 pm, about 5 pm, about 4 pm, about 3 pm, about 2 pm, or about 1 pm. In some cases, the first average thickness of the first layer of the perforated membrane is at most about 5,000 pm, about 1,000 pm, about 500 pm, about 200 pm, about 100 pm, about 50 pm, about 20 pm, about 10 pm, about 5 pm.

[0047] In some cases, the plurality of first channels comprises individual channels of having a first average diameter. In some cases, the plurality of first channels comprises individual channels of having a first average diameter that is sub stantially consistent among the individual channels of the plurality of first throughout the first layer of the perforated membrane. In some cases, the first average diameter is at least about 1 pm. In some cases, the first average diameter is no more than about 20 pm. In some cases, the first average diameter is from about 1 pm toabout 20 pm, from about 3 pm to about 10 pm, from about 4 pm to about 8 pm, or from about 5 pm to about 7 pm.

[0048] The first channels also have an aspect ratio, defined as the ratio of the first average diameter and the first average thickness of the first channels. In some cases, the first average diameter is about half of the first average thickness. In some cases, the first average diameter is about one-third of the first average thickness of the first channels.

[0049] In some cases, the plurality of first channels disposed through the first layer comprises a first array of channels. In some cases, the first array of channels comprises individual channels of having a first average diameter. In some cases, the first array of channels comprises individual channels of having a first average diameter that is substantially consistent among the individual channels of the array of channels throughout the first layer of the perforated membrane.

[0050] The first layer of the perforated membrane is made of materials or chemical compositions. In some cases, the first layer of the perforated membrane comprises materials that give the first layer a rigid structure. Alternatively, in some cases, the first layer of the perforated membrane comprises materials that give the first layer a flexible structure. In some cases, the first layer of the perforated membrane comprises a first polymer material, a silicon material, an aluminum oxide material, glass, a ceramic material, gold, copper, or other biomaterials. In some cases, the first layer comprises a first polymer material. In some cases, the first layer comprises a first thermoplastic material, first light-reactive polymer, first crosslinking polymer, first synthetic polymer, a first thermosetting polymer, a first light-curing, a first thermal-curing polymer, or a first photoresist. The first layer preferably comprises a first photoresist. In some cases, the first photoresist comprises a positive photoresist. In some cases, the first photoresist comprises a negative photoresist. In some cases, the first photoresist comprises a first SU -8 photoresist, such as a first SU-8 3000-series photoresist. In some cases, the first layer comprises a SU-8 3010 photoresist or a SU-8 3005 photoresist. In some cases, the first layer comprises a first SU-8 2000-series photoresist, such as a SU-8 2005 photoresist, for example. It is conceived that the first layer of the perforated membrane can comprise any positive photoresist or negative photoresist can be appropriately used to provide a first layer having a plurality of channels, a thickness, a length, and a width.Second layer

[0051] In some cases, the perforated membrane comprises a second layer, wherein the second layer in contact with the first layer of the perforated membrane and comprises a plurality of second channels disposed through the second layer.

[0052] The second layer of the perforated membrane has a second average thickness (i.e. height or distance alone z-axis). In some cases, the second average thickness of the second layer isdifferent from the first average thickness of the first layer of the perforated membrane. In some cases, the first average thickness of the first layer is larger than the second average thickness of the second layer. For example, the first average thickness of the first layer is at least twice or at least three times larger than the second average thickness of the second layer. In some instances, the second average thickness of the second layer is at least about 100 nm. In some instances, the second average thickness of the second layer is no more than about 10 pm. In some instances, the second average thickness of the second layer is from about 300 nm to about 7 pm, from about 500 nm to about 5 pm, from about 600 nm to about 3 pm, from about 700 nm to about 2 pm, or from about 700 nmto about 1.5 pm. In some cases, the second average thickness of the second layer of the perforated membrane is about 700 nm, 800 nm, 1 pm, about 2 pm, about 3 pm, or about 4 pm.

[0053] In some cases, the plurality of second channels comprises individual channels of having a second average diameter. In some cases, the plurality of second channels comprises individual channels of having a second average diameter that is substantially consistent among the individual channels of the plurality of second channels throughout the second layer of the perforated membrane. In some instances, the second average diameter of the second layer is at least about 100 nm. In some instances, the second average diameter of the second layer is no more than about 5 pm. In some cases, the second average diameter of the second layer is from about 100 nm to about 5 pm. In some cases, the second average diameter of the second layer is from about 300 nm to about 1.5 pm, from about 400 nm to about 1 .2 pm, or preferably from about 600 nm to about 1 pm.

[0054] The second channels also have an aspect ratio, defined as the ratio of the second average diameter and the second average thickness of the second channels. In some cases, the second average diameter is no more than half of the second average thickness of the second layer of the perforated membrane. In some cases, wherein the second average diameter is no more than one- third of the second average thickness. In some cases, the second average diameter is from about one-half to about one-third of the second average thickness or from about one-third to about one- fourth of the second average thickness. In some cases, the second average diameter is no more than half or no more than about 25% of the second average thickness.

[0055] In some cases, the plurality of second channels disposed through the second layer comprises a second array of channels. In some cases, the second array of channels comprises individual channels of having a second average diameter. In some cases, the second array of channels comprises individual channels of having a second average diameter that is substantially consistent among the individual channels of the array of channels throughout the second layer of the perforated membrane.

[0056] In some cases, the first average diameter of the first channel of the first layer of the perforated membrane is different from the second average diameter of the second channel of the second layer of the perforate membrane. In some cases, the first average diameter of the first channel of the first layer is larger than the second average diameter of the second channel of the second layer. For example, the first average diameter can be at least twice, three times, or four times larger than the second average diameter of the second channel of the second layer. There can also be a range of variability in the diameter of the first channels of the first layer and the second channels of the second layer. For example, the first average diameter of the first channel of the first layer can be from about twice to about three times, or about three times to about four times larger than the second average diameter of the second channel of the second layer of the perforated membrane.

[0057] The second layer of the perforated membrane is made of materials or chemical compositions. In some cases, the second layer of the perforated membrane comprises materials that give the second layer a rigid structure. Alternatively, in some cases, the second layer of the perforated membrane comprises materials that give the second layer a flexible structure. In some cases, the second layer of the perforated membrane comprises a second polymer material, a silicon material, an aluminum oxide material, glass, a ceramic material, gold, copper, or other biomaterials. In some cases, the second layer comprises a second polymer material. In some cases, the second layer comprises a second thermoplastic material, second light-reactive polymer, second crosslinking polymer, second synthetic polymer, a second thermosetting polymer, a second light-curing, a second thermal-curing polymer, or a second photoresist. The second layer preferably comprises a second photoresist. In some cases, the second photoresist comprises a positive photoresist. In some cases, the second photoresist comprises a negative photoresist. In some cases, the second polymer material comprises a SU-8 TF 6000-series photoresist, such as a SU-8 TF 6002 photoresist. In some cases, the second polymer material comprises a second SU-8 2000-series photoresist, including but not limited to a SU-8 2002 photoresist.Differences between the first layer and the second layer

[0058] Some embodiments of the perforated membrane include the first layer of the perforated membrane being distinct from the second layer. For example, in some cases, the first layer comprises at least one ingredient different from ingredients of the second layer materials. In some cases, the first layer is made of materials different from those of the second layer. Such differences give the first layer and second layer distinct structural properties. For example, in some cases, a first tensile strength of the first layer is larger than a second tensile strength of the second layer.

[0059] For example, in some instances, the first layer comprises the first photoresist, and the second layer comprises the second photoresist. More specifically, the first layer comprises the first negative photoresist, and wherein the second layer comprises the second negative photoresist. For instance, the first layer comprises the first SU-8 photoresist, and the second layer comprises the second SU-8 photoresist. A variety of combinations of first SU-8 photoresists of the first layer and second SU-8 photoresists of the second layer are compatible in accordance with the embodiments of the perforated membrane of this disclosure. In some cases, the first layer comprises the SU-8 3010 photoresist, the SU-8 3005 photoresist, or the SU-8 2005 photoresist, and wherein the second layer comprises the SU-8 TF 6002 photoresist or the SU-8 2002 photoresist. It is also conceived that the first layer comprises the first positive photoresist, and wherein the second layer comprises the second positive photoresist.Similarities between the first layer and the second layer

[0060] Alternatively, according to some embodiments, the first layer and the second layer of the perforated membrane have similar features. In some cases, the first layer and the second layer are made from photoresist polymers. In some cases, the first layer is made of the same materials as those of the second layer. For instance, in some embodiments, the first layer and the second layer comprise the first light-reactive polymer, the first crosslinking polymer, the first synthetic polymer, or the first light-curing polymer.

[0061] Generally, a first channel of the plurality of first channels is in fluid communication of a second channel of the plurality of second channels. In cases where the first plurality of channels of the first layer and the second plurality of the second layer form contiguous channels that span the distance between the first surface and the second surface (i.e. thickness) of the perforated membrane.II. Methods of Manufacturing Perforated Membranes

[0062] Also described herein is a method of making a perforated membrane of the instant disclosure, including a perforated membrane having a first layer and a perforated membrane having a first layer and a second layer, as previously described. In some cases, the method comprises coating a first material onto a substrate. In some cases, spin coating, blade coating, push coating, drop casting, or spray coating methods can be used for coating the first layer of the first material onto the substrate. In some cases, the method of making a perforated membrane comprises coating a first material onto a substrate; and coating a second layer of a second material onto the first material. In some cases, spin coating, blade coating, push coating, drop casting, or spray coating methods can be used for coating the second layer of the second material onto the substrate.

[0063] Yet another aspect provided by the instant disclosure is a method of making a perforated membrane from photoresist materials, including a perforated membrane having a first layer and a perforated membrane having a first layer and a second layer, as previously described. Described herein, according to some embodiments, the method of making a perforated membrane from photoresist materials comprises coating a first layer of a first photoresist material onto a substrate; and coating a second layer of a second photoresist material onto a substrate. In some cases, spin coating, blade coating, push coating, drop casting, or spray coating methods can be used for coating the first layer of the first material onto the substrate. In some cases, spin coating blade coating, push coating, drop casting, or spray coating methods can be used for coating the second layer of the second material onto the substrate. In some cases, the first layer has the features of the first layer of a perforated membrane as described above.

[0064] The first material can be any of the aforementioned materials for making a first layer of a perforated membrane (see also Section I: Perforated Membranes describing a second layer of a perforated membrane). In some cases, the first material used to make the first layer of the perforated membrane comprises materials that give the first layer of the perforated membrane a rigid structure. Alternatively, in some cases, the first layer of the perforated membrane comprises materials that give the first layer a flexible structure. In some cases, the first material for making the first layer of the perforated membrane comprises a first polymer material, a silicon material, an aluminum oxide material, glass, a ceramic material, gold, copper, a track-etched membrane, or other biomaterials. In some cases, the first material comprises a first polymer material. In some cases, the first material comprises a first thermoplastic material, first light-reactive polymer, first crosslinking polymer, first synthetic polymer, a first thermosetting polymer, a first light-curing, a first thermal-curing polymer, or a first photoresist. The first material preferably comprises a first photoresist. In some cases, the first photoresist comprises a first positive photoresist. In some cases, the first material comprises a negative photoresist. In some cases, the first polymer material comprises a SU-8 TF 6000-series photoresist, such as a SU-8 TF 6002 photoresist. In some cases, the first polymer material comprises a first SU-8 2000-series photoresist, including but not limited to a SU-8 2002 photoresist. In some cases, the first polymer material comprises a negative photoresist, including first SU-8 TF 6000-series photoresist (e.g., a SU-8 TF 6002 photoresist, etc.) or a first SU-8 2000-series photoresist (e.g., SU-8 2002 photoresist).

[0065] The second material can be any of the aforementioned materials for making a second layer of a perforated membrane (see also Section I: Perforated Membranes describing a first layer of a perforated membrane). In some cases, the second material used to make the second layer of the perforated membrane comprises materials that give the second layer a rigid structure. Alternatively, in some cases, the second layer of the perforated membrane comprises materialsthat give the second layer a flexible structure. In some cases, the second material for making the second layer of the perforated membrane comprises a second polymer material, a track-etched membrane a silicon material, an aluminum oxide material, glass, a ceramic material, gold, copper, or other biomaterials. In some cases, the second material comprises a second polymer material. In some cases, the second material comprises a second thermoplastic material, second light-reactive polymer, second crosslinking polymer, second synthetic polymer, a second thermosetting polymer, a second light-curing, a second thermal-curing polymer, or a second photoresist. The second material preferably comprises a second photoresist. In some cases, the second photoresist comprises a second positive photoresist. In some cases, the second material comprises a negative photoresist. In some cases, the second material comprises a second SU-8 photoresist, such as a second SU-8 3000-series photoresist. In some cases, the second material comprises a SU-8 3010 photoresist or a SU-8 3005 photoresist. In some cases, the second material comprises a second SU-8 2000-series photoresist, such as a SU-8 2005 photoresist, for example. In some cases, the second negative photoresist comprises a second SU-8 3000-series photoresist (e.g., a SU-8 3010 photoresist, a SU-8 3005 photoresist, etc.) or a second SU-8 2000-series photoresist (e.g., a SU-8 2005 photoresist, etc.).

[0066] In some cases, the first material is a first photoresist material, and the second material is a second photoresist material, wherein the first photoresist material is different from the second photoresist material. For example, in some cases, the first photoresist material has a higher resolution that the second photoresist material.

[0067] In some cases, the coating the first layer of the first photoresist material onto the substrate comprises (a) spin coating a first layer of a first photoresist material onto a substrate. For example, the substrate is a silicon membrane, preferably a silicon membrane coated with a release layer that allows separation of the first photoresist material from the substrate. It is contemplated that any substrate having a rigid structure can be used. The first photoresist material can comprise any of the aforementioned photoresist materials for the first layer of the perforated membrane (see also Section I: Perforated Membranes describing a second layer of a perforated membrane).

[0068] In some cases, the release layer comprises a photoresist release layer. In some cases, the release layer comprises a SU-8 photoresist release layer. For example, the release layer can be OmniCoat, AZ 15nXT, a metal such as copper, chromium, or aluminum, silicon dioxide, or polymers, including polystyrene, PDMS (Poly dimethylsiloxane), SAMs (self-assembled molecules), OmniCoat, PMGI (Polydimethylglutarimide).

[0069] In some cases, the spin coating in (a) coats the first layer onto the release layer. In some cases, the spin coating comprises spin coating the first photoresist material at a rotational speedof from about 1,000 RPMto about 10,000 RPM. In some cases, the spin coating comprises spin coating the first photoresist material at a rotational speed of about 4,000 RPM.

[0070] In some cases, the method further comprises: (b) soft baking the first layer obtained in (a), whereby (a) is the step of spin coating a first layer of a first photoresist material onto a substrate. In some cases, the first layer obtained in (a) comprises a layer of a perforated membrane having a plurality of channels, whereby the channels of the plurality of channels have an average diameter from about 300 nm to about 1.5 pm, from about 400 nm to about 1 .2 pm, or preferably from about 600 nm to about 1 pm (e.g., smaller-diameter channel segment in FIG. 3). In some cases, the soft baking comprises soft baking the first layer for at least from about 1 minute to about 60 minutes or from about 1 minute to about 10 minutes, preferably from about 2 minutes to about 4 minutes. In some cases, the soft baking comprises soft baking the first layer for no more than about 60 minutes, no more than about 20 minutes, no more than about 10 minutes, or no more than about 5 minutes. In some cases, the soft baking comprises soft baking the first layer for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, or about 5 minutes. In some cases, the soft baking comprises soft baking the first layer for about 3 minutes.

[0071] In some cases, the soft baking comprises softbakingthe first layer at a temperature of at least about 90°C. In some cases, the soft baking comprises soft baking the first layer at a temperature of no more than about 200°C. In some cases, the soft baking comprises soft baking the first layer at a temperature of from about 90°C to about 140°C or from about 100°C to about 120°C. Preferably, the soft baking comprises softbakingthe first layer at a temperature of about 110°C.

[0072] In some cases, the method further comprises: (c) exposing the soft baked first layer obtained in (b) to a first UV radiation using a first photomask.

[0073] In some cases, the first photomask comprises fused quartz, chromium, borosilicate, glass, or polyethylene terephthalate (PET).First photomask

[0074] The first photomask comprises a width and a length (i.e. distances along the x-axis and y- axis of the first surface). In some cases, the first photomask has a length of at least about 2 cm. In some cases, the first photomask has a length of at no more than about 100 cm. In some cases, the first photomask has a length of from about 2 cm to about 30 cm or from about 5 cm to about 15 cm. In some cases, the first photomask has a length of about 7 cm. In some cases, the first photomask has a width of at least about 2 cm. In some cases, the first photomask has a width of at no more than about 100 cm. In some cases, the first photomask has a width of from about 2 cm to about 30 cm or from about 5 cm to about 15 cm. In some cases, the first photomask has a width of about 7 cm.

[0075] The first photomask comprises a first pattern. In some cases, the first pattern of the first photo mask comprises plurality of first holes. The first holes can, for example, comprise a circular shape, a square shape, a rectangular shape, a triangular shape, or any geometric shape. Accordingly, the individual holes of the plurality of first holes comprise a diameter. In some cases, the plurality of first holes comprises individual holes of having a first average diameter. In some cases, the plurality of first holes comprises individual holes of having a first average diameter that is substantially consistent among the individual holes of the plurality of first holes throughout the first photomask. In some instances, the first average diameter of the first photomask is at least about 100 nm. In some instances, the first diameter of the first photomask is no more than about 5 pm. In some cases, the first average diameter of the first photomask is from about 100 nm to about 5 pm. In some cases, the first average diameter of the first photomask is from about 300 nm to about 1.5 pm, from about 400 nm to about 1.2 pm, or preferably from about 600 nm to about 1 pm.

[0076] In some cases, the individual first holes of the first photomask comprise a diameter that is substantially identical to other individual first holes of the first photomask.

[0077] In some cases, the plurality of first holes of the first photomask comprises a non-patterned arrangement or non-patterned distribution of the first holes throughout the first photomask. In some cases, the plurality of first holes of the first photomask comprises a patterned distribution of the first holes throughout the first photomask. In some cases, the plurality of first holes of the first photomask comprises more than one patterned distribution of first holes throughout the first photomask, whereby a first cross-sectional portion of the first photomask has a first distance between first holes along the length and / or width of the first photomask and a second cross- sectional portion of the first photomask that has a second distance between first holes along the length and / or width of the first photomask, wherein the second distance between first holes of the second cross-sectional portion of the first photomask is different than the first distance between first holes of the first cross-sectional portion of the first photomask.

[0078] The plurality of first holes can be distributed throughout the first photomask in either homogeneous or heterogenous densities (i.e. number of individual first holes per unit surface area of the first photomask). In some cases, the plurality of first holes of the first photomask comprises a hole density of at least about 1 x 105first holes per cm2. In some cases, the plurality of first holes of the first photomask comprises a hole density of at most about 1 x 108first holes per cm2. In some cases, the plurality of first holes of the first photomask comprises a hole density from about 1 x 105first holes per cm2to about 1 x 108first holes per cm2. In some cases, the plurality of first holes of the first photomask comprises a hole density from about 5 x 105first holes per cm2to about 5 x 106first holes per cm2.

[0079] In some cases, the method of making a perforated membrane further comprises : (d) spin coating a second layer of a second photoresist material onto the first layer after (c). The second photoresist material can comprise any of the aforementioned photoresist materials for the second layer of the perforated membrane see also Section I: Perforated Membranes describing a first layer of a perforated membrane).

[0080] In some cases, the spin coating in (d) coats the second layer onto the first layer. In some cases, the spin coating in (d) comprises spin coating the second photoresist material at a rotational speed of from about 1,000 RPM to about 10,000 RPM. In some cases, the spin coating comprises spin coating the second photoresist material at a rotational speed of about 4,000 RPM.

[0081] In some cases, the method of making the perforated membrane further comprises: after (c) but before (d), conducting another post exposure bake.

[0082] In some cases, the conducting another post exposure bake comprises post exposure baking the first layer for at least from about 1 minute to about 60 minutes or from about 1 minute to about 10 minutes, preferably from about 1 minutes to about 4 minutes. In some cases, the another post exposure baking comprises post exposure baking the first layer for no more than 60 minutes, no more than 20 minutes, no more than 10 minutes, no more than 5 minutes. In some cases, the post exposure baking comprises post exposure baking the first layer for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, or about 5 minutes. In some cases, the post exposure baking comprises post exposure baking the first layer for about 3 minutes.

[0083] In some cases, the post exposure baking comprises post exposure baking the first layer at a temperature of at least about 90°C. In some cases, the post exposure baking comprises post exposure baking the first layer at a temperature of no more than about 200°C. In some cases, the post exposure baking comprises post exposure baking the first layer at a temperature of from about 90°C to about 140°C or from about 100°C to about 120°C. Preferably, the post exposure baking comprises post exposure baking the first layer at a temperature of about 110°C.

[0084] In some cases, the method of making the perforated membrane further comprises: after (c) but before (d), coating a barrier layer on top of the first layer. The barrier layer can, for example, comprise silicon oxide, gold, silver, or any other barrier layer known in the art.

[0085] In some cases, the method of making the perforated membrane further comprises: (e) soft baking the second layer obtained in (d).

[0086] In some cases, the soft baking in (e) comprises soft baking the second layer for at least from about 1 minute to about 60 minutes or from about 1 minute to about 10 minutes, preferably from about 5 minutes to about 9 minutes. In some cases, the soft baking in (e) comprises soft baking the second layer for no more than 60 minutes, no more than 20 minutes, no more than 10minutes, or no more than 8 minutes. Preferably, the soft baking in (e) comprises soft baking the second layer for about 7 minutes.

[0087] In some cases, the soft baking in (e) comprises soft baking the first layer at a temperature of at least about 70°C. In some cases, the soft baking in (e) comprises soft baking the first layer at a temperature of no more than about 200°C. In some cases, the soft baking in (e) comprises softbakingthe first layer at a temperature of from about 85°C to about 105°C. Preferably, the soft baking in (e) comprises soft baking the first layer at a temperature of about 95°C.

[0088] In some cases, the method of making the perforated membrane further comprises: (f) exposing the soft baked second layer obtained in (e) to another UV radiation using a second photomask.Second photomask

[0089] The second photomask comprises a width and a length (i.e. distances along the x-axis and y-axis of the second surface). In some cases, the second photomask has a length of at least about 2 cm. In some cases, the second photomask has a length of at no more than about 100 cm. In some cases, the second photomask has a length of from about 2 cm to about 30 cm or from about 5 cm to about 15 cm. In some cases, the second photomask has a length of about 7 cm. In some cases, the second photomask has a width of at least about 2 cm. In some cases, the second photomask has a width of at no more than about 100 cm. In some cases, the second photomask has a width of from about 2 cm to about 30 cm or from about 5 cm to about 15 cm. In some cases, the second photomask has a width of about 7 cm. In some cases, the second photo mask comprises a width and a length that are substantially the same to the width and the length of the second photomask.

[0090] The second photomask comprises a second pattern. The second photomask comprises a second pattern. In some cases, the second pattern of the second photomask comprises plurality of second holes. Accordingly, the individual holes of the plurality of second holes comprise a diameter. In some cases, the plurality of second holes comprises individual holes of having a second average diameter. In some cases, the plurality of second holes comprises individual holes of having a second average diameter that is substantially consistent among the individual holes of the plurality of second holes throughout the second photomask. The second holes can, for example, comprise a circular shape, a square shape, a rectangular shape, a triangular shape, or any geometric shape. Preferably, the second holes of the second photomask have substantially similar shapes to the first holes of the first photomask.

[0091] In some cases, the plurality of second holes comprises individual holes of having a second average diameter. In some cases, the plurality of second holes comprises individual holes of having a second average diameter that is substantially consistent among the individual holes of the plurality of second throughout the second layer of the perforated membrane. In somecases, the second average diameter is at least about 1 gm. In some cases, the second average diameter is no more than about 20 gm. In some cases, the second average diameter is from about 1 gm to about 20 gm, from about 3 gm to about 10 gm, from about 4 gm to about 8 gm, or from about 5 gm to about 7 gm.

[0092] In some cases, the plurality of second holes of the second photomask comprises a nonpatterned arrangement or non-pattemed distribution of the second holes throughout the second photomask. In some cases, the plurality of second holes of the second photomask comprises a patterned distribution of the second holes throughout the second photomask. In some cases, the plurality of second holes of the second photomask comprises more than one patterned distribution of second holes throughout the second photomask, whereby a second cross-sectional portion of the second photomask has a second distance between second holes along the length and / or width of the second photomask and a second cross-sectional portion of the second photomask that has a second distance between second holes along the length and / or width of the second photomask, wherein the second distance between second holes of the second cross-sectional portion of the second photomask is different than the second distance between second holes of the second cross- sectional portion of the second photomask. Preferably, the individual second holes of the plurality of second holes comprise a diameterthat is substantially identical to other individual second holes of the second photomask.

[0093] The plurality of second holes can be distributed throughout the second photomask in either homogeneous or heterogenous densities (i.e. number of individual second holes per unit surface area of the second photomask). In some cases, the plurality of second holes of the second photomask comprises a hole density of at least about 1 x 105second holes per cm2. In some cases, the plurality of second holes of the second photomask comprises a hole density of at most about 1 x 108second holes per cm2. In some cases, the plurality of second holes of the second photomask comprises a hole density from about 1 x 105second holes per cm2to about 1 x io8second holes per cm2. In some cases, the plurality of second holes of the second photomask comprises a hole density from about 5 x io5second holes per cm2to about 5 x io6second holes per cm2. Preferably, the distribution and density of the individual second holes of the second photomask are substantially identical to the distribution and density of the individual first holes of the first photomask.

[0094] In some cases, the method of making the perforated membrane further comprises: (g) conducting a post exposure bake of the second layer. In some cases, the conducting the post exposure bake in (g) comprises post exposure baking the second layer for at least from about 0.5 minutes to about 60 minutes or from about 0.5 minutes to about 10 minutes, preferably from about 1 minutes to about 3 minutes. In some cases, the s post exposure baking comprises postexposure baking the second layer for no more than 60 minutes, no more than 20 minutes, no more than 10 minutes, no more than 5 minutes. In some cases, the post exposure baking comprises post exposure baking the second layer for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, or about 5 minutes. In some cases, the post exposure baking comprises post exposure baking the second layer for about 3 minutes.

[0095] In some cases, the post exposure baking comprises post exposure baking the second layer at a temperature of at least about 50°C. In some cases, the conducting the post exposure bake in(g) comprises post exposure baking the second layer at a temperature of no more than about 200°C. In some cases, the conducting the post exposure bake in (g) comprises a first step of post exposure baking the second layer comprising post exposure baking for about 0.5 minutes to about 2 minutes at a temperature of from about 50°C to about 90°C or from about 60°C to about 80°C; and a second step for post exposure baking the second layer comprising post exposure baking for about 1 minutes to about 5 minutes at a temperature of from about 70°C to about 100°C or from about 85°C to about 105°C, or preferably for about 2 minutes at about 95°C.

[0096] In some cases, the method of making the perforated membrane further comprises: (h) developing both the first layer and the second layer after (g). In some cases, the developing comprises developing the first photoresist material and the second photoresist material simultaneously. The developer solution can comprise an alkaline substance, a promoter substance, or an inhibitor substance.

[0097] In some cases, the first pattern of the first photomask is configured to produce a plurality of first channels disposed in the first layer after (h), and wherein the second pattern of the first photomask is configured to produce a plurality of second channels disposed in the second layer after (h).

[0098] In some cases, the method of making the perforated membrane further comprises: after(h), removing the release layer.

[0099] In some cases, the method of making the perforated membrane results in the perforated membrane, wherein the perforated membrane comprises a top layer comprising a plurality of top channels disposed through the top layer; and a bottom layer in contact with the top layer, the bottom layer comprising a plurality of bottom channels disposed through the bottom layer; wherein: a first average thickness of the top layer is different from a second average thickness of the bottom layer; a first channel of the plurality of top channels is in fluid communication of a second channel of the plurality of bottom channels; a first average diameter of the first channel is different from a second average diameter of the second channel.

[0100] In some cases, the method of making a perforated membrane from photoresist materials, comprises: (a) spin coating a first layer of a first photoresist material onto a substrate; (b) softbaking the first layer obtained in (a); (c) exposing the soft baked first layer obtained in (b) to a first UV radiation using a first photomask; (d) spin coating a second layer of a second photoresist material onto the firstlayer after (c); (e) soft baking the second layer obtained in (d); (f) exposing the soft baked second layer obtained in (e) to another UV radiation using a second photomask; (g) conducting a post exposure bake; and (h) developing both the first layer and the second layer after (g), thereby making a perforated membrane comprising two layers.Polymer Membranes by Sacrificial Template Imprinting

[0101] This disclosure further provides a method of making a perforated membrane from a sacrificial template. The method of making a perforated membrane from a sacrificial template comprises: (a) providing a female mold comprising a female pattern; (b) preparing a sacrificial male mold based on the female mold; and (c) preparing a perforated membrane based on the sacrificial male mold, wherein the perforated membrane comprises another pattern.

[0102] The method can produce a perforated membrane, such as a polymer membrane, having channels disposed through the perforated membrane, an average thickness.

[0103] The female mold comprising a female pattern has the desired features of the perforated membrane, such as those described of perforated membrane provided above. In some cases, the pattern of the female mold is essentially the same as the pattern of the perforated membrane.

[0104] Generally, the female mold is a flattened structure having a first surface for contacting the sacrificial male mold material and a second surface opposed the first surface. The first surface and the second surface are generally substantively parallel to one another, and the planes of the first surface and the second surface are generally perpendicular to the height of the female mold. In some cases, the female mold is a rigid female mold. Alternatively, in some cases, the female mold is a flexible female mold. Generally, the female mold comprises a plurality of channels that can generate the channels of the desired perforated membrane that can pass a substance from the first surface to the second surface of the perforated membrane (i.e. through the perforated membrane). Individual channels of the female mold can comprise a cylindrical shape, a cuboid shape, or any other three-dimensional shape that can generate a perforated membrane having channels that allow passage of a substance, voltage, or electrical current from the first surface to the second surface of the perforated membrane.

[0105] Accordingly, the individual channels of the plurality of channels comprise a diameter. In some cases, the channels have a diameter of at least about 100 nanometers (nm), 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1,000 nm, 1,100 nm, 1,200 nm, 1,300 nm, 1,400 nm, l,500 nm, 1,600 nm, 1,700 nm, 1,800 nm, 1,900 nm, 2,000 nm. In some cases, the channels have a diameter of at least about 100 nanometers (nm)to about 2,000 nm. In some cases, the channels have a diameter of at least about 200 nanometers (nm) to about 2,000 nm. In somecases, the channels have a diameter of at least about 200 nanometers (nm) to about 1 ,000 nm. In some cases, the channels have a diameter of at least about 200 nanometers (nm) to about 500 nm. In some cases, the channels have a diameter of at least about 200 nanometers (nm) to about 1,500 nm. In some cases, the channels have a diameter of at least about 1 ,000 nanometers (nm) to about 2,000 nm. In some cases, the channels have variable diameters throughout the female mold. In some cases, the channels have consistent diameters throughout the female mold. In some cases, the channels are nanochannels comprising a diameter of from about 100 nm to about 2,000 nm. In some cases, the channels comprise a diameter of no more than about 20 pm, no more than about 15 pm, about 10 pm, no more than about 8 pm, no more than about 5 pm, no more than about 4 pm, no more than about 3 pm, more than about 2 pm, or no more than about 1.5 pm.

[0106] In some cases, the channels comprise a diameter of from about 0.3 pm to about 50 pm, from about 0.3 pm to about 20 pm, from about 0.4 pm to about 10 pm, from about 0.4 pm to about 5 pm, from about 0.4 pm to about 2 pm, or preferably from about 0.4 pm to about 1.5 pm. In some cases, the channels are microchannels comprising a diameter from about 0.8 pm to about 10 pm, from about 0.8 pm to about 5 pm, or from about 1 pm to about 5 pm. In some cases, the individual channels of the plurality of channels comprise a diameter that is substantially constant throughout the length of the individual channels of the plurality of channels. It is also conceived that the individual channels of the plurality of channels can have complex three-dimensional shapes. In some cases, the individual channels of the plurality of channels comprise a plurality of segments having different diameters, such at a diameter of a first segment is different than a diameter of a second segment.

[0107] In some cases, the plurality of channels of the female mold comprises a non -patterned arrangement or non-pattemed distribution of the channels throughout the female mold. In some cases, the plurality of channels of the female mold comprises a patterned distribution of the channels throughout the female mold. In some cases, the plurality of channels of the female mold comprises more than one patterned distribution of channels throughout the female mold, whereby a first cross-sectional portion of the female mold has a first distance between channels along the length and / or width of the female mold and a second cross-sectional portion of the female mold that has a second distance between channels along the length and / or width of the female mold, wherein the second distance between channels of the second cross-sectional portion of the female mold is different than the first distance between channels of the first cross-sectional portion of the female mold.

[0108] The plurality of channels can be distributed throughout the female mold in either homogeneous or heterogenous densities (i.e. number of individual channels per unit surface area of the female mold). In some cases, the plurality of channels of the female mold comprises achannel density of at least about 1 x 105channels per cm2. In some cases, the plurality of channels of the female mold comprises a channel density of at most about 1 x 108channels per cm2. In some cases, the plurality of channels of the female mold comprises a channel density from about 1 x IQs channels per cm2to about 1 x io8channels per cm2. In some cases, the plurality of channels ofthe female mold comprises a channel density from about 5 x 105channels per cm2to about 5 x io6channels per cm2.

[0109] The female mold has size dimensions definedby a width and a length of the first surface distances (i.e. distances along the x-axis and y-axis of the first surface). In some cases, the first surface of the female mold has a length of at least about 2 cm. In some cases, the first surface of the female mold has a length of at no more than about 100 cm. In some cases, the first surface of the female mold has a length of from about 2 cm to about 30 cm or from about 5 cm to about 15 cm. In some cases, the first surface of the female mold has a length of about 7 cm. In some cases, the first surface of the female mold has a width of at least about 2 cm. In some cases, the first surface of the female mold has a width of at no more than about 100 cm. In some cases, the first surface of the female mold has a width of from about 2 cm to about 30 cm or from about 5 cm to about 15 cm. In some cases, the first surface of the female mold has a width of about 7 cm.

[0110] In some cases, distances alongthe x-axis and y-axis of the second surface of the female mold are substantially identical to the distances along the x-axis and y-axis of the first surface. [OHl] In some cases, the female mold comprising comprises a single layer, such that the female mold is made of a single material or a single chemical composition. In some cases, the female mold comprising comprises a single layer, such that the female mold is made of a homogeneous concentration of the single material or single chemical composition.

[0112] In some cases, the preparing a sacrificial male mold based on the female mold in (b) comprises casting a male template material onto the female mold. In some cases, the male template material comprises a polymer. In some cases, the male template material comprises a water-soluble polymer. For example, the male template material can comprise a water-soluble polymer, such as poly(vinyl alcohol) (PVA), cellulose, polyacrylamide (PAM), polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), carrageenan, a chitosan derivative, guar gum, or poly(acrylic acid). In some cases, the water-soluble polymer is polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyvinylpyrrolidone / vinyl alcohol copolymer (PVP / VA copolymer). In some cases, the male template material can comprise a water-soluble polymer, such as poly(vinyl alcohol) (PVA).

[0113] The preparing a perforated membrane based on the sacrificial male mold in (c) comprises applying a polymer resin (e.g., perforated membrane material) to the sacrificial male mold. Insome cases, the polymer resin is a thermal curable resin, such as an epoxy resin, a polyester resin, a polyimide resin, a polyurethane resin, a bisimide, or a benzoxazine.

[0114] In some cases, the preparing a perforated membrane based on the sacrificial male mold in (c) further comprises curing the polymer resin. In some cases, the preparing a perforated membrane based on the sacrificial male mold in (c) further comprises thermal curing the polymer resin.

[0115] In some cases, the preparing a perforated membrane based on the sacrificial male mold in (c) further comprises photo curing the polymer resin. For example, the photo curing the polymer resin includes exposing the polymer resin to ultraviolet (UV) light. In some cases, the polymer resin is a photo-curable resin. For example, the photo-curable resin can be BAPR-a, BAPR-P, BAPR-y, BAPR-5, poly (ethylene glycol) diacrylate (PEGDA), Poly (ethylene glycol) dimethacrylate (PEGDMA), or poly (propylene fumarate) / diethyl fumarate (PPF / DEF).

[0116] The preparing a perforated membrane based on the sacrificial male mold in (c) can comprise imprinting. In some cases, the imprinting comprises contacting a perforated membrane material onto the sacrificial male mold. Optionally, the imprinting may comprise positioning a weight above the perforated membrane material when the perforated membrane material is contacting the sacrificial male mold to form the perforated membrane with an array of well- defined channels disposed through the perforated membrane. In some cases, the imprinting comprising providing a substrate and a weight.

[0117] The imprinting can further include applying a release layer onto a soft substrate. In some cases, the substrate can be a rubbery layer. In some cases, the imprinting comprises coating the substrate (e.g., rubbery layer, etc.) with the release layer. The release layer can comprise a releasable polymer. In some cases, the release layer comprises a silicone -based polymer, a fluoropolymer (e.g., polytetrafluoroethylene (PTFE)), a polyolefin, a polymer with long alkyl chains, and poly(vinyl alcohol). In some cases, the polyolefine comprises polyethylene (low- density, high-density, and linear low-density polyethylene), polypropylene, and polybutene (polybutene-1 and polyisobutylene). In some cases, the polymer with long alkyl chains can be polyethylene (PE), polypropylene (PP), poly(vinyl chloride) (PVC), poly(methyl methacrylate) (PMMA), polyisoprene (natural rubber), polybutadiene, or a synthetic rubber with long alkyl side chains. In some cases, the releasable polymer can also comprise a water-soluble polymer, such as poly(vinyl alcohol) (PVA), cellulose, polyacrylamide (PAM), polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), carrageenan, a chitosan derivative, guar gum, or poly(acrylic acid). In some cases, the water-soluble polymer is polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyvinylpyrrolidone / vinyl alcohol copolymer (PVP / VA copolymer).

[0118] In some cases, the (c)preparing a perforated membrane based on the sacrificial male mold comprises casting a perforated membrane material onto the sacrificial male mold. In some cases, the perforated membrane material comprises a polymer, such as a polymer resin. In some cases, the polymer resin can include polydimethylsiloxane (PDMS) resin or acrylic resin. In some cases, the perforated membrane material comprises a silicon polymer, such as polycarbonate (PC), polymethylmethacrylate (PMMA), polyvinylchloride (PVC), polyethylene (PE), or polydimethylsiloxane (PDMS).

[0119] In some cases, the contacting the perforated membrane material onto the sacrificial male mold can comprise spin coating, blade coating, push coating, or spray coating the perforated membrane material onto the sacrificial male mold. In some cases, the (c) preparing a perforated membrane based on the sacrificial male mold can comprise spin coating from about 1000 RPM to about 10,000 RPM for at least about 1 minute or from about 1 minute to about 10 minutes.

[0120] The (c)preparing a perforated membrane based on the sacrificial male mold further comprises: removing the sacrificial male mold. The removing the sacrificial male mold comprises exposing the releasable layer on the cured polymer resin to water vapor at a release temperature of at least about 50°C to dissolve the release layer from the cured polymer resin, thereby making the perforated membrane form a sacrificial template. In some cases, the release temperature is from about 50°C to about 100°C, or from about 65°C to about 85°C. In some cases, the release temperature is about 75 °C.

[0121] The method can further comprise, before (a) the providing a female mold comprising a female pattern, (d) preparing a female mold. In some cases, (d) can comprise performing soft lithography. In some cases, the female mold is complementary to a master mold. In some cases, the female mold is made of a female mold material, such as a polymer. In some cases, the female mole material comprises a silicon polymer, such as polycarbonate (PC), polymethylmethacrylate (PMMA), polyvinylchloride (PVC), polyethylene (PE), or polydimethylsiloxane (PDMS).

[0122] The method can further comprise, before (d) preparing the female mold, (e) preparing the master mold. In some cases, (e) comprises fabricating the master mold, such as a silicon master mold. In some cases, (e) can comprise deep reactive ion etching (DRIE) of the master mold.III. High-throughput Cell Electroporation System with Dual-Layer Perforated Membrane and Spacer Array

[0123] Described herein is a system for high-throughput cell electroporation. In some cases, the system for high-throughput cell electroporation comprises: (a) a perforated membrane, wherein the perforated membrane comprises: (i) a first layer comprising a plurality of first channels disposed through the first layer; and (ii) a second layer in contact with the first layer, the second layer comprising a plurality of second channels disposed through the second layer, wherein: afirst average thickness of the first layer is different from a second average thickness of the second layer; a first channel of the plurality of first channels is in fluid communication of a second channel of the plurality of second channels; a first average diameter of the first channel is different from a second average diameter of the second channel; and (b) at least one donor cell in contact with the perforated membrane.

[0124] In some cases, the perforated membrane comprises any of the perforated membranes and embodiments thereof previously described in this disclosure. For example, the system can comprise the perforated membrane comprising a first layer and a second layer, according to the embodiments provided in Section I: Perforated Membranes of the instant disclosure.

[0125] The first average thickness of the first layer can be any of the embodiments described above, such as those provided in Section I: Perforated Membranes. In some cases, the first average thickness is from about 1 pm to about 200 pm.

[0126] The second average thickness of the second layer can be any of the embodiments previously described, such as those in Section I: Perforated Membranes . In some cases, the second average thickness is from about 100 nm to about 10 pm.

[0127] In some cases, the first average thickness of the first layer of the perforated membrane is larger than the second average thickness of the second layer of the perforated membrane. In some cases, the first average thickness is at least twice or at least three times of the second average thickness.

[0128] The first average diameter of the first layer can be any of the embodiments described above, such as those provided in Section I: Perforated Membranes. In some cases, the first average diameter is from about 1 pm to about 20 pm.

[0129] The plurality of first channels of the first layer have an aspect ratio, wherein the first average diameter is about half or about one third of the first average thickness of the first layer.

[0130] The second average diameter of the second layer can be any of the embodiments described above, such as those provided in Section I: Perforated Membranes. In some cases, the second average diameter is from about 100 nm to about 5 pm.

[0131] The plurality of second channels of the second layer have an aspect ratio, wherein the second average diameter is no more than half or no more than one third of the second average thickness. In some cases, the second average diameter is from about one-half to about one-third of the second average thickness or from about one-third to about one-fourth of the second average thickness. The second average diameter can be about half of the second average thickness of the second layer of the perforated membrane. In some cases, the second average diameter is no more than half of the second average thickness.

[0132] In some cases, the first average diameter is larger than the second average diameter. In some cases, the first average diameter is at least twice or at least three times of the second average diameter. In some embodiments, the first average diameter is from about twice to about three times of the second average diameter. The first average diameter can be, for example, from about three times to about four times larger than the second average diameter.

[0133] The first layer can comprise a first polymer material according to any of the embodiments described previously, such as those provided in Section I: Perforated Membranes. In some cases, the first layer comprises a first polymer material. In some cases, the first polymer material comprises a first light-reactive polymer, a first crosslinking polymer, a first synthetic polymer, a first thermosetting polymer, a first light-curing polymer, a first thermal-curing polymer, or a first photoresist. In some cases, the first polymer material comprises a first positive photoresist or a first negative photoresist. In some cases, the first polymer material comprises a first SU-8 photoresist, such as a first SU-8 3000-series photoresist (e.g., a first SU-8 3010 photoresist, a first SU-8 3005 photoresist, etc.) or a first SU-8 2000-series photoresist (e.g., a first SU-8 2005 photoresist, etc.)

[0134] The second layer can comprise a second polymer material according to any of the embodiments described previously, such as those provided in Section I: Perforated Membranes. In some cases, the second layer comprises a second polymer material. In some cases, the second polymer material comprises a second light -reactive polymer, a second crosslinking polymer, a second synthetic polymer, a second thermosetting polymer, a second light-curing polymer, a second thermal-curing polymer, or a second photoresist. In some cases, the second polymer material comprises a second positive photoresist or a second negative photoresist. In some cases, the second polymer material comprises a second SU-8 photoresist, such as a second SU-8 TF 6000-series photoresist (e.g., a SU-8 TF 6002 photoresist, etc.) or a second SU-8 2000-series photoresist (e.g., a SU-8 2002 photoresist).

[0135] In some embodiments, the first layer comprises at least one ingredient different from ingredients of the second materials. In some cases, the first layer is made of materials different from those of the second layer. According to some embodiments, the first layer comprises the first photoresist, and the second layer comprises the second photoresist. In some cases, the first layer comprises the first negative photoresist, and wherein the second layer comprises the second negative photoresist. In some cases, the first layer comprises the first SU-8 photoresist, and wherein the second layer comprises the second SU-8 photoresist. Preferably, the first layer comprises the SU-8 3010 photoresist, the SU-8 3005 photoresist, or the SU-8 2005 photoresist, and wherein the second layer comprises the SU-8 TF 6002 photoresist or the SU-8 2002photoresist. According to some embodiments, a first tensile strength of the first layer is larger than a second tensile strength of the second layer.

[0136] The first layer and the second layer can be made from photoresist polymers. In some cases, the first layer is made of the same materials as those of the second layer, such as instances when the first layer and the second layer comprise the first light -reactive polymer, the first crosslinking polymer, the first synthetic polymer, or the first light-curing polymer.Donor cell

[0137] The system for high-throughput cell electroporation also comprises a donor cell. The donor cell of the system is in contact with the perforated membrane. In some cases, the donor cell is adhered to a first surface of the perforated membrane. In some cases, donor cells of a plurality of donor cells are in contact with the first surface of the perforated membrane. In some cases, the donor cells of a plurality of donor cells are adhered to the first surface of the perforated membrane. In some cases, the donor cells of a plurality of donor cells are adhered to the first surface of the perforated membrane as a monolayer of donor cells. For example, the donor cell can be in contact with a first surface of the perforated membrane or a portion thereof. In some cases, the donor cell is adhered to the perforated membrane, such as the first surface of the perforated membrane. In some cases, the donor cell is in contact with an opening of at least one channel of the perforated membrane. Preferably, the donor cell is in contact with an opening of at least one channel of the first surface of the perforated membrane, where the at least one channel has an average diameter of from about 0.3 pm to about 50 pm, from about 0.3 pm to about 20 pm, from about 0.4 pm to about 10 pm, from about 0.4 pm to about 5 pm, from about 0.4 pm to about 2 pm, or from about 0.4 pm to about 1.5 pm. Preferably, the donor cell is in contact with an opening of at least one channel of the first surface of the perforated membrane, where the at least one channel has an average diameter of from about 0.4 pm to about 1.5 pm.

[0138] The donor cell can be any type of cell. In some cases, the donor cell is a cell that secretes oris capable of secreting extracellular vesicles. In some cases, the donor cell is a eukaryotic cell (e.g., mammalian cell, human cell, non-human mammalian cell, rodent cell, mouse cell, etc.). In some instances, the donor cell is a cell from a cell line, stem cell, primary cell, or differentiated cell. In some embodiments, the donor cell is a primary cell. In some instances, the donor cell is a mouse embryonic fibroblast (MEF), a human embryonic fibroblast (HEF), a human dermal fibroblast (HDF), a dendritic cell, a mesenchymal stem cell, a bone marrow-derived dendritic cell, a bone marrow derived stromal cell, an adipose stromal cell, an enucleated cell, a neural stem cell, an immature dendritic cell, or an immune cell. The donor cell may be an adherent cell. In some cases, the donor cell is an adherent cell. In some cases, the donor cell is a suspensioncell. In some cases, the donor cell is a cell of a suspension cell line. In some cases, the donor cell is a suspension primary cell. In some cases, the donor cell is a human cell.

[0139] The system for high-throughput cell electroporation, in some cases, is configured to culture the donor cell.

[0140] The system for high-throughput cell electroporation, in some cases, is configured to electroporate a donor cell with a transfection reagent.Transfection reagents

[0141] The transfection reagent can be any type of biomolecule. In some cases, the transfection agents are at least one heterologous polynucleotide such as a vector (e.g., plasmid, DNA). In specific cases, the at least one heterologous polynucleotide encodes at least one polypeptide. In certain cases, the at least one polypeptide is therapeutic. In certain cases, the at least one polypeptide is for targeted delivery of the extracellular vesicle. In certain cases, the at least one polypeptide is both therapeutic and for targeted delivery of the extracellular vesicle. In other cases, the transfection reagent can be a therapeutic compound (e.g., a therapeutic DNA, therapeutic RNA, therapeutic mRNA, therapeutic miRNA, therapeutic tRNA, therapeutic rRNA, therapeutic siRNA, therapeutic shRNA, therapeutic SRP RNA, therapeutic tmRNA, therapeutic gRNA, or therapeutic crRNA), a therapeutic non-coding polynucleotide (e.g., non-coding RNA, IncRNA, piRNA, snoRNA, snRNAs, exRNA, or scaRNA), a drug, or a combination thereof. In other cases, the transfection reagent can be a non -therapeutic compound (e.g., non -therapeutic polynucleotide). In some cases, the transfection reagent is loaded in an electroporation chamber, as further described below.First spacer array

[0142] The system for high-throughput cell electroporation further comprises at least one spacer array (e.g., a first spacer array). When the system is assembled, the first spacer array has holes that can be used to form an array having multiple cell culture wells, or chambers, thus providing a multi-well electroporation device that enables electroporation of one or more types of donor cells. Generally, the perforated membrane stretches across a cell culture chamber such that a compartment suitable for a buffer (e.g., electroporation buffer, etc.) is situated below the membrane (or on one side of the perforated membrane) and a compartment is situated above the membrane (such as a cell culture chamber), such that donor cells can be seeded and cultured on the top of the perforated membrane (or on the side of the perforated membrane that is opposite of the side facing the buffer chamber). The spacer can also serve to separate the chambers. For example, the chambers can be separated by a length of 5 mm, as shown in FIG. 8, FIG. 9A, and FIG. 9B. Generally, at least a portion of the perforated membrane is situated within one or more of the cell culture chambers. In some cases, the first spacer array is in contact with the firstsurface of the perforated membrane. In some cases, the first spacer array is affixed to the perforated membrane by Van der Waals interactions. In some cases, the first spacer array is affixed to the perforated membrane by an adhesive material. In some cases, the first spacer array is affixed to the perforated membrane by a bioadhesive material. In some cases, the first spacer array is in contact with the first surface of the perforated membrane having a larger first average diameter of first channels than the second average diameter of the second channels of the second layer of the perforated membrane.

[0143] Generally, the first spacer array is a flat sheet having a certain thickness (i.e. height, or a distance between a first surface of the first spacer array and a second surface of the first spacer array that is opposite the first surface of the first spacer array). In some cases, the thickness of the first spacer array is greater than a diameter of a donor cell. In some cases, the thickness of the first spacer array is at least about 1 mm. In some cases, the thickness of the first spacer array is no more than about 5 cm, no more than about 2 cm, no more than about 1 cm, no more than about 8 mm, or no more than about 5 mm.

[0144] In some cases, the thickness of the first spacer array is from at least about 1 mm to at least about 5 cm, from about 1 mm to about 1.5 cm, from about 1 mm to about 1 cm, from about 1 mm to about 5 mm. In some cases, the thickness of the first spacer array is about 3 mm. In some cases, the thickness of the first spacer array is from about 1 mm to about 20 mm, from about 1 mm to about 30 mm, about 1 mm to about 40 mm, or about 1 mm to about 50 mm.

[0145] The first spacer array also comprises a plurality of first holes, wherein a cross-sectional area of the individual first holes is defined by an absence a first material of the first spacer array. In some cases, at least one hole of the first spacer array is covered by the perforated membrane. In some cases, the plurality of first holes of the first spacer array are covered by the perforated membrane. In some cases, the plurality of first holes are arranged in a non-patterned arrangement or distribution throughout the first spacer array. In some cases, the plurality of first holes are arranged in a patterned arrangement throughout the first spacer array. In some cases, the distribution of first holes of the plurality of first holes are distributed homogenously throughout the first spacer array. In some cases, the distribution of first holes of the plurality of first holes are not distributed homogenously throughout the first spacer array.

[0146] The first holes of the plurality of first holes are defined by an ab sence of a first material of the first spacer array and as having walls defined by the first material of the first spacer array that surrounds each of the first holes. Accordingly, a first hole of the first spacer array comprises a first hole width and a second hole width orthogonal to the first hole width, wherein the first hole has a cross-sectional area defined by the first hole width and the second hole width of a first hole (i.e., an individual hole) of the first spacer array. In some cases, the first hole width and thesecond hole width of holes of the array of holes of the first spacer array do not exceed a surface area of a surface of the perforated membrane having openings of the first plurality of channels.

[0147] In some cases, the first hole width of the first hole is at least about 1 mm. In some cases, the first hole width of the first hole is no more than about 30 cm, no more than about 10 cm, no more than about 5 cm, or no more than about 1 cm. In some cases, the first hole width of the first hole is from at least about 1 mm to about 10 cm. In some cases, the first hole width of the first hole is about 1 cm, about 9 mm, or about 5 mm. In some cases, the first hole width canbe at least about 1 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, or 50 mm. In some cases, the first hole width can be from at least about 1 mm to about 50 mm.

[0148] In some cases, the second hole width of the first hole is at least about 1 mm. In some cases, the second hole width of the first hole is no more than about 30 cm, no more than about 10 cm, no more than about 5 cm, or no more than about 1 cm. In some cases, the second hole width of the first hole is from at least about 1 mm to about 10 cm. In some cases, the second hole width of the first hole is about 1 cm, about 9 mm, or about 5 mm. In some cases, the second hole width can be at least about 1 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, or 50 mm. In some cases, the second hole width can be from at least about 1 mm to about 50 mm. In some cases, the first hole width of the first hole is substantially identical to the second hole width of the first hole. In some cases, the first hole width of the first hole is different the second hole width of the first hole.

[0149] In some cases, the cross-sectional area of the first hole of the first spacer array (e.g., the first holes formed by the holes in the first spacer array) canbe, for example, 0.1 cm2, 0.5 cm2, 1 cm2, 5 cm2, 10 cm2, or values greater than or less than such values. In some cases, the cross- sectional area of the first hole of the first spacer array is from about 0.1 cm2to about 5 cm2, from 1 cm2to about 2 cm2, or from about 0.5 cm2to about 4 cm2. In some cases, the cross-sectional area of the first hole is no more than about 10 cm2, no more than about 5 cm2, no more than about 3 cm2, no more than about 2 cm2, or no more than about 1 cm2.

[0150] In some cases, the cross-sectional area of the first hole is less than, greater than, or equal to about 10 mm x about 10 mm and other dimensions such as this. In some cases, the cross- sectional area of the first hole is about 2 mm x 2 mm or about 50 mm x 50 mm.

[0151] The first spacer array also comprises a plurality of first holes, whereby a first hole of the plurality of first holes is separated from other first holes adjacent to the first hole of the first spacer array by a first inter-hole distance. In some cases, the first hole of the first spacer array is separated from other first holes adjacent to the first hole of the first spacer array by an first interhole distance of at least about 1 mm. In some cases, the first hole of the first spacer array is separated from other first holes adjacent to the first hole of the first spacer array by an first inter-hole distance by no more than about 10 cm. In some cases, the first hole of the first spacer array is separated from other first holes adjacent to the first hole of the first spacer array by an first inter-hole distance of from about 1 mm to about 5 cm or from about 1 mm to about 1.5 cm. In some cases, the first hole of the first spacer array is separated from other first holes adjacent to the first hole of the first spacer array by an first inter-hole distance of about 5 mm. In some cases, the first hole of the first spacer array is separated from other first holes adjacent to the first hole of the first spacer array by an first inter-hole distance of about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, or about 20 mm.

[0152] In some cases, the first inter-hole distance is substantially the same for each first hole of the first spacer array. In some cases, the first inter-hole distance of the first hole is different than the first inter-hole distance of another first hole of the first spacer array.

[0153] The first spacer array also comprises an outer perimeter surrounding the plurality of first holes. The outer perimeter of the first spacer array is defined by an outer side width (e.g., a length of a first outer side of the second spacer array measured in a plane parallel to a surface of the second spacer array) and an outer side length (e.g., a second length of a second outer side of the second spacer array orthogonal to the first outer side of the second spacer array and measured in a plane parallel to a surface of the second spacer array). In some cases, the outer side width of the of an outer side of the first spacer array is at least about 1 cm. In some cases, the outer side width of the of an outer side of the first spacer array is no more than about 30 cm. In some cases, the outer side width of the of an outer side of the first spacer array is from about 1 cm to about 30 cm, from about 2 cm to about 30 cm, from about 2 cm to about 20 cm, or from about 2 cm to about 10 cm. In some cases, the outer side width of the of an outer side of the first spacer array is about 10 cm, about 9 cm, about 8 cm, about 7 cm, about 6 cm, about 5 cm, about 4 cm, about 3 cm, or about 2 cm. In some cases, the length is greater than 1 mm. In some cases, the outer side width of the of an outer side is at least about 1 millimeter (mm) to about 5 mm. In some cases, the outer side width of the of an outer side is at least about 1 millimeter (mm) to about 10 mm. In some cases, the length is at least about 1 millimeter (mm) to about 20 mm. In some cases, the first hole of the first spacer array does not exceed a surface area of a surface of the perforated membrane having openings of a first plurality of channels.

[0154] The first spacer array comprises a number of first holes. In some cases, the first spacer array comprises at least one first hole or at least 2 first holes. In some cases, the first spacer array comprises no more than about 2,500 first holes. In some cases, the first spacer array comprises from about 2 first holes to about 500 first holes, from about 2 first holes to about 100 first holes,or from about 2 first holes to about 30 first holes. In some cases, the number of first holes comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 16, about 18, about 20, about 24, about 25, about 30, about 36, about 40, about 48, about 49, about 50, about 56, about 64, about 81, about 96, about 100, about 121, about 144, about 225, about 256, or about 384 first holes. The first spacer array can have any number of first holes arranged into any desired number of rows and number of columns of first holes.

[0155] In some cases, the plurality of first holes of the first spacer array have a particular shape. The shape can be, e.g., a square shape, a circle shape, a triangular shape, or any geometrical shape. In some cases, the shape is a square, rectangle, diamond, polygon, circle, or oval). In some cases, the shape is a shape with at least one angle that is less than or greater than 90 degrees. In some cases, a shape of a first hole can be different from a shape of another first hole of the plurality of first holes. In some cases, the shape of the first hole can be substantially identical to a shape of another first hole of the plurality of first holes.

[0156] In some cases, the first spacer array comprises a plurality of first holes that are arranged in an array having rows and columns. In some cases, the array format of first holes comprises an array of row and columns of first holes. In some cases, the first spacer array comprises 1 >< 1 rows and columns offirstholes, 2 x 2 rows and columns of first holes, 3 x 2 rows and columns of first holes, 6 x 4 rows and columns of first holes, 12 x 8 rows and columns of first holes, 2 x 2 rows and columns of first holes, 3 x 3 rows and columns of first holes, 4 x 3 rows and columns of first holes, 4 x 4 rows and columns of first holes, 5 x 5 rows and columns of first holes, 6 x 6 rows and columns of first holes, 7 x 7 rows and columns of first holes, 8 x 8 rows and columns of first holes, 8 x 12 rows and columns of first holes, 9 x 9 rows and columns of first holes, 10 x 10 rows and columns of first holes, 11 x 11 rows and columns of first holes, 12 x 12 rows and columns of first holes, 16 x 16 rows and columns of first holes, 16 x 24 rows and columns of first holes, 24 x 24 rows and columns of first holes, or 48 x 60 rows and columns of first holes. The first spacer array can have any first of rows and columns of first holes arranged into any desired number of rows and number of columns of rows and columns of first holes.

[0157] In some cases, the first spacer array comprises at least 1 row or more than one row. In some cases, the second spacer array comprises at least 2 rows. In some cases, the first spacer array comprises no more than 100 rows. In some cases, the first spacer array comprises from about 1 to about 60 rows, from about 1 to about 30 rows, from about 1 to about 20 rows, or from about 1 to about 10 rows. In some cases, the first spacer array comprises about 5 rows.

[0158] In some cases, the first spacer array comprises at least 1 column or more than one column. In some cases, the first spacer array comprises at least 2 columns. In some cases, the first spacerarray comprises no more than 100 columns. In some cases, the first spacer array comprises from about 1 to about 60 columns, from about 1 to about 30 columns, from about 1 to about 20 columns, or from about 1 to about 10 columns. In some cases, the first spacer array comprises about 5 columns. In some cases, the first holes of the first spacer array comprise: (a) at least 2 rows of first holes of the first spacer array; and (b) at least 2 columns of first holes of the first spacer array.

[0159] The first spacer array is made of a first spacer array material. In some cases, the first spacer array is non-porous. In some cases, the first spacer material can be a material that can bind well with another smooth surface like a perforated membrane (e.g., track-etched membrane, silicon perforated membrane, a photoresist perforated membrane, or any polymer perforated membrane). In some cases, the spacer comprises or is made of rubber. For example, the spacer may be a polymer, such as a silicon rubber. In some cases, the spacer is made of a polymer. In some cases, the first spacer array comprises a thermoplastic polymer, a crosslinking polymer, a light-curing polymer, a thermal-curing polymer, a light-reactive polymer, a silicon polymer, a rubber, a thermoplastic elastomer, or a photoresist polymer. In some cases, the first spacer array can comprise a water-soluble polymer (e.g., polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), etc.) or a curable polymer, such as a curable polymer resin (e.g. acrylic, polydimethylsiloxane (PDMS), etc.). In some cases, the first spacer array comprises polyethylene terephthalate (PET), polycarbonate, poly dimethylsiloxane (PDMS), polydimethylsiloxane (PDMA), polyvinyl alcohol (PVA), polyethylene, polypropylene, polystyrene, polyacrylamide, or polyacrylic acid.

[0160] As described above, the holes of the first spacer array can be used to form an array of cell culture chambers when in contact with the first surface of the perforated membrane.Accordingly, the cell culture chambers are configured for culturing donor cells. The number of donor cells that can be cultured within the cell culture chambers depends on the cross-sectional area of the cell culture chambers, as defined by any embodiments of the first hole width and second hole width of the first holes of the first spacer array. In some cases, an average cell culture chamber can contain at least about 10,000 channels of the perforated membrane per cm2. In some cases, an average cell culture chamber can contain from about 10,000 to about 1x108channels of the perforated membrane per cm2. In some cases, an average cell culture chamber can contain from about lxl05to about IxlO8channels of the perforated membrane per cm2. Preferably, an average cell culture chamber containsfrom about 5xl05to about 5xl06channels of the perforated membrane per cm2.

[0161] In some embodiments, the multi-well cell culture chamber has dimensions matching or approximating standard 6-, 12-, 24-, 48-, or 96-well cell culture plates, or any of theaforementioned dimensions described of the first holes of the first spacer array. The multi-well cell culture chamber can facilitate liquid handling using automated robotic multi-channel pipettes and dispenser, for the purpose of cell loading, washing, EV collection, etc. The multi-well cell culture chambers can, in some cases, increase the mechanical strength of the perforated membrane. The multi-well cell culture chamber can ensure that each well is physically isolated from the next and prevents well-to-well cross-contamination. The multi-well cell culture chamber can efficiently assay multiple conditions or repeat identical conditions.Second spacer array

[0162] The system for high-throughput cell electroporation can further comprise a second array spacer, which has holes that can be used to form an array having multiple electroporation reagent wells, or chambers. In such cases, the system can be assembled such that the second spacer array is in contact with a second surface of the perforated membrane that is opposite the first surface of the perforated membrane in contact with the first spacer array. This forms a “sandwich” structure whereby the perforated membrane is positioned between the first spacer array and the second spacer array, as shown in FIG. 11A and FIG. 11B. In some cases, the first spacer array is in contact with the first surface of the perforated membrane, and the second spacer array is in contact with the second surface of the perforated membrane, wherein the first average diameter of the first channel of the first layer is larger than the second average diameter of the second channel of the second layer. Whereas the first spacer array is used to form an array having multiple cell culture wells, the holes of the second spacer array are used to form multiple electroporation reagent wells, or chambers. Similar to the first spacer array, the second spacer array can also serve to separate the second holes to form multiple electroporation reagent chambers. Generally, the first spacer array and the second spacer array are aligned to form an array of “miniature” cell culture / electroporation chambers, whereby the individual culture / electroporation chambers are fluidically isolated from other culture / electroporation chambers of the multi -well electroporation device described herein. Moreover, each individual chamber of the multi-well electroporation device is configured for cell electroporation, whereby the cell culture chamber provided by the first spacer array and the electroporation reagent chamber provided by the second spacer array are fluidically coupled via the channels of the perforated membrane positions between the first spacer array and the second spacer array.

[0163] Generally, the perforated membrane stretches across at least one cell culture chamber such that a compartment suitable for a buffer is situated below the membrane (or on one side of the perforated membrane) and a compartment is situated above the membrane (such as a cell culture compartment), such that cells can be seeded and cultured on the top of the perforated membrane (or on the side of the perforated membrane that is opposite of the side facing thebuffer chamber). In some cases, reagents (e.g., DNA, RNA, plasmids, vectors, polynucleotides) present in the buffer compartment travel to the cell compartment via the pores in the perforated membrane, particularly when an electric field is applied to the electroporation device. In some embodiments, the multi-well electroporation / nanoelectroporation (CEP / CNP) device comprises cell culture chambers, often spaced apart by spacers. Accordingly, the system comprises, in part, a multi-well electroporation device that enables electroporation of one or more types of donor cells with at least one or more transfection reagents, such as any one or combination of the transfection reagents described above. In some cases, reagents (e.g., DNA, RNA, plasmids, vectors, polynucleotides) present in the buffer compartment travel to the cell compartment vi a the pores in the perforated membrane, particularly when an electric field is applied to the electroporation device. In some cases, the multi-well electroporation device comprises a reservoir or buffer well or buffer chamber to contain buffer solutions and polynucleotides (e.g., DNA, RNA, DNA plasmids, microRNA) to be electroporated into the donor cells that are in the cellular cell culture wells. In some cases, the electroporation reagent chamber has a depth considered to be about the distance between a front face ofthenon-perforated membrane and a face of the spacer (e.g., thickness of the spacer). In some cases, the depth of the electroporation reagent chamber is at least about 1 mm to about 5 mm. In some cases, the depth of the electroporation reagent chamber has a thickness of at least about 1 mm to about 10 mm. In some cases, the depth of the electroporation reagent chamber has a thickness of at least about 1 mm to about 20 mm, at least about 1 mm to about 30 mm, at least about 1 mm to about 40 mm, or at least about 1 mm to about 50 mm. In some cases, the depth of the electroporation reagent chamber is the same distance as the depth of the cell culture wells.

[0164] In some cases, a 12-well plate Transwell (cell growth area 1.12 cm2), a >6-well plate Transwell perforated membrane (cell growth area 4.67 cm2), or a » 100 mm petri dish sized Transwell perforated membrane (cell growth area 44 cm2) is used in the device provided herein.

[0165] In some cases, the second spacer array is in contact with the second surface of the perforated membrane. In some cases, the second spacer array is affixed to the perforated membrane by Van derWaals interactions. In some cases, the second spacer array is affixed to the perforated membrane by an adhesive material. In some cases, the second spacer array is affixed to the perforated membraneby a bioadhesive material. In some cases, the second spacer array is in contact with the second surface of the perforated membrane having a larger second average diameter of second channels than the second average diameter of the second channels of the second layer of the perforated membrane.

[0166] Generally, the second spacer array is a flat sheet having a certain thickness (i.e. height, or a distance between a first surface of the second spacer array and a second surface of the secondspacer array that is opposite the first surface of the second spacer array). In some cases, the thickness of the second spacer array is greater than a diameter of a donor cell. In some cases, the thickness of the second spacer array is at least about 1 mm. In some cases, the thickness of the second spacer array is no more than about 5 cm, no more than about 2 cm, no more than about 1 cm, no more than about 8 mm, or no more than about 5 mm. In some cases, the thickness of the second spacer array is from at least about 0.5 cm to at least about 5 cm .

[0167] In some cases, the thickness of the second spacer array is from at least about 1 mm to at least about 5 cm, from about 1 mm to about 1.5 cm, from about 1 mm to about 1 cm, from about 1 mm to about 5 mm. In some cases, the thickness of the second spacer array is about 3 mm. In some cases, the thickness of the second spacer array is from about 1 mm to about 20 mm, from about 1 mm to about 30 mm, about 1 mm to about 40 mm, or about 1 mm to about 50 mm.

[0168] The second spacer array also comprises a plurality of second holes, wherein a cross- sectional area of the individual second holes is defined by an absence a second material of the second spacer array. In some cases, at least one hole of the second spacer array is covered by the perforated membrane. In some cases, the plurality of second holes of the second spacer array are covered by the perforated membrane. In some cases, the plurality of second holes is arranged in a non-pattemed arrangement or distribution throughout the second spacer array. In some cases, the plurality of second holes is arranged in a patterned arrangement throughout the second spacer array. In some cases, the distribution of second holes of the plurality of second holes are distributed homogenously throughout the second spacer array. In some cases, the distribution of second holes of the plurality of second holes are not distributed homogenously throughout the second spacer array.

[0169] The second holes of the plurality of second holes are defined by an absence of a second material of the second spacer array and as having walls defined by the second material of the second spacer array that surrounds each of the second holes. Accordingly, a second hole of the second spacer array comprises a first hole width and a second hole width orthogonal to the first hole width, wherein the second hole has a cross-sectional area defined by the first hole width and the second hole width of a second hole (i.e., an individual hole) of the second spacer array. In some cases, the first hole width and the second hole width of holes of the array of holes of the second spacer array do not exceed a surface area of a surface of the perforated membrane having openings of the second plurality of channels.

[0170] In some cases, the first hole width of the second hole is at least about 1 mm. In some cases, the first hole width of the second hole is no more than about 30 cm, no more than about 10 cm, no more than about 5 cm, or no more than about 1 cm. In some cases, the first hole width of the second hole is from at least about 1 mm to about 10 cm. In some cases, the first hole width ofthe second hole is about 1 cm, about 9 mm, or about 5 mm. In some cases, the first hole width can be at least about 1 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, or 50 mm. In some cases, the first hole width can be from at least about 1 mm to about 50 mm. In some cases, the first hole width of the second hole of the second spacer array is about 1 cm.

[0171] In some cases, the second hole width of the second hole is at least about 1 mm. In some cases, the second hole width of the second hole is no more than about 30 cm, no more than about 10 cm, no more than about 5 cm, or no more than about 1 cm. In some cases, the second hole width of the second hole is from at least about 1 mm to about 10 cm. In some cases, the second hole width of the second hole is about 1 cm, about 9 mm, or about 5 mm. In some cases, the second hole width can be at least about 1 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, or 50 mm. In some cases, the second hole width can be from at least about 1 mm to about 50 mm. In some cases, the second hole width of the second hole of the second spacer array is about 1 cm. In some cases, the first hole width of the second hole is substantially identical to the second hole width of the second hole. In some cases, the first hole width of the second hole is different the second hole width of the second hole.

[0172] In some cases, the cross-sectional area of the second hole of the second spacer array (e.g., the second holes formed by the holes in the second spacer array) can be, for example, 0.1 cm2, 0.5 cm2, 1 cm2, 5 cm2, 10 cm2, or values greater than or less than such values. In some cases, the cross-sectional area of the second hole of the second spacer array is from about 0.1 cm2to about 5 cm2, from 1 cm2to about 2 cm2, or from about 0.5 cm2to about 4 cm2. In some cases, the cross-sectional area of the second hole is no more than about 10 cm2, no more than about 5 cm2, no more than about 3 cm2, no more than about 2 cm2, or no more than about 1 cm2.

[0173] In some cases, the cross-sectional area of the second hole is less than, greater than, or equal to about 10 mm x about 10 mm and other dimensions such as this. In some cases, the cross-sectional area of the second hole is about 2 mm x 2 mm or about 50 mm x 50 mm.

[0174] The second spacer array also comprises a plurality of second holes, whereby a second hole of the plurality of second holes is separated from other second holes adjacent to the second hole of the second spacer array by a second inter-hole distance. In some cases, the second hole of the second spacer array is separated from other second holes adjacent to the second hole of the second spacer array by a second inter-hole distance of at least about 1 mm. In some cases, the second hole of the second spacer array is separated from other second holes adjacent to the second hole of the second spacer array by a second inter-hole distance by no more than about 10 cm. In some cases, the second hole of the second spacer array is separated from other second holes adjacent to the second hole of the second spacer array by a second inter-hole distance offrom about 1 mm to about 5 cm or from about 1 mm to about 1.5 cm. In some cases, the second hole of the second spacer array is separated from other second holes adjacent to the second hole of the second spacer array by a second inter-hole distance of about 5 mm. In some cases, the second hole of the second spacer array is separated from other second holes adjacent to the second hole of the second spacer array by a second inter-hole distance of about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, or about 20 mm.

[0175] In some cases, the second inter-hole distance is substantially the same for each second hole of the second spacer array. In some cases, the second inter-hole distance of the second hole is different than the second inter-hole distance of another second hole of the second spacer array.

[0176] The second spacer array also comprises an outer perimeter surrounding the plurality of second holes. The outer perimeter of the second spacer array is defined by an outer side width and an outer side length. In some cases, the outer side width of the of an outer side of the second spacer array is at least about 1 cm. In some cases, the outer side width of the of an outer side of the second spacer array is no more than about 30 cm. In some cases, the outer side width of the of an outer side of the second spacer array is from about 1 cm to about 30 cm, from about 2 cm to about 30 cm, from about 2 cm to about 20 cm, or from about 2 cm to about 10 cm. In some cases, the outer side width of the of an outer side of the second spacer array is about 10 cm, about 9 cm, about 8 cm, about 7 cm, about 6 cm, about 5 cm, about 4 cm, about 3 cm, or about 2 cm. In some cases, the length is greater than 1 mm. In some cases, the outer side width of the of an outer side is at least about 1 millimeter (mm) to about 5 mm. In some cases, the outer side width of the of an outer side is at least about 1 millimeter (mm) to about 10 mm. In some cases, the length is at least about 1 millimeter (mm) to about 20 mm. In some cases, the second hole of the second spacer array does not exceed a surface area of a surface of the perforated membrane having openings of a second plurality of channels.

[0177] The second spacer array comprises a number of second holes. In some cases, the second spacer array comprises at least one second hole or at least 2 second holes. In some cases, the second spacer array comprises no more than about 2,500 second holes. In some cases, the second spacer array comprises from about 2 second holes to about 500 second holes, from about 2 second holes to about 100 second holes, or from about 2 second holes to about 30 second holes. In some cases, the number of second holes comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 16, about 18, about 20, about 24, about 25, about 30, about 36, about40, about 48, about 49, about 50, about 56, about 64, about 81, about 96, about 100, about 121, about 144, about 225, about 256, or about 384second holes. The second spacer array can have any number of second holes arranged into any desired number of rows and number of columns of second holes.

[0178] In some cases, the plurality of second holes of the second spacer array have a particular shape. The shape can be, e.g., a square shape, a circle shape, a triangular shape, or any geometrical shape. In some cases, the shape is a square, rectangle, diamond, polygon, circle, or oval). In some cases, the shape is a shape with at least one angle that is less than or greater than 90 degrees. In some cases, a shape of a second hole can be different from a shape of another second hole of the plurality of second holes. In some cases, the shape of the second hole can be substantially identical to a shape of another second hole of the plurality of second holes.

[0179] In some cases, the plurality of second holes of the second spacer array have a particular shape. The shape can be, e.g., a square shape, a circle shape, a triangular shape, or any geometrical shape. In some cases, the shape is a square, rectangle, diamond, polygon, circle, or oval). In some cases, the shape is a shape with at least one angle that is less than or greater than 90 degrees. In some cases, a shape of a second hole can be different from a shape of another second hole of the plurality of second holes. In some cases, the shape of the second hole can be substantially identical to a shape of another second hole of the plurality of second holes.

[0180] In some cases, the second spacer array comprises a plurality of second holes that are arranged in an array having rows and columns. In some cases, the array format of second holes comprises an array of row and columns of second holes. In some cases, the second spacer array comprises 1 >< 1 rows and columns of second holes, 2 x 2 rows and columns of second holes, 3 x 2 rows and columns of second holes, 6 x 4 rows and columns of second holes, 12 x 8 rows and columns of second holes, 2 x 2 rows and columns of second holes, 3 x 3 rows and columns of second holes, 4 x 3 rows and columns of second holes, 4 x 4 rows and columns of second holes, 5 x 5 rows and columns of second holes, 6 x 6 rows and columns of second holes, 7 x 7 rows and columns of second holes, 8 x 8 rows and columns of second holes, 8 x 12 rows and columns of second holes, 9 x 9 rows and columns of second holes, 10 x 10 rows and columns of second holes, 11 x 11 rows and columns of second holes, 12 x 12 rows and columns of second holes, 16 x 16 rows and columns of second holes, 16 x 24 rows and columns of second holes, 24 x 24 rows and columns of second holes, or 48 x 60 rows and columns of second holes. The second spacer array can have any second of rows and columns of second holes arranged into any desired number of rows and number of columns of rows and columns of second holes.

[0181] In some cases, the second spacer array comprises at least 1 column or more than one column. In some cases, the second spacer array comprises at least 2 columns. In some cases, the second spacer array comprises no more than 100 columns. In some cases, the second spacer array comprises from about 1 to about 60 columns, from about 1 to about 30 columns, from about 1 toabout 20 columns, or from about 1 to about 10 columns. In some cases, the second spacer array comprises about 5 columns. In some cases, the second holes of the second spacer array comprise: (a) at least 2 rows of second holes of the second spacer array; and (b) at least 2 columns of second holes of the second spacer array.

[0182] The second spacer array is made of a second spacer array material. In some cases, the second spacer array is non-porous. In some cases, the second spacer material can be a material that can bind well with another smooth surface like a perforated membrane (e.g., track-etched membrane, silicon perforated membrane, a photoresist perforated membrane, or any polymer perforated membrane). In some cases, the spacer comprises or is made of rubber. For example, the spacer may be a polymer, such as a silicon rubber. In some cases, the spacer is made of a polymer. In some cases, the second spacer array comprises a thermoplastic polymer, a crosslinking polymer, a light-curing polymer, a thermal-curing polymer, a light-reactive polymer, a silicon polymer, a rubber, a thermoplastic elastomer, or a photoresist polymer. In some cases, the second spacer array can comprise a water-soluble polymer (e.g., polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), etc.) or a curable polymer, such as a curable polymer resin (e.g. acrylic, poly dimethylsiloxane (PDMS), etc.). In some cases, the second spacer array comprises polyethylene terephthalate (PET), polycarbonate, polydimethylsiloxane (PDMS), poly dimethylsiloxane (PDMA), polyvinyl alcohol (PVA), polyethylene, polypropylene, polystyrene, polyacrylamide, or polyacrylic acid.

[0183] As described above, the holes of the second spacer array can be used to form an array of electroporation reagent chambers when in contact with the second surface of the perforated membrane. Accordingly, the electroporation reagent chambers are configured for electroporating donor cells. The number of donor cells that can be electroporated within the cell culture chambers also depends on the cross-sectional area of the electroporation reagent chamber to which it is fluidically coupled, as defined by any embodiments of the first hole width and second hole width of second holes of the second spacer array. In some cases, an average electroporation reagent chamber can contain at least about 10,000 channels of the perforated membrane per cm2. In some cases, an average electroporation reagent chamber can contain from about 10,000 to about IxlO8channels of the perforated membrane per cm2. In some cases, an average electroporation reagent chamber can contain from about IxlO5to about IxlO8channels of the perforated membrane per cm2. Preferably, an average electroporation reagent chamber contains from about 5xl05to about 5xl06channels of the perforated membrane per cm2.

[0184] In some embodiments, the multi-well electroporation reagent chamber has dimensions matching or approximating standard 6-, 12-, 24-, 48-, or 96-well electroporation reagent plates, or any of the aforementioned dimensions described of the first holes of the first spacer array. Themulti-well electroporation reagent chamber can facilitate liquid handling using automated robotic multi-channel pipettes and dispenser, for the purpose of cell loading, washing, EV collection, loading of transfection reagents, etc. The multi-well electroporation reagent chambers can, in some cases, increase the mechanical strength of the perforated membrane. The multi-well electroporation reagent chamber can ensure that each well is physically isolated from the next and prevents well-to-well cross-contamination. The multi-well electroporation chamber can efficiently assay multiple conditions or repeat identical conditions.First spacer array vs second spacer array comparisons

[0185] Some embodiments of the system for high-throughput electroporation include the first spacer array being distinct from the second spacer array. In some cases, the thickness of the second spacer array is different than the thickness of the first spacer array . In some embodiments, the second inter-hole distance of the second spacer array is different than the first inter-hole distance of the first spacer array. For example, the second inter-hole distance between two second holes of the second spacer array can be different than the first inter-hole distance between two first holes of the first array of holes of the first spacer array. In some embodiments, the first hole width of the second hole of the second spacer array can be different from the first hole width of the first hole of the first spacer array. It is also conceived that the second hole width of the second hole of the second spacer array can be different from the second hole width of the first hole of the first spacer array. In some cases, the number of first holes of the first spacer array is different than the number of second holes of the second spacer array. Additionally, it is contemplated that the first spacer array comprises at least one ingredient different from ingredients of the second spacer array materials. In some cases, the first spacer array is made of materials different from those of the second spacer array.

[0186] Alternatively, according to some embodiments of the system for high-throughput electroporation, the first spacer array and the second spacer array. In some cases, the thickness of the second spacer array is the same as the thickness of the first spacer array . The second spacer array can, for example, be identical to the first spacer array. For example, the second inter-hole distance between two second holes of the second spacer array can be substantially the same as, or identical to, the first inter-hole distance between two first holes of the first array of holes of the first spacer array. In some embodiments, the first hole width of the second hole of the second spacer array can be substantially the same as, or identical to, the first hole width of the first hole of the first spacer array. It is also conceived that the second hole width of the second hole of the second spacer array can be substantially the same as, or identical to, the second hole width of the first hole of the first spacer array. In some cases, the number of first holes of the first spacer array is substantially the same as, or identical to, the number of second holes of the secondspacer array. Additionally, it is contemplated that the first spacer array is made from at least one of the same materials as the second spacer array materials. In some cases, the first spacer array is made of identical materials as the second spacer array.Cathode array

[0187] The system for high-throughput electroporation can also comprise a cathode. The cathode can be made of any material that is electrically conductive and non-toxic to the at least one donor cell, such as gold, silver, platinum, or aluminum. In some cases, the cathode has a three- dimensional plate shape. Here, the cathode is configured to be disposed within a first hole of the first spacer array (e.g., a cell culture chamber). In some cases, the cathode is configured to contact a surface of the first spacer array or the second spacer array. Accordingly, the cathode plate can have a length and width that is substantially the length and the width of the outer sides of the first spacer array or a length and width that is substantially the length and the width of the outer sides of the second spacer array. The cathode plate may have a length and width that covers only a portion of the first spacer array or second spacer array. In some cases, the cathode plate has a cross-sectional length of at least about 1 mm. In some cases, the cathode plate has a cross- sectional length of no more than about 50 cm, or no more than about 10 cm. In some cases, the cathode plate has a cross-sectional length from about 1 mm to about 20 cm or from about 1 mm to about 10 cm. Placement of the anode into the first spacer array or the second spacer array will depend on the electrical charge of a substance disposed within the first spacer array or the second spacer array. Therefore, in some cases, it is appropriate to contact the cathode plate with the first spacer array or the second spacer array. The cathode plate may also be in electrical communication with the first spacer array or the second spacer array via a solution.

[0188] In other cases, the cathode can have a rod shape or a wire shape, including a wire formed into a coil (e.g., a cathode coil). For example, the cathode can be a cathode coil to configured to be disposed within the first hole of the first spacer array or the second hole of the second spacer array.

[0189] Moreover, to accommodate the multi-well configuration of the high-throughput electroporation systems and devices provided herein, the cathode can comprise an array of cathode coils. For example, the array of cathode coils is configured to position a cathode coil within the first hole of the first spacer array or the second hole of the second spacer array. In some cases, the array of cathode coils is configured to position individual cathode coils within at least 2, at least 3, or at least 5 first holes of the first spacer array. In some cases, the array of cathode coils is configured to position individual cathode coils within a portion of the first holes of the first spacer array. In some cases, the array of cathode coils is configured to position individual cathode coils within all first holes of the first spacer array.

[0190] In some cases, the array of cathode coils is configured to position individual cathode coils within at least 2, at least 3, or at least 5 second holes of the second spacer array. In some cases, the array of cathode coils is configured to position individual cathode coils within a portion of the second holes of the second spacer array. In some cases, the array of cathode coils is configured to position individual cathode coils within all second holes of the second spacer array.

[0191] The cathode wire can have a diameter that is less than the cross-sectional area of the first hole of the first spacer array or is less than the cross-sectional area of the second hole of the second spacer array. In some cases, the cathode wire has a diameter of at least about 1 mm. In some cases, the cathode wire has a diameter of no more than about 5 cm or no more than about 1 cm. In some cases, the cathode wire has a diameter from about 1 mm to about 20 cm, from about 1 mm to about 1 cm, or from about 1 mm to about 5 mm.

[0192] The cathode array comprises a number of cathodes. In some cases, the cathode array comprises at least one cathode or at least 2 cathodes. In some cases, the cathode array comprises no more than about 2,500 cathodes. In some cases, the cathode array comprises from about 2 cathodes to about 500 cathodes, from about 2 cathodes to about 100 cathodes, or from about 2 cathodes to about 30 cathodes. In some cases, the number of cathodes comprises at least about 1, about 2, about 3, about4, about 5, about 6, about ?, about 8, about 9, about 10, about 12, about 16, about 18, about 20, about 24, about 25, about 30, about 36, about 40, about 48, about 49, about 50, about 56, about 64, about 81, about 96, about 100, about 121, about 144, about 225, about 256, or about 384 cathodes. The cathode array can have any desired number of cathodes.

[0193] In some cases, the cathodes of the cathode array have a particular shape. The shape can be, e.g., a square shape, a circle shape, a triangular shape, or any geometrical shape. In some cases, the shape is a square, rectangle, diamond, polygon, circle, or oval). In some cases, the shape is a shape with at least one angle that is less than or greater than 90 degrees. In some cases, a shape of a first hole can be different from a shape of another first hole of the plurality of cathodes. In some cases, the shape of the first hole can be substantially identical to a shape of another first hole of the plurality of cathodes.

[0194] In some cases, the cathode array comprises a plurality of cathodes that are arranged in an array having rows and columns. In some cases, the array format of cathodes comprises an array of rows and columns of cathodes. In some cases, the cathode array comprises 1 >< 1 rows and columns of cathodes, 2 x 2 rows and columns of cathodes, 3 x 2 rows and columns of cathodes, 6 x 4 rows and columns of cathodes, 12 x 8 rows and columns of cathodes, 2 x 2 rows and columns of cathodes, 3 x 3 rows and columns of cathodes, 4 x 3 rows and columns of cathodes, 4 x 4 rows and columns of cathodes, 5 x 5 rows and columns of cathodes, 6 x 6 rows and columns of cathodes, 7 x 7 rows and columns of cathodes, 8 x 8 rows and columns of cathodes,8 x 12 rows and columns of cathodes, 9 x 9 rows and columns of cathodes, 10 x 10 rows and columns of cathodes, 11 x H rows and columns of cathodes, 12 x 12 rows and columns of cathodes, 16 x 16 rows and columns of cathodes, 16 x 24 rows and columns of cathodes, 24 x 24 rows and columns of cathodes, or 48 x 60 rows and columns of cathodes. The cathode array can have any number of cathodes arranged into any desired number of rows and number of columns of cathodes. The cathodes of the cathode array can be cathode wires, cathode coils, or have any shape. A cathode of the cathode array can have different shapes from other cathodes of the cathode array.

[0195] In some cases, the cathode array comprises at least 1 row or more than one row. In some cases, the second spacer array comprises at least 2 rows of cathodes. In some cases, the cathode array comprises no more than 100 rows of cathodes. In some cases, the cathode array comprises from about 1 to about 60 rows of cathodes, from about 1 to about 30 rows of cathodes, from about 1 to about 20 rows of cathodes, or from about 1 to about 10 rows of cathodes. In some cases, the cathode array comprises about 5 rows of cathodes.

[0196] In some cases, the cathode array comprises at least 1 column or more than one column of cathodes. In some cases, the cathode array comprises at least 2 columns of cathodes. In some cases, the cathode array comprises no more than about 100 columns of cathodes. In some cases, the cathode array comprises from about 1 to about 60 columns of cathodes, from about 1 to about 30 columns of cathodes, from about 1 to about 20 columns of cathodes, or from about 1 to about 10 columns of cathodes. In some cases, the cathode array comprises about 5 columns of cathodes. In some cases, the cathodes of the cathode array comprise: (a) at least 2 rows of cathodes of the cathode array; and (b) at least 2 columns of cathodes.

[0197] In some cases, the cathode wires are structurally fixed into a common casing. In some cases, the cathode wires are reversibly connected to the array of cathode wires. In some cases, the array of cathode wires is customizable to add, remove, or reposition the cathode wires to, for example, accommodate any of the forementioned embodiments of the multi-well system for high-throughput electroporation.Anode array

[0198] The system for high-throughput electroporation can also comprise an anode. The anode can be made of any material that is electrically conductive and non-toxic to the at least one donor cell, such as gold, silver, platinum, or aluminum. In some cases, the anode has a three- dimensional plate shape. Here, the anode is configured to be disposed within a second hole of the second spacer array (e.g., an electroporation reagent chamber). In some cases, the anode is configured to contact a surface of the first spacer array or the second spacer array. Accordingly, the anode plate can have a length and width that is substantially the length and the width of theouter sides of the first spacer array or a length and width that is substantially the length and the width of the outer sides of the second spacer array. The anode plate may have a length and width that covers only a portion of the first spacer array or second spacer array. In some cases, the anode plate has a cross-sectional length of at least about 1 mm. In some cases, the anode plate has a cross-sectional length of no more than about 50 cm, or no more than about 10 cm. In some cases, the anode plate has a cross-sectional length from about 1 mm to about 20 cm or from about 1 mm to about 10 cm. Placement of the anode into the first spacer array or the second spacer array will depend on the electrical charge of a substance disposed within the first spacer array or the second spacer array. Therefore, in some cases, it is appropriate to contact the cathode plate with the first spacer array or the second spacer array. The cathode plate may also be in electrical communication with the first spacer array or the second spacer array via a solution.

[0199] In other cases, the anode can have a rod shape or a wire shape, including a wire formed into a coil (e.g., an anode coil). For example, the anode can be an anode coil to configured to be disposed within the first hole of the first spacer array or the second hole of the second spacer array.

[0200] Moreover, to accommodate the multi-well configuration of the high-throughput electroporation systems and devices provided herein, the anode can comprise an array of anode coils. For example, the array of anode coils is configured to position an anode coil within the first hole of the first spacer array or the second hole of the second spacer array. In some cases, the array of anode coils is configured to position individual anode coils within at least 2, at least 3, or at least 5 first holes of the first spacer array. In some cases, the array of anode coils is configured to position individual anode coils within a portion of the first holes of the first spacer array. In some cases, the array of anode coils is configured to position individual anode coils within all first holes of the first spacer array.

[0201] In some cases, the array of anode coils is configured to position individual anode coils within at least 2, at least 3, or at least 5 second holes of the second spacer array. In some cases, the array of anode coils is configured to position individual anode coils within a portion of the second holes of the second spacer array. In some cases, the array of anode coils is configured to position individual anode coils within all second holes of the second spacer array.

[0202] The anode wire can have a diameter that is less than the cross-sectional area of the first hole of the first spacer array or is less than the cross-sectional area of the second hole of the second spacer array. In some cases, the anode wire has a diameter of at least about 1 mm. In some cases, the anode wire has a diameter of no more than about 5 cm or no more than about 1 cm. In some cases, the anode wire has a diameter from about 1 mm to about 20 cm, from about 1 mm to about 1 cm, or from about 1 mm to about 5 mm.

[0203] The anode array comprises a number of anodes. In some cases, the anode array comprises at least one anode or at least 2 anodes. In some cases, the anode array comprises no more than about 2,500 anodes. In some cases, the anode array comprises from about 2 anodes to about 500 anodes, from about 2 anodes to about 100 anodes, or from about 2 anodes to about 30 anodes. In some cases, the number of anodes comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about ?, about 8, about 9, about 10, about 12, about 16, about 18, about 20, about 24, about 25, about 30, about 36, about40, about48, about49, about 50, about 56, about 64, about 81, about 96, about 100, about 121, about 144, about 225, about 256, or about 384 anodes. The anode array can have any desired number of anodes.

[0204] In some cases, the anodes of the anode array have a particular shape. The shape can be, e.g., a square shape, a circle shape, a triangular shape, or any geometrical shape. In some cases, the shape is a square, rectangle, diamond, polygon, circle, or oval). In some cases, the shape is a shape with at least one angle that is less than or greater than 90 degrees. In some cases, a shape of a first hole can be different from a shape of another first hole of the plurality of anodes. In some cases, the shape of the first hole can be substantially identical to a shape of another first hole of the plurality of anodes.

[0205] In some cases, the anode array comprises a plurality of anodes that are arranged in an array having rows and columns. In some cases, the array format of anodes comprises an array of rows and columns of anodes. In some cases, the anode array comprises 1 >< 1 rows and columns of anodes, 2 x 2 rows and columns of anodes, 3 x 2 rows and columns of anodes, 6 x 4 rows and columns of anodes, 12 x 8 rows and columns of anodes, 2 x 2 rows and columns of anodes, 3 x 3 rows and columns of anodes, 4 x 3 rows and columns of anodes, 4 x 4 rows and columns of anodes, 5 x 5 rows and columns of anodes, 6 x 6 rows and columns of anodes, 7 x 7 rows and columns of anodes, 8 x 8 rows and columns of anodes, 8 x 12 rows and columns of anodes, 9 x 9 rows and columns of anodes, 10 x 10 rows and columns of anodes, 11 x 11 rows and columns of anodes, 12 x 12 rows and columns of anodes, 16 x 16 rows and columns of anodes, 16 x 24 rows and columns of anodes, 24 x 24 rows and columns of anodes, or 48 x 60 rows and columns of anodes. The anode array can have any number of anodes arranged into any desired number of rows and number of columns of anodes. The anodes of the anode array can be anode wires, anode coils, or have any shape. An anode of the anode array can have a different shape than other anodes of the anode array.

[0206] In some cases, the anode array comprises at least 1 row or more than one row. In some cases, the second spacer array comprises at least 2 rows of anodes. In some cases, the anode array comprises no more than 100 rows of anodes. In some cases, the anode array comprises from about 1 to about 60 rows of anodes, from about 1 to about 30 rows of anodes, from about 1 toabout 20 rows of anodes, or from about 1 to about 10 rows of anodes. In some cases, the anode array comprises about 5 rows of anodes.

[0207] In some cases, the anode array comprises at least 1 column or more than one column of anodes. In some cases, the anode array comprises at least 2 columns of anodes. In some cases, the anode array comprises no more than about 100 columns of anodes. In some cases, the anode array comprises from about 1 to about 60 columns of anodes, from about 1 to about 30 columns of anodes, from about 1 to about 20 columns of anodes, or from about 1 to about 10 columns of anodes. In some cases, the anode array comprises about 5 columns of anodes. In some cases, the anodes of the anode array comprise: (a) at least 2 rows of anodes of the anode array; and (b) at least 2 columns of anodes.

[0208] In some cases, the anode wires are structurally fixed into a common casing. In some cases, the anode wires are reversibly connected to the array of anode wires. In some cases, the array of anode wires is customizable to add, remove, or reposition the anode wires to, for example, accommodate any of the forementioned embodiments of the multi-well system for high-throughput electroporation.Electrical pulse generation system

[0209] The system for high-throughput electroporation can also comprise an electric pulse generation system configured to provide an electrical voltage or an electrical current to the donor cell via the cathodes and anodes described above (e.g., configured to electroporate the donor cell). Here, the electric pulse generation system comprises: (a) an electrical pulse generator; (b) a shock pulse converter; and (c) an electrical current output device.

[0210] The electric pulse generation system can be automated or manually operated. Preferably, the electric pulse generation system is configured to adjust a feature of an electrical pulse applied to the at least one donor cell in contact with the perforated membrane. An adjustable feature of an electrical pulse can include a voltage, an electrical current, a pulse duration, an inter-pulse interval, a pulse frequency, or a pulse delay. Any one or combination of the pulse features can be adjusted to optimized the system for high-throughput cell electroporation of the donor cells. In some cases, the electric pulse generation system is configured to adjust a voltage applied to the at least one donor cell in contact with the perforated membrane.Pulse features

[0211] In some cases, the electric pulse generation system delivers electrical pulses to the cathode and anode of the system for generating an electric field within at least one or more of the multi-well cell culture / electroporation chambers described above. In some cases, the voltage generated by the cathode and anode driven by the electric pulse generation system comprises a voltage that is between about 10 V to about 500 V.

[0212] In some cases, the voltage generated by the cathode and anode driven by the electric pulse generation system comprises a voltage that is between about 10 V to about 25 V, about 10 V to about 50 V, about 10 V to about 100 V, about 10 V to about 125 V, about 10 V to about 150 V, about 10 V to about 175 V, about 10 V to about 200 V, about 10 V to about 225 V, about 10 V to about250 V, about 10 V to about 300 V, about 10 V to about 500 V, about 25 V to about 50 V, about 25 V to about 100 V, about 25 V to about 125 V, about25 V to about 150 V, about 25 V to about 175 V, about 25 V to about 200 V, about 25 V to about 225 V, about 25 V to about 250 V, about 25 V to about 300 V, about 25 V to about 500 V, about 50 V to about 100 V, about 50 V to about 125 V, about 50 V to about 150 V, about 50 V to about 175 V, about 50 V to about 200 V, about 50 V to about 225 V, about 50 Vto about 250 V, about 50 V to about 300 V, about 50 V to about 500 V, about 100 V to about 125 V, about 100 V to about 150 V, about 100 V to about 175 V, about 100 V to about200 V, about 100 V to about 225 V, about 100 V to about 250 V, about 100 V to about 300 V, about 100 Vto about 500 V, about 125 V to about 150 V, about 125 V to about 175 V, about 125 V to about200 V, about 125 V to about225 V, about 125 V to about 250 V, about 125 V to about 300 V, about 125 V to about 500 V, about 150 V to about 175 V, about 150 V to about 200 V, about 150 Vto about 225 V, about 150 Vto about 250 V, about 150 V to about 300 V, about 150 V to about 500 V, about 175 V to about 200 V, about 175 V to about 225 V, about 175 V to about250 V, about 175 V to about 300 V, about 175 V to about 500 V, about 200 V to about 225 V, about 200 V to about 250 V, about 200 V to about 300 V, about 200 V to about 500 V, about 225 V to about 250 V, about 225 V to about 300 V, about 225 V to about 500 V, about 250 V to about 300 V, about250 V to about 500 V, or about 300 V to about 500 V. In some cases, the voltage generated by the cathode and anode driven by the electric pulse generation system comprises a voltage that is between about 10 V, about25 V, about 50 V, about 100 V, about 125 V, about 150 V, about 175 V, about 200 V, about 225 V, about 250 V, about 300 V, or about 500 V. In some cases, the voltage generated by the cathode and anode driven by the electric pulse generation system comprises a voltage that is between at least about 10 V, about 25 V, about 50 V, about 100 V, about 125 V, about 150 V, about 175 V, about 200 V, about 225 V, about 250 V, or about 300 V. In some cases, the voltage generated by the cathode and anode driven by the electric pulse generation system comprises a voltage that is between at most about 25 V, about 50 V, about 100 V, about 125 V, about 150 V, about 175 V, about 200 V, about 225 V, about 250 V, about 300 V, or about 500 V.

[0213] In some cases, the electric field generated by the cathode and anode driven by the electric pulse generation system comprises an electric strength from about 0.1 volt / mm to about 50,000 volt / mm. In some cases, the electric field generated by the cathode and anode driven by the electric pulse generation system comprises an electric field strength from about 0.1 volt / mm toabout 0.5 volt / mm, about 0.1 volt / mm to about 1 volt / mm, about 0.1 volt / mm to about 5 volt / mm, about 0.1 volt / mm to about 10 volt / mm, about 0.1 volt / mm to about 50 volt / mm, about 0.1 volt / mm to about 100 volt / mm, about 0.1 volt / mm to about 500 volt / mm, about 0.1 volt / mm to about 1,000 volt / mm, about 0. 1 volt / mm to about 5,000 volt / mm, about 0. 1 volt / mm to about 10,000 volt / mm, about 0.1 volt / mm to about 50,000 volt / mm, about 0.5 volt / mm to about 1 volt / mm, about 0.5 volt / mm to about 5 volt / mm, about 0.5 volt / mm to about 10 volt / mm, about 0.5 volt / mm to about 50 volt / mm, about 0.5 volt / mm to about 100 volt / mm, about 0.5 volt / mm to about 500 volt / mm, about 0.5 volt / mm to about 1,000 volt / mm, about 0.5 volt / mm to about 5,000 volt / mm, about 0.5 volt / mm to about 10,000 volt / mm, about 0.5 volt / mm to about 50,000 volt / mm, about 1 volt / mm to about 5 volt / mm, about 1 volt / mm to about 10 volt / mm, about 1 volt / mm to about 50 volt / mm, about 1 volt / mm to about 100 volt / mm, about 1 volt / mm to about 500 volt / mm, about 1 volt / mm to about 1,000 volt / mm, about 1 volt / mm to about 5,000 volt / mm, about 1 volt / mm to about 10,000 volt / mm, about 1 volt / mm to about 50,000 volt / mm, about 5 volt / mm to about 10 volt / mm, about 5 volt / mm to about 50 volt / mm, about 5 volt / mm to about 100 volt / mm, about 5 volt / mm to about 500 volt / mm, about 5 volt / mm to about 1,000 volt / mm, about 5 volt / mm to about 5,000 volt / mm, about 5 volt / mm to about 10,000 volt / mm, about 5 volt / mm to about 50,000 volt / mm, about 10 volt / mm to about 50 volt / mm, about 10 volt / mm to about 100 volt / mm, about 10 volt / mm to about 500 volt / mm, about 10 volt / mm to about 1,000 volt / mm, about 10 volt / mm to about 5,000 volt / mm, about 10 volt / mm to about 10,000 volt / mm, about 10 volt / mm to about 50,000 volt / mm, about 50 volt / mm to about 100 volt / mm, about 50 volt / mm to about 500 volt / mm, about 50 volt / mm to about 1,000 volt / mm, about 50 volt / mm to about 5,000 volt / mm, about 50 volt / mm to about 10,000 volt / mm, about 50 volt / mm to about 50,000 volt / mm, about 100 volt / mm to about 500 volt / mm, about 100 volt / mm to about 1,000 volt / mm, about 100 volt / mm to about 5,000 volt / mm, about 100 volt / mm to about 10,000 volt / mm, about 100 volt / mm to about 50,000 volt / mm, about 500 volt / mm to about 1,000 volt / mm, about 500 volt / mm to about 5,000 volt / mm, about 500 volt / mm to about 10,000 volt / mm, about 500 volt / mm to about 50,000 volt / mm, about 1,000 volt / mm to about 5, 000 volt / mm, about 1,000 volt / mm to about 10,000 volt / mm, about 1,000 volt / mm to about 50,000 volt / mm, about 5,000 volt / mm to about 10,000 volt / mm, about 5,000 volt / mm to about 50,000 volt / mm, or about 10,000 volt / mm to about 50,000 volt / mm In some cases, the electric field generated by the cathode and anode driven by the electric pulse generation system comprises an electric field strength from about 0.1 volt / mm, about 0.5 volt / mm, about 1 volt / mm, about 5 volt / mm, about 10 volt / mm, about 50 volt / mm, about 100 volt / mm, about 500 volt / mm, about 1,000 volt / mm, about 5,000 volt / mm, about 10,000 volt / mm, or about 50,000 volt / mm In some cases, the electric field generated by the cathode and anode driven by the electric pulsegeneration system comprises an electric field strength from at least about 0.1 volt / mm, about 0.5 volt / mm, about 1 volt / mm, about 5 volt / mm, about 10 volt / mm, about 50 volt / mm, about 100 volt / mm, about 500 volt / mm, about 1,000 volt / mm, about 5,000 volt / mm, or about 10,000 volt / mm In some cases, the electric field generated by the cathode and anode driven by the electric pulse generation system comprises an electric field strength from at most about 0.5 volt / mm, about 1 volt / mm, about 5 volt / mm, about 10 volt / mm, about 50 volt / mm, about 100 volt / mm, about 500 volt / mm, about 1,000 volt / mm, about 5,000 volt / mm, about 10,000 volt / mm, or about 50,000 volt / mm.

[0214] In some instances, the electric field generated by the cathode and anode driven by the electric pulse generation system comprises a plurality of pulses with pulse duration from about 0.01 millisecond / pulse to about 5,000 millisecond / pulse. In some instances, the electric field generated by the cathode and anode driven by the electric pulse generation system comprises a plurality of pulses with pulse duration from about 0.01 millisecond / pulse to about 0.05 millisecond / pulse, about 0.01 millisecond / pulse to about 0.1 millisecond / pulse, about 0.01 millisecond / pulse to about 0.5 millisecond / pulse, about 0.01 millisecond / pulse to about 1 millisecond / pulse, about 0.01 millisecond / pulse to about 5 millisecond / pulse, about 0.01 millisecond / pulse to about 10 millisecond / pulse, about 0.01 millisecond / pulse to about 50 millisecond / pulse, about 0.01 millisecond / pulse to about 100 millisecond / pulse, about 0.01 millisecond / pulse to about 500 millisecond / pulse, about 0.01 millisecond / pulse to about 1,000 millisecond / pulse, about 0.01 millisecond / pulse to about 5,000 millisecond / pulse, about 0.05 millisecond / pulse to about 0.1 millisecond / pulse, about 0.05 millisecond / pulse to about 0.5 millisecond / pulse, about 0.05 millisecond / pulse to about 1 millisecond / pulse, about 0.05 millisecond / pulse to about 5 millisecond / pulse, about 0.05 millisecond / pulse to about 10 millisecond / pulse, about 0.05 millisecond / pulse to about 50 millisecond / pulse, about 0.05 millisecond / pulse to about 100 millisecond / pulse, about 0.05 millisecond / pulse to about 500 millisecond / pulse, about 0.05 millisecond / pulse to about 1,000 millisecond / pulse, about 0.05 millisecond / pulse to about 5,000 millisecond / pulse, about 0.1 millisecond / pulse to about 0.5 millisecond / pulse, about 0.1 millisecond / pulse to about 1 millisecond / pulse, about 0.1 millisecond / pulse to about 5 millisecond / pulse, about 0.1 millisecond / pulse to about 10 millisecond / pulse, about 0.1 millisecond / pulse to about 50 millisecond / pulse, about 0.1 millisecond / pulse to about 100 millisecond / pulse, about 0.1 millisecond / pulse to about 500 millisecond / pulse, about 0.1 millisecond / pulse to about 1,000 millisecond / pulse, about 0.1 millisecond / pulse to about 5,000 millisecond / pulse, about 0.5 millisecond / pulse to about 1 millisecond / pulse, about 0.5 millisecond / pulse to about 5 millisecond / pulse, about 0.5 millisecond / pulse to about 10 millisecond / pulse, about 0.5 millisecond / pulse to about 50millisecond / pulse, about 0.5 millisecond / pulse to about 100 millisecond / pulse, about 0.5 millisecond / pulse to about 500 millisecond / pulse, about 0.5 millisecond / pulse to about 1,000 millisecond / pulse, about 0.5 millisecond / pulse to about 5,000 millisecond / pulse, about 1 millisecond / pulse to about 5 millisecond / pulse, about 1 millisecond / pulse to about 10 millisecond / pulse, about 1 millisecond / pulse to about 50 millisecond / pulse, about 1 millisecond / pulse to about 100 millisecond / pulse, about 1 millisecond / pulse to about 500 millisecond / pulse, about 1 millisecond / pulse to about 1,000 millisecond / pulse, about 1 millisecond / pulse to about 5,000 millisecond / pulse, about 5 millisecond / pulse to about 10 millisecond / pulse, about 5 millisecond / pulse to about 50 millisecond / pulse, about 5 millisecond / pulse to about 100 millisecond / pulse, about 5 millisecond / pulse to about 500 millisecond / pulse, about 5 millisecond / pulse to about 1,000 millisecond / pulse, about 5 millisecond / pulse to about 5,000 millisecond / pulse, about 10 millisecond / pulse to ab out 50 millisecond / pulse, about 10 millisecond / pulse to about 100 millisecond / pulse, about 10 millisecond / pulse to about 500 millisecond / pulse, about 10 millisecond / pulse to about 1,000 millisecond / pulse, about 10 millisecond / pulse to about 5,000 millisecond / pulse, about 50 millisecond / pulse to about 100 millisecond / pulse, about 50 millisecond / pulse to about 500 millisecond / pulse, about 50 millisecond / pulse to about 1,000 millisecond / pulse, about 50 millisecond / pulse to about 5,000 millisecond / pulse, about 100 millisecond / pulse to about 500 millisecond / pulse, about 100 millisecond / pulse to about 1,000 millisecond / pulse, about 100 millisecond / pulse to about 5,000 millisecond / pulse, about 500 millisecond / pulse to about 1,000 millisecond / pulse, about 500 millisecond / pulse to about 5,000 millisecond / pulse, or about 1,000 millisecond / pulse to about 5,000 millisecond / pulse. In some instances, the electric field generated by the cathode and anode driven by the electric pulse generation system comprises a plurality of pulses with pulse duration from about 0.01 millisecond / pulse, about 0.05 millisecond / pulse, about 0.1 millisecond / pulse, about 0.5 millisecond / pulse, about 1 millisecond / pulse, about 5 millisecond / pulse, about 10 millisecond / pulse, about 50 millisecond / pulse, about 100 millisecond / pulse, about 500 millisecond / pulse, about 1,000 millisecond / pulse, or about 5,000 millisecond / pulse. In some instances, the electric field generated by the cathode and anode driven by the electric pulse generation system comprises a plurality of pulses with pulse duration from at least about 0.01 millisecond / pulse, about 0.05 millisecond / pulse, about 0.1 millisecond / pulse, about 0.5 millisecond / pulse, about 1 millisecond / pulse, about 5 millisecond / pulse, about 10 millisecond / pulse, about 50 millisecond / pulse, about 100 millisecond / pulse, about 500 millisecond / pulse, or about 1,000 millisecond / pulse. In some instances, the electric field generated by the cathode and anode driven by the electric pulse generation system comprises a plurality of pulses with pulse duration fromat most about 0.05 millisecond / pulse, about 0.1 millisecond / pulse, about 0.5 millisecond / pulse, about 1 millisecond / pulse, about 5 millisecond / pulse, about 10 millisecond / pulse, about 50 millisecond / pulse, about 100 millisecond / pulse, about 500 millisecond / pulse, about 1,000 millisecond / pulse, or about 5,000 millisecond / pulse. In some cases, the cathode and anode are driven by the electric pulse generation system to generate comprises 1 pulse, 2 pulses, 3 pulses, 4 pulses, 5 pulses, 6 pulses, 7 pulses, 8 pulses, 9 pulses, 10 pulses, 11 pulses, 12 pulses, 13 pulses, 14 pulses, 15 pulses, 16 pulses, 17 pulses, 18 pulses, 19 pulses, 20 pulses, or more to each multiwell cell culture / electroporation chamber of the multi-well electroporation device or system for high-throughput electroporation of the donor cells.

[0215] Preferably, the electric pulse generation system is configured to sequentially deliver an electrical pulse to a first cathode of the cathode array and a first anode of the anode array, a second cathode of the cathode array and a second anode of the anode array, a third cathode of the cathode array and a third anode of the anode array, etc. until electrical pulses are delivered to each of the culture / electroporation wells of the multi-well electroporation device or system. In some cases, the sequential delivery of the electrical pulse can include delivering the electrical pulse or plurality of electrical pulses to each culture / electroporation well one at a time in sequence. In some cases, the sequential delivery of the electrical pulse or plurality of pulses can include electroporating culture / electroporation wells along sequential rows or along sequential columns of the multi-well electroporation device or system, optionally wherein the electrical pulses are delivered simultaneously to all culture / electroporation wells of a first row of wells, and then delivered simultaneously to all culture / electroporation wells of a second row of wells, until an electrical pulse or plurality of electrical pulses is delivered to a desired portion of all culture / electroporation wells or all of the all culture / electroporation wells. As another example, the sequential delivery of the electrical pulse can include delivering the electrical pulse or plurality of electrical pulses simultaneously to more than one culture / electroporation wells that are not adjacent to one another (e.g., a partial checkerboard pattern). In yet another nonlimiting example, the sequential delivery of the electrical pulse can include delivering the electrical pulse or plurality of electrical pulses simultaneously to a first group of more than one culture / electroporation wells that are not adjacent to one another, then delivering an electrical pulse or plurality of electrical pulses simultaneously to a second group of culture / electroporation wells that are not adjacent to one another after then electrical pulses have been delivered to the first group, and optionally delivering an electrical pulse or a plurality of electrical pulse to a third group and / or a fourth group of culture / electroporation wells in sequence after the electrical pulse(s) of the previous group have concluded. In some cases, the electric pulse generation system is configured to execute a design raster pattern determine by a user to deliver theelectrical pulse or plurality of electrical pulses to at least one culture / electroporation well, more than one culture / electroporation well, a first portion of culture / electroporation wells, a second portion of culture / electroporation wells, a third portion culture / electroporation wells, a fourth portion or culture / electroporation wells, any desired combination of culture / electroporation wells, or all culture / electroporation wells of the multi-well electroporation device or system.

[0216] In some cases, the electric pulse generation system is configured to execute an automated routine to deliver an electrical pulse to the anode wire or at least one of the individual wire anodes of the array of wire anodes.Accessory device for automation

[0217] The system for high-throughput electroporation can also comprise a liquid handling or liquid distribution device configured to: (a) dispose the at least one donor cell within at least one individual hole of the first spacer array; (b) add or remove an electroporation buffer within at least one individual hole of the second spacer array; (c) add or remove a cell culture medium within at least one individual hole of the first spacer array.

[0218] In some cases, the liquid handling or liquid distribution device is configured to accomplish (a), (b), and (c) in an automated fashion. Alternatively, the liquid handling or liquid distribution device can be manually operated.

[0219] In some cases, the liquid handling or liquid distribution device is configured to add a cell culture medium to at least one, more than one, a portion of, or to all cell culture chambers of the multi-well electroporation device or system. The liquid handling or liquid distribution device can also be configured to remove a cell culture medium from at least one, more than one, a portion of, or from all cell culture chambers of the multi-well electroporation device or system. The liquid handling or liquid distribution device can also be configured to replace a cell culture medium of at least one, more than one, a portion of, or of all cell culture chambers of the multi - well electroporation device or system.

[0220] In some cases, the liquid handling or liquid distribution device is configured to dispose at least one donor cell or a plurality of donor cells to at least one, more than one, a portion of, or to all cell culture chambers of the multi-well electroporation device or system.

[0221] The multi-well electroporation device or system is also configured to culture more than one type of donor cell, including at least two, at least three, at least four, at least five, at least eight, at least ten, or more types of donor cells. For example, a first type of donor is disposed and cultured within a first cell culture chamber, a second type of donor is disposed and cultured within a second cell culture chamber, a third type of donor is disposed and cultured within a third cell culture chamber, a fourth type of donor is disposed and cultured within a fourth cell culture chamber, etc. In some cases, the multi-well electroporation device or system is also configured toculture from one type of donor cell to about one hundred types of donor cells, from one type of donor cell to about 500 types of donor cells, from one type of donor cell to about 50 types of donor cells, from one type of donor cell to about 25 types of donor cells.

[0222] In some cases, the liquid handling or liquid distribution device is configured to add an electroporation buffer to at least one, more than one, a portion of, or to all electroporation reagent chambers of the multi-well electroporation device or system. The liquid handling or liquid distribution device can also be configured to remove an electroporation buffer from at least one, more than one, a portion of, or from all electroporation reagent chambers of the multi - well electroporation device or system. The liquid handling or liquid distribution device can also be configured to replace an electroporation buffer of at least one, more than one, a portion of, or of all electroporation reagent chambers of the multi-well electroporation device or system.

[0223] The system for high-throughput electroporation can also be configured to perform multiplex cell electroporation. For example, each electroporation reagent chamber of the multiwell electroporation device or system can be loaded with a different electroporation buffer comprising one or any combination of the transfection reagents described above. As another example, an electroporation buffer of a first electroporation reagent chamber can comprise a different concentration of a transfection reagent than a second concentration of a transfection reagent a second electroporation buffer of a second electroporation reagent chamber, than a third concentration of a transfection reagent third electroporation buffer of a third electroporation reagent chamber, etc. Accordingly, the multi-well electroporation device or system is configured to titrate a transfection reagent across a plurality of cell culture / electroporation wells of the multi-well electroporation device or system.

[0224] In some cases, the electroporation buffer of a first electroporation reagent chamber can be the same as a second electroporation buffer of a second electroporation reagent chamber, as a third electroporation buffer of a third electroporation reagent chamber, etc. if desired. In some cases, the first electroporation buffer can be added to all electroporation reagent chambers of the multi-well electroporation device or system.

[0225] The system for high-throughput electroporation can also comprise a power supply. The power supply of the system or multi-well electroporation device can be configured to have a voltage output of at least about 1 V, from about IV to about 500 V, or from about 10 V to about 300 V, from about 20 V to about 300 V, or preferably from about 50 V to about 200 V. In some embodiments, the power supply is configured to delivery an electrical current from about 0.1 A to about 20 A, from about 1 A to about 20 A, from about 5 A to about 15 A, or preferably from about 8 A to about 12 A. In some cases, the power supply is configured to deliver an electrical current of about 10 A to the multi-well electroporation device or system.

[0226] In some cases, the second spacer array is in contact with a surface of the perforated membrane and defines an array of electroporation chambers; wherein individual cell electroporation chambers of the array of electroporation chambers are isolated from neighboring electroporation chambers by the second spacer array; and wherein the individual cell electroporation chambers are configured to contain at least one electroporation buffer, optionally wherein the individual cell electroporation chambers are configured to contain a different electroporation buffer, a different transfection reagent, or a different concentration of a transfection reagent than other individual cell electroporation chambers. In some cases, the first spacer array is in contact with a first surface of the perforated membrane opposite the surface of the perforated membrane in contact with the first spacer array; wherein the first spacer array defines an array of cell culture chambers; wherein individual cell culture chambers of the array of cell culture chambers are isolated from neighboring cell culture chambers by the first spacer array; wherein individual cell culture chambers are fluidically coupled to an individual electroporation chamber via the channels of the perforated membrane; and wherein the individual cell culture chambers are configured to contain at least one cell culture medium and the at least one donor cell, optionally wherein the individual cell culture chambers are configured to contain a different cell culture medium or a different donor cell type than other individual cell culture chambers.Device for use in producing EVs

[0227] The systems for high-throughput electroporation provided herein can be used to produce extracellular vesicles (EVs) containing any one or more of the transfection reagents described above. The system can also be used to collect the electroporated extracellular vesicles (e.g., extracellular vesicles (EVs) containing any one or more of the transfection reagents, such as a DNA, a DNA plasmid, RNA, protein, etc.) secreted from donors cells that are cultured and electroporated within an isolated cell culture / electroporation chamber of the multi-well electroporation device. Any perforated membrane described herein and known in the art can be used.

[0228] In some cases, the multi-well electroporation device is capable of stimulating production of extracellular vesicles (EVs) from donor cells within a cell culture chamber. In some cases, the multi-well electroporation device can result in an increase of EV production. In some cases, the multi-well electroporation device can result in at least about a 1 -fold, 2-fold, 3-fold, 4-fold, 5- fold, 10-fold, 100-fold, 1,000-fold, 2,000-fold, 3,000-fold, 4,000-fold, or 5,000-fold increase of EV production from the donor cells within the cell culture chamber. In some cases, the multiwell electroporation device is capable of increasing production of extracellular vesicles (EVs) from donor cells within a cell culture chamber relative to a Transwell electroporation device. Insome cases, the multi-well electroporation device is capable of increasing production of extracellular vesicles (EVs) from donor cells within a cell culture chamber relative to a 6 -well Transwell electroporation device. In some cases, the multi-well electroporation device is capable of increasing production of extracellular vesicles (EVs) from donor cells within a cell culture chamber relative to a 12 -well Transwell electroporation device. In some cases, the multi -well electroporation device is capable of increasing production of extracellular vesicles (EVs) from donor cells within a cell culture chamber by at least about a 50 -fold increase relative to a 6 -well Transwell electroporation device. In some cases, the multi-well electroporation device is capable of increasing production of extracellular vesicles (EVs) from donor cells within a cell culture chamber by at least about 2,000-fold increase relative to a 12-well Transwell electroporation device.IV. High-throughput Cell Electroporation System with Dual-Spacer Array Configuration

[0229] The instant disclosure further provides a system for high-throughput cell electroporation. The system for high-throughput cell electroporation comprises: (a) a perforated membrane, wherein the perforated membrane comprises channels disposed through the perforated membrane; (b) a first array spacer; (c) a second array spacer; and (d) at least one donor cell, wherein the first array spacer, the second array spacer, and the at least one donor cell are in contact with the perforated membrane.

[0230] The perforated membrane can comprise a polymer membrane, a silicon membrane, an Aluminum Oxide membrane, or track-etched membrane. In some cases, the perforated membrane comprises a polymer microporous membrane, a microfabricated silicon membrane, an Aluminum Oxide membrane, a water-soluble polymer membrane, or a track-etched membrane). In some cases, the perforated membrane comprises any of the perforated membranes and embodiments thereof previously described in this disclosure. For example, the system can comprise the perforated membrane comprising a first layer and a second layer, according to the embodiments provided in Section I: Perforated Membranes of the instant disclosure. The system can, as another example, comprise a perforated membrane made according to the methods and embodiments provided in Section IE Methods of Manufacturing Perforated Membranes, such as a perforated membrane made by sacrificial template imprinting.

[0231] In some cases, the perforated membrane comprises a micropore patterned silicon membrane, a nanopore patterned silicon membrane, a ceramic micropore membrane, a ceramic nanopore membrane, other porous materials, or a combination thereof. In some cases, the perforated membrane is made of a rigid material. Alternatively, the perforated membrane can be made of a flexible material.

[0232] A perforated track-etch membrane can be made of a polymer, such as a thermoplastic polymer (e.g., polyethylene terephthalate (PET), polycarbonate, etc.). The perforated track-etch membrane comprises a thickness. In some cases, the thickness of the track-etch membrane is at least about 10 pm. In some cases, the thickness of the track-etch membrane is no more than about 100 pm. In some cases, the thickness of the track-etch membrane is from about 1 pm to about 100 pm, from about 1 pm to about 50 pm, from about 1 pm to about 30 pm, from about 5 pm to about 30 pm, or from about 5 pm to about 15 pm. In some cases, the thickness of the track-etch membrane is about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, or about 30 pm. The perforated track-etch membrane can have channels with features previously described in Section I: Perforated Membranes of the instant disclosure.

[0233] In some cases, the perforated membrane comprises: (i) a first layer comprising a plurality of first channels disposed through the first layer; and (ii) a second layer in contact with the first layer, the second layer comprising a plurality of second channels disposed through the second layer, wherein: a first average thickness of the first layer is different from a second average thickness of the second layer; a first channel of the plurality of first channels is in fluid communication of a second channel of the plurality of second channels; a first average diameter of the first channel is different from a second average diameter of the second channel. The features of such a perforated membrane are provided above, such as in Section I: Perforated Membranes of the instant disclosure.

[0234] Embodiments of the channels disposed through the perforated membrane can be any of the features of channels previously discussed of channels disposed in perforated membranes of this disclosure. Such features of channels for perforated membranes are provided in, for example, Section I: Perforated Membranes of the instant disclosure and channels of perforated membranes made according to the methods of Section II: Methods of Manufacturing Perforated Membranes, such as a perforated membrane made by sacrificial template imprinting. In some cases, the channels are distributed throughout the perforated membrane in a patterned arrangement or a non-pattemed arrangement.

[0235] The first spacer array comprises a first thickness, a plurality of holes defined by an absence of the first material of the first spacer array, an outer perimeter having a length and a width, and other features and embodiments thereof described above in Section III: High- throughput Cell Electroporation System with Dual-Lay er Perforated Membrane and Spacer Array of this disclosure. In some cases, the first spacer array comprises: (a) a thickness greater than a diameter of a donor cell; and (b) a plurality of first holes, wherein a first hole of the plurality of first holes comprises a first hole width and a second hole width orthogonal to the first width, wherein the first hole of the first spacer array has a cross-sectional area defined by a material ofthe first spacer array. In some cases, the first hole width and the second hole width of the first hole of the first spacer array is less than a surface area of a surface of the perforated membrane having openings of a first plurality of channels. As previously described, the first spacer array comprises a plurality of first holes. In some cases, a first hole of the first spacer array is covered by the perforated membrane.

[0236] The first holes of the first spacer array have a first hole width and a second hole width, as previously described. In some cases, the first hole width of the first hole is at least about 1 mm. In some cases, the first hole width of the first hole is from at least about 1 mm to about 10 cm. In some cases, the first hole width of the first hole is about 1 cm. In some cases, the second hole width of the first hole is at least about 5 mm. In some cases, the second hole width of the first hole is from at least about 1 mm to about 10 cm. In some cases, the second hole width of the first hole is about 1 cm.

[0237] The first holes of the first spacer array also have an inter-hole distance defined by the presence of the material of the first spacer array that separates a first hole and another first hole that is adjacent to the first hole. In some cases, the first hole is separated from other first holes adjacent to the first hole of the first spacer array by a first inter-hole distance. In some cases, the first hole of the first spacer array is separated from other first holes adjacent to the first hole of the first spacer array by a first inter-hole distance of at least about 1 mm. In some cases, the first hole of the first spacer array is separated from other first holes adjacent to the first hole of the first spacer array by a first inter-hole distance of from about 1 mm to about 1 .5 cm or from about 1 mm to about 5 cm. In some cases, the first hole of the first array spacer is separated from other first holes adjacent to the first hole of the first spacer array by a first inter-hole distance of about 5 mm.

[0238] As previously provided, the first spacer array also has a first thickness. In some cases, the thickness of the first spacer array is from about 0.1 cm to about 5 cm.

[0239] The first spacer array also has an out perimeter defined by a length and a width of an outer side and in a plane that is parallel to a surface of the first spacer array in contact with the perforated membrane. In some cases, a width of an outer side of the first spacer array is from about 1 cm to about 30 cm.

[0240] The first spacer array also has a number of first holes. In some cases, the first spacer array comprises at least about 2 first holes. In some cases, the first spacer array comprises from about 2 first holes to about 500 first holes, from about 2 first holes to about 100 first holes, or from about 2 first holes to about 30 first holes. The first spacer array can have any number of first holes.

[0241] As outlined above, the plurality of first holes of the first spacer array can be arranged into an array having rows and columns. In some cases, the plurality of first holes of the first spacer array comprises: (a) at least 2 rows of first holes of the plurality of first holes; and (b) at least 2 columns of first holes of the plurality of first holes. The first spacer array can have any number of first holes arranged into any desired number of rows and number of columns of first holes.

[0242] As described above, the first spacer array is made of a material, such as any of the materials described of spacer arrays in this disclosure. For example, the first spacer array can comprise a polymer. In some cases, the first spacer array comprises a thermoplastic polymer, a crosslinking polymer, a light-curing polymer, a thermal-curing polymer, a light-reactive polymer, a silicon polymer, a rubber, or a photoresist polymer. In some cases, the first array spacer comprises polyethylene terephthalate (PET), polycarbonate, poly dimethylsiloxane (PDMS), poly dimethylsiloxane (PDMA), polyvinyl alcohol (PVA), polyethylene, polypropylene, polystyrene, polyacrylamide, or polyacrylic acid.

[0243] The second spacer array comprises a second thickness, a plurality of holes defined by an absence of the second material of the second spacer array, an outer perimeter having a length and a width, and other features and embodiments thereof described above in Section III: High- throughput Cell Electroporation System with Dual-Lay er Perforated Membrane and Spacer Array of this disclosure. The second spacer array of the system comprises: (a) a second thickness greater than a diameter of a donor cell; and (b) a plurality of second holes, wherein a second hole of the second spacer array comprises a first hole width and a second hole width orthogonal to the first width; wherein the second hole of the second spacer array has a cross-sectional area defined by a material of the second spacer array. In some cases, the second spacer array is in contact with the perforated membrane. In some cases, the second hole of the second spacer array does not exceed a surface area of a surface of the perforated membrane having openings of a second plurality of channels. In some cases, the second hole width and the second hole width of the second hole of the second spacer array is less than a surface area of a surface of the perforated membrane having openings of a second plurality of channels. As previously described, the second spacer array comprises a plurality of second holes. In some cases, a second hole of the second spacer array is covered by the perforated membrane.

[0244] The second holes of the second spacer array have a first hole width and a second hole width, as previously described. In some cases, the first hole width of the second hole is at least about 1 mm. In some cases, the first hole width of the second hole is from at least about 1 mm to about 10 cm. In some cases, the first hole width of the second hole is about 1 cm. In some cases, the second hole width of the second hole is at least about 5 mm. In some cases, the second hole width of the second hole is from at least about 1 mm to about 10 cm. In some cases, the secondhole width of the second hole is about 1 cm. In some cases, the first hole width and the second hole width of the second hole of the second spacer array are the same as the first hole width and the second hole width of the first hole of the first spacer array . In some cases, the first hole width and the second hole width of the second hole of the second spacer array are different than the first hole width and the second hole width of the first hole of the first spacer array.

[0245] The second holes of the second spacer array also have an inter-hole distance defined by the presence of the material of the second spacer array that separates a second hole and another second hole that is adjacent to the second hole. In some cases, the second hole is separated from other second holes adjacent to the second hole of the second spacer array by a second inter -hole distance. In some cases, the second hole of the second spacer array is separated from other second holes adjacent to the second hole of the second spacer array by a second inter -hole distance of at least about 1 mm. In some cases, the second hole of the second spacer array is separated from other second holes adjacent to the second hole of the second spacer array by a second inter-hole distance of from about 1 mm to about 1.5 cm or from about 1 mm to about 5 cm. In some cases, the second hole of the second array spacer is separated from other second holes adjacent to the second hole of the second spacer array by a second inter-hole distance of about 5 mm. In some cases, the second inter-hole distance of the second spacer array is different than the first inter-hole distance of the first spacer array. In some cases, the second inter-hole distance of the second spacer array is substantially the same as the first inter-hole distance of the first spacer array. In some cases, the second inter-hole distance of the second spacer array is identical to the first inter-hole distance of the first spacer array. In some cases, the second interhole distance separating the second hole from other second holes adjacent to the second hole of the second spacer array is different than the first inter-hole distance separating the first hole from the other first holes adjacent to the first hole of the first array of holes of the first spacer array. In some cases, the second inter-hole distance separating the second hole from other second holes adjacentto the second hole of the second spacer array is substantially the same as the first interhole distance separating the first hole from the other first holes adjacent to the first hole of the first array of holes of the first spacer array. In some cases, the second inter-hole distance separating the second hole from other second holes adjacent to the second hole of the second spacer array is identical to the first inter-hole distance separating the first hole from the other first holes adjacent to the first hole of the first array of holes of the first spacer array .

[0246] As previously provided, the second spacer array of this system also has a second thickness. In some cases, the thickness of the second spacer array is from about 0. 1 cm to about 5 cm. In some cases, the second thickness of the second spacer array is the same as the firstthickness of the first spacer array . In some cases, the second thickness of the second spacer array is different than the first thickness of the first spacer array.

[0247] The second spacer array also has an out perimeter defined by a length and a width of an outer side and in a plane that is parallel to a surface of the second spacer array in contact with the perforated membrane. In some cases, a width of an outer side of the second spacer array is from about 1 cm to about 30 cm.

[0248] The second spacer array also has a number of second holes. In some cases, the second spacer array comprises at least about 2 second holes. In some cases, the second spacer array comprises from about 2 second holes to about 500 second holes, from about 2 second holes to about 100 second holes, or from about 2 second holes to about 30 second holes. The second spacer array can have any number of second holes.

[0249] As outlined above, the plurality of second holes of the second spacer array can be arranged into an array having rows and columns. In some cases, the plurality of second holes of the second spacer array comprises: (a) at least 2 rows of second holes of the plurality of second holes; and (b) at least 2 columns of second holes of the plurality of second holes. The second spacer array can have any number of second holes arranged into any desired number of rows and number of columns of second holes.

[0250] In some cases, the second spacer array is identical to the first spacer array. In some cases, the second spacer array is substantively the same as the first spacer array. In some cases, the second spacer array is different than the first spacer array by at least one aspect.

[0251] As described above, the second spacer array is made of a material, such as any of the materials described of spacer arrays in this disclosure. For example, the second spacer array can comprise a polymer. In some cases, the second spacer array comprises a thermoplastic polymer, a crosslinking polymer, a light-curing polymer, a thermal -curing polymer, a light-reactive polymer, a silicon polymer, a rubber, or a photoresist polymer. In some cases, the second array spacer comprises polyethylene terephthalate (PET), polycarbonate, poly dimethylsiloxane (PDMS), poly dimethylsiloxane (PDMA), polyvinyl alcohol (PVA), polyethylene, polypropylene, polystyrene, polyacrylamide, or polyacrylic acid.

[0252] The system for high-throughput electroporation can also comprise a cathode. The cathode is made of a material that is electrically conductive and non -toxic to the at least one donor cell and has a three-dimensional shape, among other features described within this disclosure, such as in Section III: High-throughput Cell Electroporation System with Dual-Layer Perforated Membrane and Spacer Array. The cathode can have any one or combination of the embodiments provided throughoutthe disclosure. As previously outlined, the cathode can be configured to be disposed within a first hole of the first spacer array (e.g., a cell culture chamber). In some cases,the cathode comprises a shape, such as a three-dimensional (3D) cathode plate or a rod shape. In some cases, the cathode is configured to contact a surface of the first spacer array or the second spacer array. In some embodiments, the cathode comprises a cathode coil to configured to be disposed within the first hole of the first spacer array or the second hole of the second spacer array. The cathode can comprise an array of cathode coils having any of the features described above.

[0253] The system for high-throughput electroporation can also comprise an anode. The anode is made of a material that is electrically conductive and non-toxic to the at least one donor cell and has a three-dimensional shape, among other features described within this disclosure, such as in Section III: High-throughput Cell Electroporation System with Dual -Lay er Perforated Membrane and Spacer Array. The anode can have any one or combination of the embodiments provided throughout the disclosure. As previously outlined, the anode can be configured to be disposed within a first hole of the first spacer array (e.g., a cell culture chamber). In some cases, the anode comprises a shape, such as a three-dimensional (3D) anode plate or a rod shape. In some cases, the anode comprises a wire anode. In some cases, the anode is configured to contact a surface of the first spacer array or the second spacer array. In some embodiments, the anode comprises an anode coil to configured to be disposed within the first hole of the first spacer array or the second hole of the second spacer array. The anode can comprise array of wire anodes configured to position individual wire anodes of the array of wire anodes within the individual holes of the first spacer array or second spacer array. The array of anodes can have any of the features of anode arrays described above.

[0254] The system for high-throughput electroporation can also comprise an electric pulse generation system configured to provide an electrical voltage or an electrical current to the donor cell via the cathodes and anodes described above (e.g., configured to electroporate the donor cell). As described above in Section III: High-throughput Cell Electroporation System with Dual-Layer Perforated Membrane and Spacer Array of this disclosure, the electric pulse generation system comprises: (a) an electrical pulse generator; (b) a shock pulse converter; and (c) an electrical current output device. The electric pulse generation system can be automated or manually operated. Preferably, the electric pulse generation system is configured to adjust a feature of an electrical pulse applied to the at least one donor cell in contact with the perforated membrane. The electric pulse generation system of this system for high-throughput cell electroporation can possess any of the features of an electric pulse generation system described throughout this disclosure.

[0255] As outlined above, the system for high-throughput electroporation provided herein can also comprise a liquid handling or liquid distribution device configured to: (a) dispose the at leastone donor cell within at least one individual hole of the first spacer array; (b) add or remove an electroporation buffer within at least one individual hole of the second spacer array; (c) add or remove a cell culture medium within at least one individual hole of the first spacer array. The liquid handling or liquid distribution device suitable for this system for high-throughput electroporation can have any one or combination of the features and embodiments of liquid handling or liquid distribution devices described previously.

[0256] Furthermore, the donor cell of the system for high-throughput electroporation is in contact with the perforated membrane, such as in contact with an opening of at least one channel of the perforated membrane, and has other features, including a cell type. The donor cell of the system can have any of the features and embodiments described of the donor cells of this disclosure, such as in Section III: High-throughput Cell Electroporation System with Dual -Layer Perforated Membrane and Spacer Array.V. Methods of high-throughput Cell Electroporation

[0257] Provided herein is a method of producing and collecting extracellular vesicles. The method comprises: (a) providing an electroporation device comprising multiple wells, such device comprising a perforated membrane situated between a first spacer array and, optionally, a second spacer array, wherein: the spacer array(s) comprise complementary arrays of holes defining cross-sectional area of the wells; the wells comprise a portion of the perforated membrane; and the portion of the perforated membrane separates a cell culture chamber from an electroporation buffer chamber such that pores within the perforated membrane fluidically couple the cell culture chamber with the electroporation buffer chamber; (b) introducing donors cells into at least one cell culture chamber; (c) introducing a polynucleotide, DNA, RNA, vector, or plasmid into at the least electroporation buffer chamber; (d) applying an electric field, a current, or a voltage to one or more of the electroporation reagent chambers of the multi-well electroporation device, thereby electroporating the donor cells; and (e) collecting extracellular vesicles (EVs) produced by the donors cells. In some cases, the method comprises sequentially electroporating the donor cells with more than one transfection reagent, such as any combination of transfection reagents provided above. Alternatively, in some cases, delivering more than one transfection reagent to the donor cells can comprise simultaneously introducing at least 2 transfection regents to the donor cells. In some cases, delivery more than one transfection reagent to the donor cells can comprise repeating the method from step (b) to step (e), from step (c) to step (d), or repeating only step (d), whereby one or a portion of the transfection agents to be electroporated into the donor cells is / are introduced to the electroporation reagent chamber, and the method is repeated until all transfection reagents are electroporated into the donor cells. A method of producing extracellular vesicles is provided in U.S. Patent No. 11,674,130, the detailsof which are incorporated by reference. In some cases, the method from step (a) to step (e), or from step (a) to step (d), can be repeated at least once, twice, three times, or 5 times.

[0258] The extracellular vesicles (EVs) produced by the method can be extracellular vesicles are any membrane-bound particle (e.g., a vesicle with a lipid bilayer). Often, the extracellular vesicles provided herein are secreted by a cell. In some instances, the extracellular vesicles are membrane - bound particles produced in vitro. In some cases, the extracellular vesicles are produced and secreted by an extracellular vesicle donor cell transfected with at least one heterologous polynucleotide. In some instances, the extracellular vesicle is an exosome, a microvesicle, a retrovirus-like particle, an apoptotic body, an apoptosome, an oncosome, an exopher, an enveloped virus, an exomere, or other very large extracellular vesicle such as a large oncosome. In some cases, the extracellular vesicle is an exosome.

[0259] In some cases, the method produces at least about a 3-fold increase in the number of extracellular vesicles (EVs) secreted by the electroporated donor cells compared to the number of EVs secreted by an unelectroporated population of donor cells. In some cases, the method produces at least about a 5 -fold, 10-fold, 100-fold, 1,000-fold, 5,000-fold, 10,000-fold, 20,000- fold, 50,000-fold, or 100,000-fold increase in the number of extracellular vesicles (EVs) secreted by the electroporated donor cells compared to the number of EVs secreted by an unelectroporated population of donor cells. In some cases, the method produces from about a 3- fold increase to about a 100,000-fold increase, from about a 10-fold increase to about a 10,000- fold increase, from about a 10-fold increase to about a 1 ,000-fold increase, from about a 10-fold increase to about a 1,000-fold increase, from about a 10-fold increase to about a 100-fold increase, from about a 100-fold increase to about a 100,000-fold increase, from about a 100-fold increase to about a 10,000-fold increase, from about a 100-fold increase to about a 1,000-fold increase, from about a 100-fold increase to about a 100,000-fold increase, from about a 1,000- fold increase to about a 5,000-fold increase, from about a 5,000-fold increase to about a 10,000- fold increase, or from about a 100-fold increase to about a 20,000-fold increase in the number of extracellular vesicles (EVs) secreted by the electroporated donor cells compared to the number of EVs secreted by an unelectroporated population of donor cells.

[0260] In some cases, the method produces at least about a 1.1 -fold increase in the number of extracellular vesicles (EVs) secreted by the electroporated donor cells compared to the baseline amount of secreted extracellular vesicles (EVs) than the number of EVs secreted by a population of donor cells electroporated on a track-etch membrane. In some cases, the method produces at least about a 1.2-fold, 1.5-fold, 2-fold,, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, 20,000-fold, 50,000- fold, or 100,000-fold increase in the number of extracellular vesicles (EVs) secreted by theelectroporated donor cells compared to the baseline amount of secreted extracellular vesicles (EVs) than the number of EVs secreted by a population of donor cells electroporated on a tracketch membrane. In some cases, the method produces from about a 1.2-fold increase to about a 50-fold increase, from about a 1.2-fold increase to about a 20-fold increase, from about a 1.2- fold increase to about a 10-fold increase, from about a 1.2-fold increase to about a 20-fold increase, from about a 1.2-fold increase to about a 10-fold increase, from about a 2-fold increase to about a 100-fold increase in the number of extracellular vesicles (EVs) secreted by the electroporated donor cells compared to the baseline amount of secreted extracellular vesicles (EVs) than the number of EVs secreted by a population of donor cells electroporated on a track etch membrane. In some cases, the method produces at least about a 1.5 -fold increase in the number of extracellular vesicles (EVs) secreted by the electroporated donor cells compared to the baseline amount of secreted extracellular vesicles (EVs) than the number of EVs secreted by a population of donor cells electroporated on a track-etch membrane.

[0261] In some cases, the method produces at least about a 1.1 -fold increase in the number of extracellular vesicles (EVs) secreted by the electroporated donor cells compared to the baseline amount of secreted extracellular vesicles (EVs) than the number of EVs secreted by a population of donor cells electroporated on an electroporation membrane lacking a patterned array of channels. In some cases, the method produces at least about a 1.2-fold, 1.5-fold, 2-fold„ 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, 20,000-fold, 50,000-fold, or 100,000-fold increase in the number of extracellular vesicles (EVs) secreted by the electroporated donor cells compared to the baseline amount of secreted extracellular vesicles (EVs) than the number of EVs secreted by a population of donor cells electroporated on an electroporation membrane lacking a patterned array of channels. In some cases, the method produces from about a 1.2-fold increase to about a 50-fold increase, from about a 1.2-fold increase to about a 20-fold increase, from about a 1.2-fold increase to about a 10-fold increase, from about a 1.2-fold increase to about a 20-fold increase, from about a 1.2-fold increase to about a 10-fold increase, from about a 2-fold increase to about a 100-fold increase in the number of extracellular vesicles (EVs) secreted by the electroporated donor cells compared to the baseline amount of secreted extracellular vesicles (EVs) than the number of EVs secreted by a population of donor cells electroporated on an electroporation membrane lacking a patterned array of channels. In some cases, the method produces at least about a 1.5-fold increase in the number of extracellular vesicles (EVs) secreted by the electroporated donor cells compared to the baseline amount of secreted extracellular vesicles (EVs) than the number of EVs secreted by a population of donor cells electroporated on an electroporation membrane lacking a patterned array of channels.

[0262] In some cases, the method produces at least about a 3-fold increase in the number of extracellular vesicles (EVs) secreted by the electroporated donor cells compared to the baseline amount of secreted extracellular vesicles (EVs) than the number of EVs secreted by a population of donor cells transfected by lipofectamine transfection. In some cases, the method produces at least about a 5 -fold, 10-fold, 100-fold, 1,000-fold, 5,000-fold, 10,000-fold, 20,000-fold, 50,000- fold, or 100,000-fold increase in the number of extracellular vesicles (EVs) secreted by the electroporated donor cells compared to the baseline amount of secreted extracellular vesicles (EVs) than the number of EVs secreted by a population of donor cells transfected by lipofectamine transfection. In some cases, the method produces from about a 3-fold increase to about a 100,000-fold increase, from about a 10-fold increase to about a 10,000-fold increase, from about a 10-fold increase to about a 1,000-fold increase, from about a 10-fold increase to about a 1,000-fold increase, from about a 10-fold increase to about a 100-fold increase, from about a 100-fold increase to about a 100,000-fold increase, from about a 100-fold increase to about a 10,000-fold increase, from about a 100-fold increase to about a 1,000-fold increase, from about a 100-fold increase to about a 100,000-fold increase, from about a 1,000-fold increase to about a 5,000-fold increase, from about a 5, 000 -fold increase to about a 10,000-fold increase, or from about a 100-fold increase to about a 20,000-fold increase in the number of extracellular vesicles (EVs) secreted by the electroporated donor cells compared to the baseline amount of secreted extracellular vesicles (EVs) than the number of EVs secreted by a population of donor cells transfected by lipofectamine transfection.

[0263] In some cases, at least about 50% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the polynucleotide. In some cases, atleast about 70% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the polynucleotide. In some cases, at least about 75%, 80%, 85%, 90%, 95%, 97.5%, or 100% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the polynucleotide. In some cases, from about 50% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the polynucleotide,. In some cases, from about 70% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the polynucleotide. In some cases, from about 80% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the polynucleotide. In some cases, from about 85% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the polynucleotide.

[0264] In some cases, at least about 50% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the DNA. In some cases, at least about 70% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the DNA. Insome cases, at least about 75%, 80%, 85%, 90%, 95%, 97.5%, or 100% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the DNA. In some cases, from about 50% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the DNA,. In some cases, from about 70% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the DNA. In some cases, from about 80% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the DNA. In some cases, from about 85% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the DNA.

[0265] In some cases, at least about 50% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the RNA. In some cases, at least about 70% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the RNA. In some cases, at least about 75%, 80%, 85%, 90%, 95%, 97.5%, or 100% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the RNA. In some cases, from about 50% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the RNA,. In some cases, from about 70% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the RNA. In some cases, from about 80% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the RNA. In some cases, from about 85% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the RNA.

[0266] In some cases, at least about 50% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the vector. In some cases, at least about 70% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the vector. In some cases, at least about 75%, 80%, 85%, 90%, 95%, 97.5%, or 100% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the vector. In some cases, from about 50% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the vector,. In some cases, from about 70% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the vector. In some cases, from about 80% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the vector. In some cases, from about 85% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the vector.

[0267] In some cases, at least about 50% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the plasmid. In some cases, at least about 70% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the plasmid. In some cases, at least about 75%, 80%, 85%, 90%, 95%, 97.5%, or 100% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the plasmid. In some cases,from about 50% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the plasmid,. In some cases, from about 70% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the plasmid. In some cases, from about 80% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the plasmid. In some cases, from about 85% to about 95% of the extracellular vesicles (EVs) secreted by the electroporated donor cells comprise the plasmid.

[0268] In some cases, the introducing the polynucleotide comprises introducing at least 2 polynucleotides, at least 3 polynucleotides, at least 4 polynucleotides, at least 5 polynucleotides, or more polynucleotides into at least one electroporation buffer chamber.

[0269] In some cases, the extracellular vesiclescan have a diameter about 10 nmto about 50,000 nm. In some cases, the extracellular vesicles can have a diameter about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 50 nm, about lO nm to about 100 nm, about 10 nm to about 200 nm, about 10 nmto about 500 nm, about 10 nm to about 1,000 nm, about 10 nm to about 2,000 nm, about 10 nm to about 5,000 nm, about 10 nmto about 10,000 nm, about 10 nm to about 50,000 nm, about 20 nm to about 30 nm, about 20 nm to about 50 nm, about 20 nm to about 100 nm, about 20 nmto about 200 nm, about 20 nm to about 500 nm, about 20 nm to about 1,000 nm, about 20 nm to about 2,000 nm, about 20 nmto about 5,000 nm, about 20 nm to about 10,000 nm, about 20 nmto about 50,000 nm, about 30 nm to about 50 nm, about 30 nm to about 100 nm, about 30 nm to about 200 nm, about 30 nm to about 500 nm, about 30 nmto about 1,000 nm, about 30 nm to about 2,000 nm, about 30 nm to about 5,000 nm, about 30 nm to about 10,000 nm, about 30 nm to about 50,000 nm, about 50 nm to about 100 nm, about 50 nm to about 200 nm, about 50 nm to about 500 nm, about 50 nm to about 1,000 nm, about 50 nm to about 2,000 nm, about 50 nm to about 5,000 nm, about 50 nm to about 10,000 nm, about 50 nm to about 50,000 nm, about 100 nm to about 200 nm, about 100 nm to about 500 nm, about 100 nm to about 1,000 nm, about 100 nmto about2,000 nm, about 100 nm to about 5,000 nm, about 100 nm to about 10,000 nm, about 100 nmto about 50,000 nm, about 200 nm to about 500 nm, about 200 nm to about 1,000 nm, about 200 nm to about 2,000 nm, about 200 nm to about 5,000 nm, about 200 nm to about 10,000 nm, about 200 nmto about 50,000 nm, about 500 nm to about 1,000 nm, about 500 nm to about2,000 nm, about 500 nm to about 5,000 nm, about 500 nm to about 10,000 nm, about 500 nm to about 50,000 nm, about 1,000 nm to about 2,000 nm, about 1,000 nm to about 5,000 nm, about 1,000 nm to about 10,000 nm, about 1,000 nm to about 50,000 nm, about 2,000 nm to about 5,000 nm, about 2,000 nm to about 10,000 nm, about 2,000 nm to about 50,000 nm, about 5,000 nmto about 10,000 nm, about 5,000 nm to about 50,000 nm, or about 10,000 nm to about 50,000 nm. In some cases, the extracellular vesicles have a diameter about 10 nm, about 20 nm, about 30 nm, about 50 nm, about lOO nm, about 200 nm, about 500 nm, about 1,000 nm,about 2,000 nm, about 5,000 nm, about 10,000 nm, or about 50,000 nm. In some cases, the extracellular vesicles can have a diameter at least about lO nm, about 20 nm, about 30 nm, about 50 nm, about lOO nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm. In some cases, the extracellular vesicles can have a diameter at mo st about 20 nm, about 30 nm, about 50 nm, about lOO nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, about 10,000 nm, or about 50,000 nm.

[0270] In some cases, the extracellular vesicles (EVs) are exosomes, apoptotic bodies or microvesicles.

[0271] The providing the electroporation device comprising multiple wells can include providing the multi-well electroporation device or system as previously described throughout this disclosure.

[0272] The perforated membrane of the electroporation device comprises multiple wells can be the perforated membrane is a silicon membrane, a track-etch membrane, a single-layer polymer membrane, a dual-layer polymer membrane, or any perforated membrane having the features of perforated membranes discussed above.

[0273] The introducing the donor cells into one or more cell culture chamber(s) can include adding, positioning, or disposing the donors cells within at least one or more than one of the plurality of cell culture chambers of the multi -well electroporation device and systems for high- throughput cell electroporation described above. In some cases, the introducing the donor cells into the cell culture chamber comprises contacting or adhering the donor cells to a first surface of the perforated membranes provided throughout this disclosure.

[0274] The introducing a polynucleotide, DNA, RNA, vector, or plasmid into the electroporation buffer chamber can include introducing any one or combination of the transfection reagents provided herein to at least one or more than one of the electroporation reagent chambers (e.g., electroporation buffer chambers) of the multi-well electroporation device and systems of this disclosure.

[0275] The applying the electric field to the donor cells can comprise applying any of the described voltages, electric fields of an electric field strength, or an electrical current listed in, for example, Section III: High-throughput Cell Electroporation System with Dual -Lay er Perforated Membrane and Spacer Array. The applying the electric field to the donor cells can comprise applying any of the described electrical pulse generating routines according to any embodiments described utilizing the electrical pulse generation system of the multi-well electroporation device, such as the number of cell culture / electroporation chambers to be stimulated, the pattern of stimulation (e.g., sequential from chamber to chamber, checkerboard pattern, simultaneous stimulation of non-adjacent cell culture / electroporation chambers, etc.), and any other parametersprovided in this disclosure. In some cases, the applying the electric field to the donor cells comprises sequentially applying the electric field to at least 1, 2, 3, 4, 5, 10, 12, 16, 25, or more cell culture / electroporation chambers of the multi-well electroporation device. In some cases, the applying the electric field to the donor cells comprises sequentially applying the electric field to of the cell culture / electroporation chambers of the multi -well electroporation device until all of the electric field is applied to all of the cell culture / electroporation chambers. In some cases, the electric field applied to one cell culture / electroporation chamber can differ from the electric field applied to another cell culture / electroporation chamber of the multi-well electroporation device.

[0276] Once the applying the electric field occurs, the donor cells are electroporated donor cells that can express the transfection reagent or combination of transfection reagents. In some cases, the electroporated donor cells can produced extracellular vesicles (EVs) encapsulating an expression product (e.g., mRNA, protein, or other biomolecule) of the one or combination transfection reagent(s) that were electroporated into the donor cells. In some cases, the transfection reagent can comprise a polynucleotide, DNA, or RNA encoding a protein or biomolecule of interest, such as a collagen (e.g., CollAl) or a dystrophin. In cases when the method is reiterated, the applying the electric field of an iteration can be different than the electric field applied during another iteration of the method.

[0277] The collecting extracellular vesicles (EVs) produced by the donors cells can comprise removing the extracellular vesicles (EVs) by manual or automated liquid handling devices. The extracellular vesicles (EVs) can then be further processed by any means to concentrate the extracellular vesicles (EVs), isolate the extracellular vesicles (EVs), and quantify a parameter or feature of the extracellular vesicles (EVs).

[0278] The EVs are collected after a period of time after applying the electric field to the donor cells. In some cases, the extracellular vesicles (EVs) are collected within an hour of applying the electric field to the donor cells. In some cases, the extracellular vesicles (EVs) are collected within about 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 16, 20, 24, 30, 40, 45, 50, 72, 84, or 96 hours of applying the electric field to the donor cells. In some cases, the extracellular vesicles (EVs) are collected after at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 16, 20, 24, 30, 40, 45, 50, 72, 84, or 96 hours of applying the electric field to the donor cells. In some cases, the extracellular vesicles (EVs) are collected within about 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 16, 20, 24, 30, 40, 45, 50, 72, 84, or 96 hours of applying the electric field to the donor cells.

[0279] In cases when the method is repeated in sequential iterations to add additional electroporation reagent(s) to the donor cells, the extracellular vesicles (EVs) are collected within about 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 16, 20, 24, 30, 40, 45, 50, 72, 84, or 96 hours of applying the electric field of the last iteration of the method to the donor cells. In cases when the method isrepeated in sequential iterations, the EVs can be collected after the applying the electric field of an iteration of the method and before the next iteration of the introducing one or more types of donor cells or the adding one or more electroporation reagents to one or more of the electroporation reagent chambers of the multi-well electroporation device. In cases when the method is repeated in sequential iterations, the extracellular vesicles (EVs) are collected within about 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 16, 20, 24, 30, 40, 45, 50, 72, 84, or 96 hours of applying the electric field of the last iteration of the method to the donor cells. In cases when the method is repeated in sequential iterations, the extracellular vesicles (EVs) are collected after at least about 0.5, 1 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 16, 20, 24, 30, 40, 45, 50, 72, 84, or 96 hours of applying the electric field of the last iteration of the method to the donor cells.Definitions

[0280] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0281] Throughout this application, various cases maybe presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0282] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the terms “a channel”, “a cell culture well”, “an electroporation reagent well”, and “an inter-hole distance” include a plurality of channels, a plurality of cell culture wells, or a plurality of electroporation reagent wells.

[0283] As used herein, open terms, for example, “comprise”, “contain”, “include”,“including”, “have”, “having” and the like refer to comprising unless otherwise indicates.

[0284] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0285] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. “Exemplary” means “an example of’ and is not intended to convey an indication of a preferred or ideal aspect. “Such as” is not used in a restrictive sense, but for explanatory purposes.

[0286] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value. Similarly, when the term “about” is used before a non-numerical term that is a stand-in for a numerical value (e.g., horizontal, perpendicular, aligned), the term “about” refers to the value of the non-numerical term (e.g., 90 degrees, 180 degrees) plus or minus 10% of that value.

[0287] As used herein, the term “substantially,” when describing a relative value, a relative amount or a relative degree between two subjects, generally refers to within 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110% of each other in value, amount or degree.

[0288] Whenever the term “at least,” “greater than,” “greater than or equal to,” “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term applies to each of the numerical values in that series of numerical values, unless otherwise specified. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1 , greater than or equal to 2, or greater than or equal to 3.

[0289] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0290] As used herein, the term “about” means within 10% above or below a given value. For example, “about 10”, would include values from 9 to 11, unless otherwise indicated by the context in which the term is used.

[0291] As used herein, “or” may refer to “and”, “or,” or “and / or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.

[0292] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0293] The term “diameter” is used herein to refer to a distance between a first wall of a pore and another wall opposite the first wall of the pore.

[0294] The term “array”, “patterned arrangement”, and “patterned distribution” are herein used interchangeably to referto a consistent distance between adjacent or neighboring individual channels of the perforated membrane with respect to an individual channel. For example, an “array” of individual channels can have a consistent distance among adjacent individual channels in a first dimension (e.g., width of the perforated membrane) and a consistent distance between adjacent individual channels in a second dimension (e.g., length of the perforated membrane). As a non-limiting illustrative example, in cases where the distance between individual channels of the array of channels is constant throughout the first surface and second surface of the perforated membrane, the individual channels may be separated by a distance of approximately 5 pm along the length of the perforated membrane and by a distance of approximately 5 pm along the width of the perforated membrane. Moreover, in cases where the rows or columns of individual channels of the array of channels are staggered, the individual channels may be separated by a distance of approximately 5 pm along the length of the perforated membrane and by approximately 10 pm along the width of the perforated membrane.

[0295] The term “inter-hole width” is used herein to refer to a distance between adjacent individual holes of a spacer array that is defined by a presence of a material of the spacer array that exists between a wall of a first individual hole and a wall of another individual hole adjacent to the first individual hole.

[0296] The term “first hole width” is used herein to referto a distance between a first wall of an individual hole and a second wall of the individual hole that is opposite of the first wall of the individual hole of a spacer array along a dimension that is parallel to a first surface of the spacer array and is defined by an absence of a material of a spacer array .

[0297] The term “second hole width” is used herein to refer to a wall-to-wall distance between of an individual hole that is orthogonal to the first hole width of the individual channel of the spacer array, whereby the wall-to-wall distance is defined by an absence of a material of a spacer array. As a non-limiting example, an individual square-shaped hole of a spacer array has 4 walls defined by a material of the spacer array. The “first hole width” is the distance between a first pair of oppositely facing walls of the individual hole defined by a lack of the spacer array material. The “second hole width” is a second distance between a second pair of oppositely facing walls that are orthogonal to the first pair of oppositely facing walls of the same individual hole.

[0298] As used herein, the term “substrate” or “solid substrate” generally refers to a substance, structure, surface, material, means, or composition, which comprises a nonbiological, synthetic, nonliving, planar, spherical or flat surface. The substrate may include, for example and without limitation, semiconductors, synthetic metals, synthetic semiconductors, insulators and dopants; metals, alloys, elements, compounds and minerals; synthetic, cleaved, etched, lithographed, printed, machined and microfabricated slides, devices, structures and surfaces; industrial polymers, plastics, membranes; silicon, silicates, glass, metals and ceramics; wood, paper, cardboard, cotton, wool, cloth, woven and nonwoven fibers, materials and fabrics; nanostructures and micro structures. The substrate may comprise an immobilization matrix such as but not limited to, insolubilized substance, solid phase, surface, layer, coating, woven or nonwoven fiber, matrix, crystal, membrane, insoluble polymer, plastic, glass, biological or biocompatible or bioerodible or biodegradable polymer or matrix, microparticle or nanoparticle. Other example may include, for example and without limitation, monolayers, bilayers, commercial membranes, resins, matrices, fibers, separation media, chromatography supports, polymers, plastics, glass, mica, gold, beads, microspheres, nanospheres, silicon, gallium arsenide, organic and inorganic metals, semiconductors, insulators, microstructures and nanostructures. Micro structures and nanostructures may include, without limitation, microminiaturized, nanometer-scale and supramolecular probes, tips, bars, pegs, plugs, rods, sleeves, wires, filaments, and tubes. The substrate can exist as one or more particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, or semiconductor integrated surface, for example. The substrate can be flat or can take on alternative surface configurations. For example, the substrate can contain raised or depressed regions on which synthesis or deposition takes place. In some examples, the substrate can contain raised or depressed regions in different 3 -D shapes and / or heights. In some examples, the substrate can comprise a plurality of features. In some examples, the substrate can comprise a topographical pattern and the topographical can comprise a group of troughs, a group of bars, a group of pillars, a group of well, or a combination thereof. In some cases, the topographical pattern can be formed by at least two different materials, for example, a silicon surface covered with a layer of photoresist, or a quartz surface covered with a hydrogel, etc. In some examples, the substrate can contain raised or depressed regions in substantially the same 3 -D shapes and / or heights. In some examples, the substrate can be chosen to provide appropriate light-absorbing characteristics. For example, the substrate can be a polymerized Langmuir Blodgett film, functionalized glass (e.g., controlled pore glass), silica, titanium oxide, aluminum oxide, indium tin oxide (ITO), Si, Ge, GaAs, GaP, SiO2, SiN4, modified silicon, the top dielectric layer of a semiconductor integrated circuit (IC) surface, or any one of a variety of gels or polymers such as (poly)tetrafluoroethylene,(poly)vinylidenedifluoride, polystyrene, polycarbonate, polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA), polycyclicolefins, or combinations thereof.

[0299] The substrates can comprise polymer coatings or gels, such as a polyacrylamide gel or a PDMS gel. Gels and coatings can additionally comprise components to modify their physicochemical properties, for example, hydrophobicity. For example, a polyacrylamide gel or coating can comprise modified acrylamide monomers in its polymer structure such as ethoxylated acrylamide monomers, phosphorylcholine acrylamide monomers, betaine acrylamide monomers, and combinations thereof.

[0300] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.EXAMPLES

[0301] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.Example 1. Design of a dual-layer polymer perforated membrane

[0302] Silicon substrates for cell electroporation / nanoelectroporation (CEP / CNP) present several limitations, including high costs, brittleness, and labor-intensive cleanroom semiconductor fabrication protocols. While silicon-based electroporation / nanoelectroporation surfaces are effective for proof-of-concept research and small-scale mRNA production in extracellular vesicles (EVs), efforts to scale-up production and efficiency of cell electroporation / nanoelectroporation would benefit greatly from a low-cost, mass producible polymer alternative. The dual-layer polymer CEP / CNP surfaces also offer higher production performance of EV mRNA drugs, especially for large mRNA molecules, compared to track- etched membranes.

[0303] We invented a polymer CEP / CNP surface design based on a photoresist film-coated silicon (Si) membrane. Here, in contrast to the thin photoresist film used for patterning in the Si CEP / CNP surface production, we apply a thick photoresist film (~10 pm thickness) on the silicon membrane and the film can be released after patterning to form a standalone CEP / CNP surface. This approach eliminates the need to prepare “back-side” wells on Si membrane and expensive deep reactive ion etching (DRIE) for silicon etching. In addition to saving theproduction cost, the entire surface is available for more cell loading and EV mRNA production compared to the Si CEP / CNP surface. Although many photoresist resins may be applicable, we demonstrate this processhere using epoxy -based SU-8 positive photoresists. SU-8 is an epoxybased photoresist that can be used to lithographically fabricate micro structures with low cost for disposable perforated membranes and offers several advantages. It enables the creation of high- aspect-ratio micro / nano-scale patterns in a single photolithographic step with excellent mechanical properties, thermal stability, and chemical resistance. In this study, we demonstrate a dual-layer SU-8 membrane for polymer-based CNP surfaces using SU-8 TF 6002 and SU-8 3010 photoresist resins.

[0304] Fabrication of dual-layer perforated membrane: As shown in FIG. 1, the fabrication process involves several key steps: (1) spin coating a light-sensitive photoresist onto the silicon membrane to create the first layer of the dual-layer perforated membrane, (2) soft baking the first photoresist layer to evaporate the solvent and make the SU-8 photoresist more solid, (3) masking the photoresist with an array of ~1 -pm-diameter pores to cause negative photoresist to form the nanopores of the first layer of the dual -layer perforated membrane, (4) post-exposure baking (PEB), (5) repeating steps (1) through (4) with a second light-sensitive photoresist onto the surface of the first layer to form the second layer of the dual -lay er perforated membrane having an array of larger-diameter pores (e.g., 2 pm to about 8 pm in diameter), and (6) simultaneously developing both layers of the photoresist to dissolve the unexposed photoresist to form the smaller pores (i.e. nanochannels or ~l-pm-diameter pores) of the first layer that are contiguous with the larger-diameter pores (i.e. microchannels) of the second layer to form a dual-layer perforated membrane having “bottle”-shaped pores spanning the thickness of the two photoresist layers ( FIG. 2 and FIG. 3).

[0305] Briefly, a 2 pm thick of SU-8 TF 6002 is first spin-coated at a centrifugal speed of 4000 rpm on a Si membrane, which is coated with a release layer of OmniCoat. After spin-coating with the SU-8 TF 6002, the SU-8 TF 6002 undergoes a 3-minute soft bake at 110°C. A first mask of 1-pm pore array is used for UV exposure, followed by 2 min PEB at 110°C. Then an 8 pm thick of SU-8 3010 is spun coated on top of the exposed but yet undeveloped SU-8 TF 6002 layer, followed by 7 min soft bake at 95°C. A second mask of a larger pore array is used for UV exposure, which is aligned with the pattern of the first mask, followed by PEB (1 min at 65°C and 2 min at 95°C). After that, both layers of SU-8 are developed simultaneously. The SU-8 developer dissolves the areas that are not exposed (blocked by dark regions of the photomasks) to UV light during each of the exposure steps. Finally, the release layer of OmniCoat is removed to free the SU-8 double- layer membrane.

[0306] Design Considerations and Challenges: Two key design constraints are addressed: (1) the membrane requires a minimum pore size of 1 pm for the CNP surface, and (2) the final membrane thickness is ~10 pm to ensure sufficient mechanical strength for handling.

[0307] Achieving a 1 pm pore size in a single-layer SU-8 photoresist film thicker than 2 pm (aspect-ratio >2:1) is technically challenging. As the film thickness increases, it becomes more difficult for UV light to penetrate the entire film, especially at smaller feature sizes. To overcome this, we implement a double-layer approach. The SU-8 TF 6002 photoresist, which is designed for high-resolution thin structures, is selected for the first layer (thin) to make the nanopores having a pore diameter of ~1 -pm. Additionally, a SU-8 3010 photoresist is chosen forthe second layer (thick layer) to provide additional mechanical strength, bringing the total membrane thickness to ~10 pm. The larger-diameter micropores on the thick layer are carefully aligned with the ~l-pm nanopores on the thin layer to achieve the CEP / CNP surface design with a “bottle-like” pore structure. The bottle-like pore structure is advantageous because it reduces cell damage via electroporation through the small nanopores contacting the donor cells and minimizes plasmid DNA aggregation and / or pore (i.e., channel) blockage due to larger-diameter micropores in contact with the plasmid DNA solution. This is especially important for delivering very large transfection cargos (e.g., DNA plasmids, RNAs, and small molecules) into the donor cells.

[0308] As shown in FIG. 4A and FIG. 4B, we also fabricated a dual-layer perforated membrane using a SU-8 2002 photoresist for the thin layer to form the smaller-diameter channels with a diameter of ~1 pm and a SU-8 2005 photoresist for the thick layer to form the larger-diameter microchannels (e.g., 3-4 pm). The fabrication process of this dual -layer perforated membrane was similar to that described for the SU-8 TF 6002-SU-8 3010 dual-layer perforated membrane.

[0309] An additional benefit of using different SU-8 materials for the two layers is the temperature compatibility between their processing steps. The post-exposure bake (PEB) temperature for the SU-8 TF 6002 photoresist of the thin layer (for forming the nanochannels) is 110°C, while the SU-8 3010 photoresist of the thick layer (for forming the microchannels is processed at a lower post-exposure bake (PEB) temperature of 95°C. This temperature difference allows the thin layer to undergo an additional post-exposure bake (PEB) during the processing of the thick layer, which further improves the structurally integrity of the dual-layer perforated membrane. This dual-layer polymer design circumvents the challenges of high-aspect-ratio challenge we faced in the single layer fabrication.

[0310] It is conceived that multiple layers can be applied to create pores (i.e., channels) with more complex structures. For large silicon membranes, the UV exposure rate may not be uniform across the entire membrane surface. To address this, a thin barrier layer, such as silicon oxide(SiO2), gold, or silver, can be coated on the layer surface after UV exposure and before adding the next photoresist layer. The presence of the barrier layer prevents over-exposure into the underlying layer.Example 2. Comparison of dual-layer photoresist and silicon CEP / CNP perforated membranes for producing mRNA cargo-loaded extracellular vesicles

[0311] CEP / CNP experiments were conducted to evaluate the performance of the newly developed SU-8 dual-layer perforated membrane for producing extracellular vesicles (EVs) encapsulating mRNA therapeutic cargo in comparison to our legacy silicon CEP / CNP membranes. To test this possibility, an equal number of -24,000 human skin fibroblast cells (from ABM) were seeded on a 0.9-cm x 0.9-cm surface area of either the SU-8 dual-layer membrane or the silicon membrane.The skin fibroblasts were allowed to adhere to form contacts with the openings of the smaller- diameter channels ofthe perforated membrane surface. After cell incubation, dystrophin (DMD) plasmid DNA was electroporated / nanoelectroporated under identical conditions. The applied electric field properties during CEP / CNP were: 222 V / cm at 100V, 4.5 mm electrode-to- electrode distance, ten 10-ms pulses with a 0.1 -sec interval. The cell culture medium was collected after 24 hours. After purification of the supernatant, the mRNA copies in the collected EVs were quantified using RT-qPCR. FIG. 5 shows that both SU-8 and Si surfaces produced similar dystrophin mRNA copies per electroporated fibroblast. It should be noticed that the electric fiend strength of 222 V / cm was optimized for the silicon perforated membrane but not for the SU-8 dual-layer perforated membrane. The EV mRNA yield of the SU-8 dual-layer perforated membrane could potentially surpass the efficiency of the silicon perforated membrane if electroporation conditions are optimized.Example 3. Sacrificial template-based imprinting of polymer CEP / CNP perforated membranes

[0312] Silicon-based 3D CEP / CNP membranes are high brittle. They are also expensive because the use of silicon (Si) membranes and the associated labor-intensive semiconductor fabrication methods that require stringent cleanroom conditions. While Si-based CEP / CNP membranes are suitable for proof-of-concept research purposes and small-scale EV production, a low-cost, mass-producible polymer CEP / CNP wafter alternatives to the Si CEP / CNP membrane is essential for large-scale cell transfection and EV production for the development of cell and EV- based gene therapeutics. Polymers can be more durable and cheaper than silicon membranes. Moreover, the micro- / nano-fabrication of polymers in non-cleanroom conditions is also less costly than cleanroom semiconductor fabrication. In comparison to commercially available polymer CEP / CNP membranes with micropores and / or nanopores (e.g., track-etch membranes),silicon CEP / CNP membranes provide well-defined and uniformly distributed pore size, pattern, and density. These features are critical for efficient cell transfection in that they afford high cell viability, particularly for human primary cells. Highly efficient CEP / CNP may lead to robust and abundant EV production with improved loading of genetic cargo or transfection reagents. For polymer membranes with a large array of micropores and / or nanopores having a high aspect ratio (i.e. the ratio of pore length / pore diameter » 1), the conventional polymer fabrication methods such as injection molding and embossing works poorly because the mold / master and formed polymer membranes may break easily due to high stresses generated during fabrication, processing, and handling.

[0313] Fabrication Process: Described herein is a novel method to fabricate polymer 3D CEP / CNP membranes. Although direct imprinting can be used for producing polymer thin films with channels (e.g. microchannels, nanochannels), our experience shows that the mold does not last longbecause the extremely fragile nano / micro-pillar structures. As shown in FIG. 6, a two- step process is applied first to produce a large number of non-photoresist polymer CEP / CNP surfaces. Here, a patterned photoresist master mold on a silicon membrane with a nano / micro- well array is first designed and fabricated as that shown in FIG. 1. Many female molds are prepared by soft lithography first as transition molds. This female master mold can be used to prepare many PVA sacrificial template molds using water soluble polymers such as poly(vinyl alcohol) (PVA) as shown in FIG. 7A, whereby an aqueous solution of PVA is casted onto the SU-8 photoresist female mold. After drying, a sacrificial PVA template replicating the SU-8 mater mold can be peeled off from the master mold. Liquid imprinting of a photo-curable poly(dimethylsiloxane) (PDMS) resin is then added onto the PVA sacrificial mold to form the PDMS membrane with having a well-defined array of micropores and / or nanopores, as shown in FIG. 7B. A weight can be used to apply even force to improve the definition of the micropores and / or nanopores. A PDMS or plastic spacer layer is added on the back side of the thin layer with the channel array (e.g., microchannel array, nanochannel array) to enhance the mechanical strength of the final polymer CEP / CNP . Imprinting can also be carried out by spin-coating a liquid resin such as PDMS or acrylic resin onto the PVA sacrificial template. A thin polymer layer with patterned channels (e.g. patterned microchannels, patterned nanochannels) is formed by controlling the spin conditions to ensure that the film thickness is lightly less than the smaller- tip height on the sacrificial template. After the resin cures, the polymer layer with channel array can be released by dissolving PVA in water. In some embodiments, the channel array can comprise a microchannel array or, preferably, a nanochannel array. The PMMA nano-nozzle array (200 nm in diameter) is limited to very thin films (less than 3 -pm membrane thickness) which are too fragile to handle a large number of cells on a large surface area (S. Wang, C. Zeng,S. Lai, Y.J. Juang, Y. Yang and L. J. Lee, “Polymeric Nanonozzle Array Fabricated by Sacrificial Template Imprinting”, Advanced Materials, 17, 1182-1186 (2005)). Furthermore, the optical fiber bundle-based mold / master cannot provide the design flexibility of pore size, pattern and density.

[0314] Design Considerations and Challenges: The sacrificial molds are made from water- soluble polymers such as polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), PVP / VA copolymer, among others, while the curable polymer resins include poly dimethylsiloxane (PDMS), acrylic, among others. PVA is preferred for both sacrificial molds and release films on imprinting surfaces due to its low cost. Once the polymer resin is fully cured by heat or UV, the sacrificial mold is dissolved in water, allowing the water-soluble polymer to be recovered for reuse. PDMS is the preferred resin material for CNP surfaces because of its widely recognized biocompatibility in both in vitro and in vivo medical applications.

[0315] Spin-coating is a simple process, but the challenge lies in controlling the precise PDMS film thickness across the entire sacrificial mold surface. If the film is too thick, it covers the male structures, while if it is too thin, it misses the smaller pores. Imprinting, by contrast, provides better control over the PDMS film thickness. However, the challenge here is ensuring the complete penetration of the male structures through the partially cured PDMS resin film across the entire sacrificial mold surface. To address this, a soft substrate, such as a rubbery layer, is used during imprinting to ensure full contact with the male structures on the entire sacrificial mold surface. To protect the male structures on the sacrificial mold surface during imprinting, PVA coated on the rubbery layer as a release film can be softened by exposure to hot water vapor or heating above its glass transition temperature (Tg = 76 °C). Alternatively, a stronger water-soluble polymer such as PVP and PVP / VA copolymer (Tg = 100-175 °C depending on molecular weight and copolymer ratio) can be used for the sacrificial molds. FIG. 7A and FIG. 7B exhibit optical microscopy images of a PVA-based sacrificial template and the larger pores of a STI-prepared PDMS CEP / CNP surface.Example 4. Multi-well electroporation cell chamber array device for high-throughput cell electroporation

[0316] Cells and their derivates, extracellular veiscles (EVs), hold immense potential as nextgeneration medicine. Precise cell transfection for genetic modification / editing is crucial in cell therapy, however, existing cell transfection methods are less satisfactory. EVs outperform synthetic nanoparticles and viral vectors in mRNA therapy due to their ability to overcome physiological barriers, reduce immunogenicity, and minimize reactogenicity. Nevertheless, cells naturally secret a limited number of EVs containing few functional mRNAs. Our patented technology, cell nanoelectroporation (CNP) for gene delivery through nano- and submicron-scalepores can achieve remarkable efficacy and high precision in cell transfection. CNP also yields abundant EVs containing substantial quantities of endogenous mRNAs. Such engineered EVs have been successfully demonstrated in various mouse models including cancer therapy, anti- aging, bone repair, and are in human phase I-II clinical trials now. For future clinic uses, automated high throughput cell transfection and EV production is essential to make the CNP technology and engineered cells and their secreted EVs scalable, affordable, and sustainable.

[0317] This disclosure provides a multi-well cell culture well array attached onto a large electroporation / nanoporation perforated membrane. In conjunction with an electrode array design and automated cell loading and culture medium collection devices, an automated high - throughput CEP / CNP system is achieved. As previously described, the electroporation / nanoelectroporation surface is a perforated membrane with micropores and / or nanopores and is affixed to a polymer spacer array, which forms the boundaries of cell culture wells. Cells are cultured inside the individual wells of the multi -well cell culture spacer, and CEP / CNP is carried out when an electric pulse or plurality of electric pulses is applied to the donor cells via the openings of the channels in the perforated membrane.

[0318] FIG. 8 shows an example of a schematic and a photo of the well array layer-based CEP / CNP membrane setup with a 5 * 5 well array on a 7 cm x 7 cm PDMS spacer with 9 mm x 9 mm square wells. The membrane consists of a CEP / CNP layer, made of either a micro / nano- fabricated Si membrane with well-defined cell transfection pore array (FIG. 10A) or a commercially available perforated membrane such as track etched membranes (FIG. 10B), which is attached onto the well layer / spacer via a bioadhesive. The electric circuit for cell electroporation consists of a plate-like cathode in contact with the negatively charged reagent solution and a coil- or rod-like anode inserted into one of the cell-loaded well on the spacer. Different spacer array dimensions and well patterns can be designed. Preferred well-array layers have dimensionsmatched with the standard 6- 24-, 96-, or 384-well microtiter plates to allow for the use of commercially available bio lab equipment items for automated cell and reagent loading before electroporation, and EV collection in the cell culture medium after electroporation.

[0319] Cells are cultured inside each well of the layer / spacer array to adhere onto the surface of the CEP / CNP layer. Before electroporation, transfection reagents such as plasmid DNA or CRISPR / Cas9 reagents with a desirable concentration and composition are prepared and loaded into the reagent reservoir under the CEP / CNP layer. An electric voltage with desirable pulse length, number, and interval is then selected from an electroporation power supply to stimulate the adhered cells in a well on the chamber layer and to deliver the transfect reagents via the pores on the CEP / CNP layer. The coil- or rod-like anode is then moved from one well to another welltill cells in all wells are transfected sequentially. The design of sequential electroporation from well-to-well is to keep the electric current low during electroporation (which depends on the pore number in each cell well). Otherwise, high Joule heating caused by the high electric current would lead to severe cell death. Cell secreted EVs in the culture medium are collected after a selected incubation time after electroporation.Example 5. Multi-well electroporation chamber array device for multiplexed high- throughput cell electroporation and production of extracellular vesicles

[0320] This disclosure provides a dual-well array chamber layer-based CEP / CNP device that includes a CEP / CNP perforated membrane sandwiched between two polymer spacer arrays (FIG. HA and FIG. 11B) Here, one polymer spacer array is attached onto the surface of a large CEP / CNP perforated membrane for loading of the same or different donor cells in individual cell culture wells that are defined by the openings within the spacer array. Here, the cell culture well (i.e. first spacer array) contacts the surface of the CEP / CNP perforated membrane having the openings of the nanochannels. Additionally, a second polymer spacer array is attached onto the surface of the large CEP / CNP surface opposite the first spacer array. The holes within the second spacer array define the dimensions of isolated electroporation reagent chambers, which is then used for loading different cell transfection reagent(s) (e.g. plasmid DNAs, RNAs, CRISPR / Cas9 components, small molecules, etc.) or the same cell transfection reagent(s) at different reagent concentrations and compositions within the individual electroporation reagent wells. Together, this system allows for multiplexing cell transfection andEV production analysis using different cell types and transfection reagents on a single CNP membrane.

[0321] Unlike the single-array CEP / CNP device in FIG. 8, the plate electrode is the anode and is placed on the cell-side well array chamber layer. After the same or different cell types are loaded into individual wells and adhered as a monolayer onto the CEP / CNP layer surface, the membrane is turned upside down such that different transfection reagents or the same transfection reagents with different concentrations and / or compositions can be loaded into individual wells on the reagent-side of the chamber layer (FIG. 11B and FIG. 12). The rod- or coil-like cathode array (FIG. 13) is then inserted into the reagent wells to apply the electrical current via the channels of the perforated membrane for automated cell transfection by sequential electroporation. After a certain incubation time, the remaining transfection reagents are removed from each well followed with the removal of the reagent-side chamber layer. Individual transfected cells in each well on the cell-side array layer can be analyzed on-membrane by cell staining or off-membrane via cell removal for further analyses such as flow cytometry, PCR and / or Western blot. If necessary, the cell-side well layer / spacer array can also be removed suchthat the adhered cells on-surface can be examined more closely at higher magnification under a fluorescence microscope.Example 6. Comparison of patterned channel-array silicon CEP / CNP membrane and track etch CEP / CNP membrane for mRNA production in extracellular vesicles

[0322] To test if the CEP / NEP membrane and / or channel arrangement differently affected the level of mRNA levels of electroporated genetic cargo in extracellular vesicles, neonatal human dermal fibroblast (nHDF) cells were cultured on either a patterned channel-array silicon CEP / CNP membrane or track etch CEP / CNP membrane and then subjected to CEP / CNP with a COL1 Al DNA plasmid. The extracellular vesicles (EVs) secreted from neonatal human dermal fibroblast (nHDF) cells were collected from culture media on the silicon CEP / CNP membrane and track etch CEP / CNP membrane 24 hours after CEP / CNP treatment. The COL1 Al mRNA levels in extracellular vesicles (EVs) collected from the silicon CEP / CNP membrane and track etch CEP / CNP membrane were then quantified by qPCR. As shown in FIG. 14, the average copy number of COL1 Al mRNA in extracellular vesicles (EVs) secreted from neonatal human dermal fibroblast (nHDF) cells that underwent electroporation on the silicon membrane (Si-CNP; CNP) were -54% higher than the mean copy number of COL1 Al mRNA in EVs secreted from neonatal human dermal fibroblast (nHDF) cells that underwent electroporation on the track-etch membrane (TEP). CEP / CNP via the silicon membrane (Si-CNP; Si) proved to induce EV mRNA production more efficiently than the track-etch membrane. All data are presented as mean ± SD; CTR: control of untreated cells.Example 7. Automated system for high-throughput cell electroporation

[0323] This disclosure provides an automated system for high-throughput cell electroporation or high-throughput cell electroporation / nanoelectroporation (HIT-CEP / CNP). This system includes the double-well array layer-based CEP / CNP membrane setup shown in FIG. 15 A, a newly developed sequential CEP / CNP electrical pulse generation system that automates electric pulse output to individual wells within the double-well array layer-based CEP / CNP membrane, and optional accessory components. The CEP / CNP electrical pulse generation system can also be configured to delivery electrical pulses to cells in all or some of the electroporation chambers, but sequential electrical stimulation is preferred. This automated HIT-CEP / CNP system affords the ability to perform large-scale, multiplexed cell transfection and production of extracellular vesicles (EVs) carrying the transfection cargo of interest (e.g., DNA plasmids, RNAs, or small molecule) with minimal manual operations.

[0324] First, the prototype of a sequential CEP / CNP electrical power system that automates sequential electric pulse output to individual wells of the double- well array chamber is described. The CEP / CNP electrical power system consists of an electrical pulse generator, a shock pulseconverter, and a shock box to deliver highly controllable electrical stimulation of the donor cells to mediate CEP / CNP (FIG. 15 A). The electric pulse generator (FIG. 15B) generates continuous and / or triggered square-wave electric pulses to be applied for high-throughput cell electroporation device forHIT-CEP / CNP of donor cells. The train (or, if desired, more than one train) of electrical pulses to be used for CEP / CNP (e.g., voltage, pulse width, and pulse interval) can be adjusted to modify the parameters of HIT-CEP / CNP. The electric pulse generator is operatively coupled to and sends electrical pulses to the shock pulse converter (FIG. 15C). The shock pulse converter then converts the electrical output signals of the electrical pulse generator to pulses of electricity that are then sent to the shock box to mediate CEP / CNP. As an example, the shock pulse converter sends 10-electric-output pulses to each CEP / CNP electrode sequentially via a 25-pin connector to the shock box. The shock box (FIG. 15D) has an automated push button to initiate CEP / CNP in individual wells by the electrode array. After the cells in the first HIT-CEP / CNP chamber is transfected by CEP / CNP via the first electrode on the electrode array, the pushbutton is triggered automatically for cell transfection in the second well by the second electrode on the electrode array. The cycle is repeated till cells in all 25 wells are transfected by CEP / CNP sequentially.

[0325] Additionally, optional accessory items are also used in addition to the automated sequential electric power system and the double-well array chamber layer-based CEP / CNP membrane to further increase automation of the HIT-CEP / CNP system. For example, a commercial robotic pipetting system can be used to automate washing, cell loading, reagent loading, and EV collection within each of HIT-CEP / CNP well to reduce contamination and human error.

[0326] While preferred cases of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such cases are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the cases of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.EMBODIMENTSEmbodiment 1. A perforated membrane, comprising: a first layer comprising a plurality of first channels disposed through the first layer; anda second layer in contact with the first layer, the second layer comprising a plurality of second channels disposed through the second layer; wherein: a first average thickness of the first layer is different from a second average thickness of the second layer; a first channel of the plurality of first channels is in fluid communication of a second channel of the plurality of second channels; a first average diameter of the first channel is different from a second average diameter of the second channel.Embodiment 2. The perforated membrane of embodiment 1, wherein first average thickness is larger than the second average thickness.Embodiment 3. The perforated membrane of embodiment 2, wherein the first average thickness is from about 1 pm to about 200 pm.Embodiment 4. The perforated membrane of embodiment 2 or 3, wherein the first average thickness is at least twice of the second average thickness.Embodiment 5. The perforated membrane of any one of embodiments 2-4, wherein the first average thickness is at least three times of the second average thickness.Embodiment 6. The perforated membrane of any one of embodiments 2-5, wherein the second average thickness is from about 100 nm to about 10 pm.Embodiment 7. The perforated membrane of any one of embodiments 1 -6, wherein the first average diameter is larger than the second average diameter.Embodiment 8. The perforated membrane of any one of embodiments 1-7, wherein the first average diameter is from about 1 pm to about 20 pm.Embodiment 9. The perforated membrane of any one of embodiments 1 -7, wherein the first average diameter is at least twice of the second average diameter.Embodiment 10. The perforated membrane of any one of embodiments 1 -7, wherein the first average diameter is at least three times of the second average diameter.Embodiment 11. The perforated membrane of any one of embodiments 1 -7, wherein the first average diameter is from about twice to about three times of the second average diameter. Embodiment 12. The perforated membrane of any one of embodiments 1 -7, wherein the first average diameter is from about three times to about four times larger than the second average diameter.Embodiment 13. The perforated membrane of any one of embodiments 1 -7, wherein the second average diameter is from about 100 nm to about 5 pm.Embodiment 14. The perforated membrane of any one of embodiments 1 -7, wherein the first average diameter is about half of the first average thickness.Embodiment 15. The perforated membrane of any one of embodiments 1 -7, wherein the first average diameter is about one-third of the first average thickness.Embodiment 16. The perforated membrane of any one of embodiments 1 -7, wherein the second average diameter is no more than half of the second average thickness.Embodiment 17. The perforated membrane of any one of embodiments 1 -7, wherein the second average diameter is no more than one-third of the second average thickness.Embodiment 18. The perforated membrane of any one of embodiments 1 -7, wherein the second average diameter is from about one-half to about one-third of the second average thickness.Embodiment 19. The perforated membrane of any one of embodiments 1 -7, wherein the second average diameter is from about one-third to about one-fourth of the second average thickness.Embodiment 20. The perforated membrane of any one of embodiments 1 -7, wherein the second average diameter is about half of the second average thickness.Embodiment 21. The perforated membrane of any one of embodiments 1 -7, wherein the second average diameter is no more than half of the second average thickness.Embodiment 22. The perforated membrane of any one of embodiments 1 -7, wherein the second average diameter is no more than about 25% of the second average thickness.Embodiment 23. The perforated membrane of any one of embodiments 1 -22, wherein the first layer comprises a first polymer material.Embodiment 24. The perforated membrane of embodiment 23, wherein the first polymer material comprises a first light-reactive polymer.Embodiment 25. The perforated membrane of embodiment 23, wherein the first polymer material comprises a first crosslinking polymer.Embodiment 26. The perforated membrane of embodiment 23, wherein the first polymer material comprises a first synthetic polymer.Embodiment 27. The perforated membrane of embodiment 23, wherein the first polymer material comprises a first thermosetting polymer.Embodiment 28. The perforated membrane of embodiment 23, wherein the first polymer material comprises a first light-curing or a first thermal-curing polymer.Embodiment 29. The perforated membrane of embodiment 23, wherein the first polymer material comprises a first photoresist.Embodiment 30. The perforated membrane of embodiment 23, wherein the first polymer material comprises a first positive photoresist.Embodiment 31. The perforated membrane of embodiment 23, wherein the first polymer material comprises a first negative photoresist.Embodiment 32. The perforated membrane of embodiment 23, wherein the first polymer material comprises a first SU-8 photoresist.Embodiment 33. The perforated membrane of embodiment 23, wherein the first polymer material comprises a first SU-8 3000-series photoresist.Embodiment 34. The first perforated membrane of embodiment 23, wherein the first polymer material comprises a SU-8 3010 photoresist or a SU-8 3005 photoresist polymer.Embodiment 35. The perforated membrane of embodiment 23, wherein the first polymer material comprises a first SU-8 2000-series photoresist.Embodiment 36. The perforated membrane of embodiment 23, wherein the first polymer material comprises a SU-8 2005 photoresist.Embodiment 37. The perforated membrane of any one of embodiments 1-36, wherein the second layer comprises a second polymer material.Embodiment 38. The perforated membrane of embodiment 37, wherein the second polymer material comprises a second light-reactive polymer.Embodiment 39. The perforated membrane of embodiment37, wherein the second polymer material comprises a second crosslinking polymer.Embodiment 40. The perforated membrane of embodiment 37, wherein the second polymer material comprises a second synthetic polymer.Embodiment 41. The perforated membrane of embodiment 37, wherein the second polymer material comprises a second thermosetting polymer.Embodiment 42. The perforated membrane of embodiment 37, wherein the second polymer material comprises a second light-curing polymer or a second thermal -curing polymer.Embodiment 43. The perforated membrane of embodiment 37, wherein the second polymer material comprises a second photoresist.Embodiment 44. The perforated membrane of embodiment 37, wherein the second polymer material comprises a second positive photoresist.Embodiment 45. The perforated membrane of embodiment 37, wherein the second polymer material comprises a second negative photoresist.Embodiment 46. The perforated membrane of embodiment 37, wherein the second polymer material comprises a second SU-8 photoresist.Embodiment 47. The perforated membrane of embodiment 37, wherein the second polymer material comprises a SU-8 TF 6000-series photoresist.Embodiment 48. The perforated membrane of embodiment 37, wherein the second polymer material comprises a SU-8 TF 6002 photoresist.Embodiment 49. The perforated membrane of embodiment 37, wherein the second polymer material comprises a second SU-8 2000-series photoresist.Embodiment 50. The perforated membrane of embodiment 37, wherein the second polymer material comprises a SU-8 2002 photoresist.Embodiment 51. The perforated membrane of any one of embodiments 1 -50, wherein the first layer comprises at least one ingredient different from ingredients of the second layer materials.Embodiment 52. The perforated membrane of any one of embodiments 1 -50, wherein the first layer is made of materials different from those of the second layer.Embodiment 53. The perforated membrane of embodiment 52, wherein the first layer comprises the first photoresist, and wherein the second layer comprises the second photoresist. Embodiment 54. The perforated membrane of embodiment 52, wherein the first layer comprises the first negative photoresist, and wherein the second layer comprises the second negative photoresist.Embodiment 55. The perforated membrane of embodiment 52, wherein the first layer comprises the first SU-8 photoresist, and wherein the second layer comprises the second SU-8 photoresist.Embodiment 56. The perforated membrane of embodiment 52, wherein the first layer comprises the SU-8 3010 photoresist, the SU-8 3005 photoresist, or the SU-8 2005 photoresist, and wherein the second layer comprises the SU-8 TF 6002 photoresist or the SU-8 2002 photoresist.Embodiment 57. The perforated membrane of embodiment 52, wherein a first tensile strength of the first layer is larger than a second tensile strength of the second layer.Embodiment 58. The perforated membrane of embodiment 52, wherein the first layer and the second layer are made from photoresist polymers.Embodiment 59. The perforated membrane of any one of embodiments 1 -37, wherein the first layer is made of the same materials as those of the second layer.Embodiment 60. The perforated membrane of embodiment 59, wherein the first layer and the second layer comprise the first light-reactive polymer.Embodiment 61. The perforated membrane of embodiment 59, wherein the first layer and the second layer comprise the first crosslinking polymer.Embodiment 62. The perforated membrane of embodiment 59, wherein the first layer and the second layer comprise the first synthetic polymer.Embodiment 63. The perforated membrane of embodiment 59, wherein the first layer and the second layer comprise the first light-curing polymer.Embodiment 64. The perforated membrane of any one of embodiments 1 -63, configured to electroporate a donor cell with a transfection reagent.Embodiment 65. The perforated membrane of embodiment 64, wherein the transfection reagent comprises a DNA, a RNA, or a small molecule.Embodiment 66. A method of making a perforated membrane from photoresist materials, comprising:(a) spin coating a first layer of a first photoresist material onto a substrate;(b) soft baking the first layer obtained in (a);(c) exposing the soft baked first layer obtained in (b) to a first UV radiation using a first photomask;(d) spin coating a second layer of a second photoresist material onto the first layer after (c);(e) soft baking the second layer obtained in (d);(f) exposing the softbaked second layer obtained in (e) to another UV radiation using a second photomask;(g) conducting a post exposure bake; and(h) developing both the first layer and the second layer after (g), thereby making a perforated membrane comprising two layers.Embodiment 67. The method of embodiment 66, further comprising: after (c) but before (d), conducting another post exposure bake.Embodiment 68. The method of embodiment 66 or 67, wherein the substrate is a silicon membrane coated with a release layer.Embodiment 69. The method of embodiment 68, wherein the spin coating in (a) coats the first layer onto the release layer.Embodiment 70. The method of embodiment 69, further comprising: after (h), removing the release layer.Embodiment 71. The method of embodiment 66, wherein both the first photoresist material and the second photoresist material are negative photoresists.Embodiment 72. The method of embodiment 66, wherein the first photoresist material is different from the second photoresist material.Embodiment 73. The method of embodiment 72, wherein the first photoresist material has a higher resolution than the second photoresist material.Embodiment 74. The method of embodiment 66, wherein the first photomask comprises a first pattern, and wherein the second photomask comprises a second pattern.Embodiment 75. The method of embodiment 74, wherein the first pattern is configured to produce a plurality of first channels disposed in the first layer after (h), and wherein the second pattern is configured to produce a plurality of second channels disposed in the second layer after (h).Embodiment 76. The method of embodiment 66, further comprising: after (c) but before (d), coating a barrier layer on top of the first layer.Embodiment 77. The method of embodiment 76, wherein the barrier layer comprises silicon oxide, gold, or silver.Embodiment 78. The method of any one of embodiments 66-77, wherein the perforated membrane comprises a top layer comprising a plurality of top channels disposed through the top layer; and a bottom layer in contact with the top layer, the bottom layer comprising a plurality of bottom channels disposed through the bottom layer; wherein: a first average thickness of the top layer is different from a second average thickness of the bottom layer; a first channel of the plurality of top channels is in fluid communication of a second channel of the plurality of bottom channels; a first average diameter of the first channel is different from a second average diameter of the second channel.Embodiment 79. A method of making a perforated membrane from a sacrificial template, comprising(a) providing a female mold comprising a female pattern;(b) preparing a sacrificial male mold based on the female mold; and(c) preparing a perforated membrane based on the sacrificial male mold, wherein the perforated membrane comprises another pattern.Embodiment 80. The method of embodiment 79, wherein the another pattern is substantially the same as the female pattern.Embodiment 81 . The method of embodiment 79 or 80, wherein the preparing in (c) comprises imprinting.Embodiment 82. The method of embodiment 81, wherein the imprinting comprising providing a substrate and a weight.Embodiment 83. The method of embodiment 82, wherein the substrate is coating with a release layer.Embodiment 84. The method of embodiment 83, wherein the release layer comprises a releasable polymer.Embodiment 85. The method of embodiment 79, wherein the preparing in (c) comprises spin coating.Embodiment 86. The method of any one of embodiments 79-85, wherein the preparing in(c) comprises applying a polymer resin to the sacrificial male mold.Embodiment 87. The method of embodiment 86, wherein the polymer resin is thermal curable.Embodiment 88. The method of embodiment 87, further comprising: thermal curing the polymer resin.Embodiment 89. The method of embodiment 86, wherein the polymer resin is photo - curable.Embodiment 90. The method of embodiment 87, further comprising: photo curing the polymer resin.Embodiment 91. The method of any one of embodiments 79-90, the preparing in (c) further comprises: removing the sacrificial male mold.Embodiment 92. The method of any one of embodiments 79-91, wherein the sacrificial male mold is made from water-soluble polymers.Embodiment 93. The method of embodiment 92, wherein the water-soluble polymer is polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyvinylpyrrolidone / vinyl alcohol copolymer (PVP / VA copolymer).Embodiment 94. The method of any one of embodiment 86-93, wherein the polymer resin is poly dimethylsiloxane (PDMS) resin or acrylic resin.Embodiment 95. The method of any one of embodiments 84, or 86-94, wherein the releasable polymer is polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyvinylpyrrolidone / vinyl alcohol copolymer (PVP / VA copolymer).Embodiment 96. The method of any one of embodiments 82-84, or 86-95, wherein the substrate is a rubbery layer.Embodiment 97. The method of any one of embodiments 79-96, wherein the perforated membrane comprises: a plurality of channels disposed through the perforated membrane;an average diameter of the channels; and an average thickness.Embodiment 98. A system for high-throughput cell electroporation comprising: a) a perforated membrane, wherein the perforated membrane comprises: i. a first layer comprising a plurality of first channels disposed through the first layer; and ii. a second layer in contact with the first layer, the second layer comprising a plurality of second channels disposed through the second layer, wherein: a first average thickness of the first layer is different from a second average thickness of the second layer; a first channel of the plurality of first channels is in fluid communication of a second channel of the plurality of second channels; a first average diameter of the first channel is different from a second average diameter of the second channel; and b) at least one donor cell in contact with the perforated membrane.Embodiment 99. The system of embodiment 98, wherein the first average thickness is larger than the second average thickness.Embodiment 100. The system of embodiment 99, wherein the first average thickness is from about 1 pm to about 200 pm.Embodiment 101. The system of embodiment 99 or 100, wherein the first average thickness is at least twice of the second average thickness.Embodiment 102. The system of any one of embodiments 99-101, wherein the first average thickness is at least three times of the second average thickness.Embodiment 103. The system of any one of embodiments 99-102, wherein the second average thickness is from about 100 nm to about 10 pm.Embodiment 104. The system of any one of embodiments 98-103, wherein the first average diameter is larger than the second average diameter.Embodiment 105. The system of any one of embodiments 98-104, wherein the first average diameter is from about 1 pm to about 20 pm.Embodiment 106. The system of any one of embodiments 98-104, wherein the first average diameter is at least twice of the second average diameter.Embodiment 107. The system of any one of embodiments 98-104, wherein the first average diameter is at least three times of the second average diameter.Embodiment 108. The system of any one of embodiments 98-104, wherein the first average diameter is from about twice to about three times of the second average diameter.Embodiment 109. The system of any one of embodiments 98-104, wherein the first average diameter is from about three times to about four times larger than the second average diameter. Embodiment 110. The system of any one of embodiments 98-104, wherein the second average diameter is from about 100 nm to about 5 gm.Embodiment 111. The system of any one of embodiments 98-104, wherein the first average diameter is about half of the first average thickness.Embodiment 112. The system of any one of embodiments 98-104, wherein the first average diameter is about one-third of the first average thickness.Embodiment 113. The system of any one of embodiments 98-104, wherein the second average diameter is no more than half of the second average thickness.Embodiment 114. The system of any one of embodiments 98-104, wherein the second average diameter is no more than one-third of the second average thickness.Embodiment 115. The system of any one of embodiments 98-104, wherein the second average diameter is from about one-half to about one-third of the second average thickness. Embodiment 116. The system of any one of embodiments -104, wherein the second average diameter is from about one-third to about one-fourth of the second average thickness.Embodiment 117. The system of any one of embodiments 98-104, wherein the second average diameter is about half of the second average thickness.Embodiment 118. The system of any one of embodiments 98-104, wherein the second average diameter is no more than half of the second average thickness.Embodiment 119. The system of any one of embodiments 98-118, wherein the first layer comprises a first polymer material.Embodiment 120. The system of embodiment 119, wherein the first polymer material comprises a first light-reactive polymer.Embodiment 121. The system of embodiment 119, wherein the first polymer material comprises a first crosslinking polymer.Embodiment 122. The system of embodiment 119, wherein the first polymer material comprises a first synthetic polymer.Embodiment 123. The system of embodiment 119, wherein the first polymer material comprises a first thermosetting polymer.Embodiment 124. The system of embodiment 119, wherein the first polymer material comprises a first light-curing polymer or a first thermal -curing polymer.Embodiment 125. The system of embodiment 119, wherein the first polymer material comprises a first photoresist.Embodiment 126. The system of embodiment 119, wherein the first polymer material comprises a first positive photoresist.Embodiment 127. The system of embodiment 119, wherein the first polymer material comprises a first negative photoresist.Embodiment 128. The system of embodiment 119, wherein the first polymer material comprises a first SU-8 photoresist.Embodiment 129. The system of embodiment 119, wherein the first polymer material comprises a first SU-8 3000-series photoresist.Embodiment 130. The system of embodiment 119, wherein the first polymer material comprises a first SU-8 3010 photoresist or a first SU-8 3005 photoresist.Embodiment 131. The system of embodiment 119, wherein the first polymer material comprises a first SU-8 2000-series photoresist.Embodiment 132. The system of embodiment 119, wherein the first polymer material comprises a first SU-8 2005 photoresist.Embodiment 133. The system of embodiment 132, wherein the second layer comprises a second polymer material.Embodiment 134. The system of embodiment 133, wherein the second polymer material comprises a second light-reactive polymer.Embodiment 135. The system of embodiment 133, wherein the second polymer material comprises a second crosslinking polymer.Embodiment 136. The system of embodiment 133, wherein the second polymer material comprises a second synthetic polymer.Embodiment 137. The system of embodiment 133, wherein the second polymer material comprises a second thermosetting polymer.Embodiment 138. The system of embodiment 133, wherein the second polymer material comprises a second light-curing polymer or a second thermal-curing polymer.Embodiment 139. The system of embodiment 133, wherein the second polymer material comprises a second photoresist.Embodiment 140. The system of embodiment 133, wherein the second polymer material comprises a second positive photoresist.Embodiment 141. The system of embodiment 133, wherein the second polymer material comprises a second negative photoresist.Embodiment 142. The system of embodiment 133, wherein the second polymer material comprises a second SU-8 photoresist.Embodiment 143. The system of embodiment 133, wherein the second polymer material comprises a second SU-8 TF 6000-series photoresist.Embodiment 144. The system of embodiment 133, wherein the second polymer material comprises a SU-8 TF 6002 photoresist.Embodiment 145. The system of embodiment 133, wherein the second polymer material comprises a second SU-8 2000-series photoresist.Embodiment 146. The system of embodiment 133, wherein the second polymer material comprises a SU-8 2002 photoresist.Embodiment 147. The system of any one of embodiments 98-146, wherein the first layer comprises at least one ingredient different from ingredients of the second materials.Embodiment 148. The system of any one of embodiments 98-147, wherein the first layer is made of materials different from those of the second layer.Embodiment 149. The system of embodiment 148, wherein the first layer comprises the first photoresist, and wherein the second layer comprises the second photoresist.Embodiment 150. The system of embodiment 148, wherein the first layer comprises the first negative photoresist, and wherein the second layer comprises the second negative photoresist. Embodiment 151. The system of embodiment 148, wherein the first layer comprises the first SU-8 photoresist, and wherein the second layer comprises the second SU-8 photoresist.Embodiment 152. The system of embodiment 148, wherein the firstlayer comprises the SU- 8 3010 photoresist, the SU-8 3005 photoresist, or the SU-8 2005 photoresist, and wherein the second layer comprises the SU-8 TF 6002 photoresist or the SU-8 2002 photoresist.Embodiment 153. The system of embodiment 152, wherein a first tensile strength of the first layer is larger than a second tensile strength of the second layer.Embodiment 154. The system of embodiment 152, wherein the first layer and the second layer are made from photoresist polymers.Embodiment 155. The system of any one of embodiments 98-154, wherein the first layer is made of the same materials as those of the second layer.Embodiment 156. The system of embodiment 155, wherein the first layer and the second layer comprise the first light-reactive polymer.Embodiment 157. The system of embodiment 155, wherein the first layer and the second layer comprise the first crosslinking polymer.Embodiment 158. The system of embod...

Claims

CLAIMSWHAT IS CLAIMED IS:1 . A perforated membrane, comprising: a first layer comprising a plurality of first channels disposed through the first layer; and a second layer in contact with the first layer, the second layer comprising a plurality of second channels disposed through the second layer; wherein: a first average thickness of the first layer is different from a second average thickness of the second layer; a first channel of the plurality of first channels is in fluid communication of a second channel of the plurality of second channels; a first average diameter of the first channel is different from a second average diameter of the second channel.

2. The perforated membrane of claim 1, wherein first average thickness is larger than the second average thickness.

3. The perforated membrane of claim 2, wherein the first average thickness is from about 1 pm to about 200 pm.

4. The perforated membrane of claim 2 or 3, wherein the first average thickness is at least twice of the second average thickness.

5. The perforated membrane of any one of claims 2-4, wherein the first average thickness is at least three times of the second average thickness.

6. The perforated membrane of any one of claims 2-5, wherein the second average thickness is from about 100 nm to about 10 pm.

7. The perforated membrane of any one of claims 1 -6, wherein the first average diameter is larger than the second average diameter.

8. The perforated membrane of any one of claims 1 -7, wherein the first average diameter is from about 1 pm to about 20 pm.

9. The perforated membrane of any one of claims 1 -7, wherein the first average diameter is at least twice of the second average diameter.

10. The perforated membrane of any one of claims 1 -7, wherein the first average diameter is at least three times of the second average diameter.

11. The perforated membrane of any one of claims 1 -7, wherein the first average diameter is from about twice to about three times of the second average diameter.

12. The perforated membrane of any one of claims 1 -7, wherein the first average diameter is from about three times to about four times larger than the second average diameter.

13. The perforated membrane of any one of claims 1 -7, wherein the second average diameter is from about 100 nm to about 5 pm.

14. The perforated membrane of any one of claims 1 -7, wherein the first average diameter is about half of the first average thickness.

15. The perforated membrane of any one of claims 1 -7, wherein the first average diameter is about one-third of the first average thickness.

16. The perforated membrane of any one of claims 1 -7, wherein the second average diameter is no more than half of the second average thickness.

17. The perforated membrane of any one of claims 1 -7, wherein the second average diameter is no more than one-third of the second average thickness.

18. The perforated membrane of any one of claims 1 -7, wherein the second average diameter is from about one-half to about one-third of the second average thickness.

19. The perforated membrane of any one of claims 1 -7, wherein the second average diameter is from about one-third to about one-fourth of the second average thickness.

20. The perforated membrane of any one of claims 1 -7, wherein the second average diameter is about half of the second average thickness.

21. The perforated membrane of any one of claims 1 -7, wherein the second average diameter is no more than half of the second average thickness.

22. The perforated membrane of any one of claims 1 -7, wherein the second average diameter is no more than about 25% of the second average thickness.

23. The perforated membrane of any one of claims 1 -22, wherein the first layer comprises a first polymer material.

24. The perforated membrane of claim 23, wherein the first polymer material comprises a first light-reactive polymer.

25. The perforated membrane of claim 23, wherein the first polymer material comprises a first crosslinking polymer.

26. The perforated membrane of claim 23, wherein the first polymer material comprises a first synthetic polymer.

27. The perforated membrane of claim 23, wherein the first polymer material comprises a first thermosetting polymer.

28. The perforated membrane of claim 23, wherein the first polymer material comprises a first light-curing or a first thermal-curing polymer.

29. The perforated membrane of claim 23, wherein the first polymer material comprises a first photoresist.

30. The perforated membrane of claim 23, wherein the first polymer material comprises a first positive photoresist.

31. The perforated membrane of claim 23, wherein the first polymer material comprises a first negative photoresist.

32. The perforated membrane of claim 23, wherein the first polymer material comprises a first SU-8 photoresist.

33. The perforated membrane of claim 23, wherein the first polymer material comprises a first SU-8 3000-series photoresist.

34. The first perforated membrane of claim 23, wherein the first polymer material comprises a SU-8 3010 photoresist or a SU-8 3005 photoresist polymer.

35. The perforated membrane of claim 23, wherein the first polymer material comprises a first SU-8 2000-series photoresist.

36. The perforated membrane of claim 23, wherein the first polymer material comprises a SU-8 2005 photoresist.

37. The perforated membrane of any one of claims 1 -36, wherein the second layer comprises a second polymer material.

38. The perforated membrane of claim 37, wherein the secondpolymer material comprises a second light-reactive polymer.

39. The perforated membrane of claim 37, wherein the secondpolymer material comprises a second crosslinking polymer.

40. The perforated membrane of claim 37, wherein the secondpolymer material comprises a second synthetic polymer.

41. The perforated membrane of claim 37, wherein the secondpolymer material comprises a second thermosetting polymer.

42. The perforated membrane of claim 37, wherein the secondpolymer material comprises a second light-curing polymer or a second thermal-curing polymer.

43. The perforated membrane of claim 37, wherein the secondpolymer material comprises a second photoresist.

44. The perforated membrane of claim 37, wherein the secondpolymer material comprises a second positive photoresist.

45. The perforated membrane of claim 37, wherein the secondpolymer material comprises a second negative photoresist.

46. The perforated membrane of claim 37, wherein the second polymer material comprises a second SU-8 photoresist.

47. The perforated membrane of claim 37, wherein the second polymer material comprises a SU-8 TF 6000-series photoresist.

48. The perforated membrane of claim 37, wherein the secondpolymer material comprises a SU-8 TF 6002 photoresist.

49. The perforated membrane of claim 37, wherein the secondpolymer material comprises a second SU-8 2000-series photoresist.

50. The perforated membrane of claim 37, wherein the secondpolymer material comprises a SU-8 2002 photoresist.

51. The perforated membrane of any one of claims 1 -50, wherein the first layer comprises at least one ingredient different from ingredients of the second layer materials.

52. The perforated membrane of any one of claims 1 -50, wherein the first layer is made of materials different from those of the second layer.

53. The perforated membrane of claim 52, wherein the first layer comprises the first photoresist, and wherein the second layer comprises the second photoresist.

54. The perforated membrane of claim 52, wherein the first layer comprises the first negative photoresist, and wherein the second layer comprises the second negative photoresist.

55. The perforated membrane of claim 52, wherein the first layer comprises the first SU-8 photoresist, and wherein the second layer comprises the second SU-8 photoresist.

56. The perforated membrane of claim 52, wherein the first layer comprises the SU-8 3010 photoresist, the SU-8 3005 photoresist, or the SU-8 2005 photoresist, and wherein the second layer comprises the SU-8 TF 6002 photoresist or the SU-8 2002 photoresist.

57. The perforated membrane of claim 52, wherein a first tensile strength of the first layer is larger than a second tensile strength of the second layer.

58. The perforated membrane of claim 52, wherein the first layer and the second layer are made from photoresist polymers.

59. The perforated membrane of any one of claims 1 -37, wherein the first layer is made of the same materials as those of the second layer.

60. The perforated membrane of claim 59, wherein the first layer and the second layer comprise the first light-reactive polymer.

61. The perforated membrane of claim 59, wherein the first layer and the second layer comprise the first crosslinking polymer.

62. The perforated membrane of claim 59, wherein the first layer and the second layer comprise the first synthetic polymer.

63. The perforated membrane of claim 59, wherein the first layer and the second layer comprise the first light-curing polymer.

64. The perforated membrane of any one of claims 1 -63, configured to electroporate a donor cell with a transfection reagent.

65. The perforated membrane of claim 64, wherein the transfection reagent comprises a DNA, a RNA, or a small molecule.

66. A method of making a perforated membrane from photoresist materials, comprising:(a) spin coating a first layer of a first photoresist material onto a substrate;(b) soft baking the first layer obtained in (a);(c) exposing the soft baked first layer obtained in (b) to a first UV radiation using a first photomask;(d) spin coating a second layer of a second photoresist material onto the first layer after (c);(e) soft baking the second layer obtained in (d);(f) exposing the soft baked second layer obtained in (e) to another UV radiation using a second photomask;(g) conducting a post exposure bake; and(h) developing both the first layer and the second layer after (g), thereby making a perforated membrane comprising two layers.

67. The method of claim 66, further comprising: after (c) but before (d), conducting another post exposure bake.

68. The method of claim 66 or 67, wherein the substrate is a silicon membrane coated with a release layer.

69. The method of claim 68, wherein the spin coating in (a) coats the first layer onto the release layer.

70. The method of claim 69, further comprising: after (h), removing the release layer.

71. The method of claim 66, wherein both the first photoresist material and the second photoresist material are negative photoresists.

72. The method of claim 66, wherein the first photoresist material is different from the second photoresist material.

73. The method of claim 72, wherein the first photoresist material has a higher resolution than the second photoresist material.

74. The method of claim 66, wherein the first photomask comprises a first pattern, and wherein the second photomask comprises a second pattern.

75. The method of claim 73, wherein the first pattern is configured to produce a plurality of first channels disposed in the first layer after (h), and wherein the second pattern is configured to produce a plurality of second channels disposed in the second layer after (h).

76. The method of claim 66, further comprising: after (c) but before (d), coating a barrier layer on top of the first layer.

77. The method of claim 76, wherein the barrier layer comprises silicon oxide, gold, or silver.

78. The method of any one of claims 66-77, wherein the perforated membrane comprises a top layer comprising a plurality of top channels disposed through the top layer; and a bottom layer in contact with the top layer, the bottom layer comprising a plurality of bottom channels disposed through the bottom layer; wherein: a first average thickness of the top layer is different from a second average thickness of the bottom layer; a first channel of the plurality of top channels is in fluid communication of a second channel of the plurality of bottom channels; a first average diameter of the first channel is different from a second average diameter of the second channel.

79. A method of making a perforated membrane from a sacrificial template, comprising(a) providing a female mold comprising a female pattern;(b) preparing a sacrificial male mold based on the female mold; and(c) preparing a perforated membrane based on the sacrificial male mold, wherein the perforated membrane comprises another pattern.

80. The method of claim 79, wherein the another pattern is substantially the same as the female pattern.

81. The method of claim 79, wherein the preparing in (c) comprises imprinting.

82. The method of claim 81, wherein the imprinting comprising providing a substrate and a weight.

83. The method of claim 82, wherein the substrate is coated with a release layer.

84. The method of claim 83, wherein the release layer comprises a releasable polymer.

85. The method of claim 79, wherein the preparing in (c) comprises spin coating.

86. The method of any one of claims 79-85, wherein the preparing in (c) comprises applying a polymer resin to the sacrificial male mold.

87. The method of claim 86, wherein the polymer resin is thermal curable.

88. The method of claim 87, further comprising: thermal curing the polymer resin.

89. The method of claim 86, wherein the polymer resin is photo-curable.

90. The method of claim 87, further comprising: photo curing the polymer resin.

91. The method of any one of claims 79-90, the preparing in (c) further comprises: removing the sacrificial male mold.

92. The method of any one of claims 79-91, wherein the sacrificial male mold is made from water-soluble polymers.

93. The method of claim 92, wherein the water-soluble polymer is polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyvinylpyrrolidone / vinyl alcohol copolymer (PVP / VA copolymer).

94. The method of any one of claim 86-93, wherein the polymer resin is polydimethylsiloxane (PDMS) resin or acrylic resin.

95. The method of any one of claims 84, or 86-94, wherein the releasable polymer is polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyvinylpyrrolidone / vinyl alcohol copolymer (PVP / VA copolymer).

96. The method of any one of claims 82-84, or 86-95, wherein the substrate is a rubbery layer.

97. The method of any one of claims 79-96, wherein the perforated membrane comprises a plurality of channels disposed through the perforated membrane; an average diameter of the channels; and an average thickness.

98. A system for high-throughput cell electroporation comprising: a) a perforated membrane, wherein the perforated membrane comprises: i. a first layer comprising a plurality of first channels disposed through the first layer; and ii. a second layer in contact with the first layer, the second layer comprising a plurality of second channels disposed through the second layer, wherein: a first average thickness of the first layer is different from a second average thickness of the second layer; a first channel of the plurality of first channels is in fluid communication of a second channel of the plurality of second channels; a first average diameter of the first channel is different from a second average diameter of the second channel; and b) at least one donor cell in contact with the perforated membrane.

99. The system of claim 98, wherein the first average thickness is larger than the second average thickness.

100. The system of claim 99, wherein the first average thickness is from about 1 gm to about 200 gm.

101. The system of claim 99 or 100, wherein the first average thickness is at least twice of the second average thickness.

102. The system of any one of claims 99-101, wherein the first average thickness is at least three times of the second average thickness.

103. The system of any one of claims 99-102, wherein the second average thickness is from about 100 nm to about 10 gm.

104. The system of any one of claims 98-103, wherein the first average diameter is larger than the second average diameter.

105. The system of any one of claims 98-104, wherein the first average diameter is from about 1 gm to about 20 gm.

106. The system of any one of claims 98-104, wherein the first average diameter is at least twice of the second average diameter.

107. The system of any one of claims 98-104, wherein the first average diameter is at least three times of the second average diameter.

108. The system of any one of claims 98-104, wherein the first average diameter is from about twice to about three times of the second average diameter.

109. The system of any one of claims 98-104, wherein the first average diameter is from about three times to about four times larger than the second average diameter.

110. The system of any one of claims 98-104, wherein the second average diameter is from about 100 nm to about 5 gm.

111. The system of any one of claims 98-104, wherein the first average diameter is about half of the first average thickness.

112. The system of any one of claims 98-104, wherein the first average diameter is about one- third of the first average thickness.

113. The system of any one of claims 98-104, wherein the second average diameter is no more than half of the second average thickness.

114. The system of any one of claims 98-104, wherein the second average diameter is no more than one-third of the second average thickness.

115. The system of any one of claims 98-104, wherein the second average diameter is from about one-half to about one-third of the second average thickness.

116. The system of any one of claims 98-104, wherein the second average diameter is from about one-third to about one-fourth of the second average thickness.

117. The system of any one of claims 98-104, wherein the second average diameter is about half of the second average thickness.

118. The system of any one of claims 98-104, wherein the second average diameter is no more than half of the second average thickness.

119. The system of any one of claims 98-118, wherein the first layer comprises a first polymer material.

120. The system of claim 119, wherein the first polymer material comprises a first light- reactive polymer.

121. The system of claim 119, wherein the first polymer material comprises a first crosslinking polymer.

122. The system of claim 119, wherein the first polymer material comprises a first synthetic polymer.

123. The system of claim 119, wherein the first polymer material comprises a first thermosetting polymer.

124. The system of claim 119, wherein the first polymer material comprises a first light-curing polymer or a first thermal-curing polymer.

125. The system of claim 119, wherein the first polymer material comprises a first photoresist.

126. The system of claim 119, wherein the first polymer material comprises a first positive photoresist.

127. The system of claim 119, wherein the first polymer material comprises a first negative photoresist.

128. The system of claim 119, wherein the first polymer material comprises a first SU-8 photoresist.

129. The system of claim 119, wherein the first polymer material comprises a first SU-8 3000- series photoresist.

130. The system of claim 119, wherein the first polymer material comprises a first SU-8 3010 photoresist or a first SU-8 3005 photoresist.

131. The system of claim 119, wherein the first polymer material comprises a first SU-82000- series photoresist.

132. The system of claim 119, wherein the first polymer material comprises a first SU-8 2005 photoresist.

133. The system of claim 132, wherein the second layer comprises a second polymer material.

134. The system of claim 133, wherein the second polymer material comprises a second light- reactive polymer.

135. The system of claim 133, wherein the second polymer material comprises a second crosslinking polymer.

136. The system of claim 133, wherein the second polymer material comprises a second synthetic polymer.

137. The system of claim 133, wherein the second polymer material comprises a second thermosetting polymer.

138. The system of claim 133, wherein the second polymer material comprises a second lightcuring polymer or a second thermal-curing polymer.

139. The system of claim 133, wherein the second polymer material comprises a second photoresist.

140. The system of claim 133, wherein the second polymer material comprises a second positive photoresist.

141. The system of claim 133, wherein the second polymer material comprises a second negative photoresist.

142. The system of claim 133, wherein the second polymer material comprises a second SU-8 photoresist.

143. The system of claim 133, wherein the second polymer material comprises a second SU-8 TF 6000-series photoresist.

144. The system of claim 133, wherein the second polymer material comprises a SU-8 TF 6002 photoresist.

145. The system of claim 133, wherein the second polymer material comprises a second SU-8 2000-series photoresist.

146. The system of claim 133, wherein the second polymer material comprises a SU-8 2002 photoresist.

147. The system of any one of claims 98-146, wherein the first layer comprises at least one ingredient different from ingredients of the second materials.

148. The system of any one of claims 98-147, wherein the first layer is made of materials different from those of the second layer.

149. The system of claim 148, wherein the first layer comprises the first photoresist, and wherein the second layer comprises the second photoresist.

150. The system of claim 148, wherein the first layer comprises the first negative photoresist, and wherein the second layer comprises the second negative photoresist.

151. The system of claim 148, wherein the first layer comprises the first SU-8 photoresist, and wherein the second layer comprises the second SU-8 photoresist.

152. The system of claim 148, wherein the first layer comprises the SU-8 3010 photoresist, the SU-8 3005 photoresist, or the SU-8 2005 photoresist, and wherein the second layer comprises the SU-8 TF 6002 photoresist or the SU-8 2002 photoresist.

153. The system of claim 152, wherein a first tensile strength of the first layer is larger than a second tensile strength of the second layer.

154. The system of claim 152, wherein the first layer and the second layer are made from photoresist polymers.

155. The system of any one of claims 98-154, wherein the first layer is made of the same materials as those of the second layer.

156. The system of claim 155, wherein the first layer and the second layer comprise the first light-reactive polymer.

157. The system of claim 155, wherein the first layer and the second layer comprise the first crosslinking polymer.

158. The system of claim 155, wherein the first layer and the second layer comprise the first synthetic polymer.

159. The system of claim 155, wherein the first layer and the second layer comprise the first light-curing polymer.

160. The system of any one of claims 98-159, configured to electroporate a donor cell with a transfection reagent.

161. The system of any one of claims 98-160, further comprising a first spacer array, wherein the first spacer array comprises: a) a thickness greater than a diameter of a donor cell; and b) a plurality of first holes, wherein a first hole of the plurality of first holes comprises a first hole width and a second hole width orthogonal to the first width, wherein the first hole of the first spacer array has a cross-sectional area defined by a material of the first spacer array.

162. The system of claim 161, wherein the first spacer array is in contact with the perforated membrane.

163. The system of claim 161 or 162, wherein the first hole width and the second hole width of the first hole of the first spacer array is less than a surface area of a first surface of the perforated membrane having openings of the first plurality of channels.

164. The system of any one of claims 161-163, wherein the first hole of the first spacer array is covered by the perforated membrane.

165. The system of any one of claims 161-164, wherein the first hole width of the first hole is at least about 1 mm.

166. The system of any one of claims 161-164, wherein the first hole width of the first hole is from at least about 1 mm to about 10 cm.

167. The system of any one of claims 161-164, wherein the first hole width of the first hole is about 1 cm.

168. The system of any one of claims 161-164, wherein the second hole width of the first hole is at least about 5 mm.

169. The system of any one of claims 161-164, wherein the second hole width of the first hole is from at least about 1 mm to about 10 cm.

170. The system of any one of claims 161-164, wherein the second hole width of the first hole is about 1 cm.

171. The system of any one of claims 161-170, wherein the plurality of first holes of the first spacer array comprises: a) at least 2 rows of first holes of the plurality of first holes; and b) at least 2 columns of first holes of the plurality of first holes.

172. The system of any one of claims 161-171, wherein the first hole is separated from other first holes adjacent to the first hole of the first spacer array by a first inter-hole distance.

173. The system of claim 172, wherein the first hole of the first spacer array is separated from other first holes adj acent to the first hole of the first spacer array by a first inter-hole distance of at least about 1 mm.

174. The system of claim 172, wherein the first hole of the first spacer array is separated from other first holes adj acent to the first hole of the first spacer array by a first inter-hole distance of from about 1 mm to about 5 cm.

175. The system of claim 172, wherein the first hole of the first array spacer is separated from other first holes adj acent to the first spacer array by a first inter-hole distance of from about 1 mm to about 1.5 cm.

176. The system of claim 172, wherein the first hole of the first array spacer is separated from other first holes adj acent to the first hole of the first spacer array by a first inter-hole distance of about 5 mm.

177. The system of any one of claims 161-176, wherein the thickness of the first spacer array is from about 0. 1 cm to about 5 cm.

178. The system of any one of claims 161-177, wherein a width of an outer side of the first spacer array is from about 1 cm to about 30 cm.

179. The system of any one of claims 161-178, wherein the first spacer array comprises at least about 2 first holes.

180. The system of any one of claims 161-178, wherein the first spacer array comprises from about 2 first holes to about 500 first holes.

181. The system of any one of claims 161-178, wherein the first spacer array comprises from about 2 first holes to about 100 first holes.

182. The system of any one of claims 161-178, wherein the first spacer array comprises from about 2 first holes to about 30 first holes.

183. The system of any one of claims 161-182, wherein the first spacer array comprises a polymer.

184. The system of claim 183, wherein the first spacer array comprises a thermoplastic polymer, a crosslinking polymer, a light-curing polymer, a thermal-curing polymer, a light- reactive polymer, a silicon polymer, a rubber, or a photoresist polymer.

185. The system of claim 183, wherein the first spacer array comprises polyethylene terephthalate (PET), polycarbonate, poly dimethylsiloxane (PDMS), poly dimethylsiloxane (PDMA), polyvinyl alcohol (PVA), polyethylene, polypropylene, polystyrene, polyacrylamide, or polyacrylic acid.

186. The system of any one of claims 98-185, further comprising a second spacer array, wherein the second spacer array comprises: a) a second thickness greater than a diameter of a donor cell; b) a plurality of second holes, wherein a second hole of the second spacer array comprises a first hole width and a second hole width orthogonal to the first width; and wherein the second hole of the second spacer array has a cross-sectional area defined by a material of the second spacer array.

187. The system of claim 186, wherein the second spacer array is in contact with the perforated membrane.

188. The system of claim 186 or 187, wherein the second hole of the second spacer array does not exceed a surface area of a surface of the perforated membrane having openings of a second plurality of channels.

189. The system of any one of claims 186-188, wherein the second hole of the second spacer array is covered by the perforated membrane.

190. The system of any one of claims 186-189, wherein the first hole width of the second hole of the second spacer array is at least about 1 mm.

191. The system of any one of claims 186-189, wherein the first hole width of the second hole of the second spacer array is from at least about 1 mm to about 10 cm.

192. The system of any one of claims 186-189, wherein the first hole width of the second hole of the second spacer array is about 1 cm.

193. The system of any one of claims 186-189, wherein the second hole width of the second hole of the second spacer array is at least about 1 mm.

194. The system of any one of claims 186-189, wherein the second hole width of the second hole of the second spacer array is from about 1 mm to about 10 cm.

195. The system of any one of claims 186-189, wherein the second hole width of the second hole of the second spacer array is about 1 cm.

196. The system of any one of claims 186-195, wherein the second holesof the second spacer array comprise: a) at least 2 rows of second holes of the second spacer array; and b) at least 2 columns of second holes of the second spacer array.

197. The system of any one of claims 186-196, wherein second hole of the second spacer array are separated by a second inter-hole distance.

198. The system of any one of claims 186-196, wherein second hole of the second spacer array are separated by an inter-hole distance of at least about 1 mm.

199. The system of any one of claims 186-196, wherein second hole of the second spacer array are separated by a second inter-hole distance of from about 1 mm to about 5 cm.

200. The system of any one of claims 186-196, wherein second hole of the second spacer array are separated by a second inter-hole distance of from about 1 mm to about 1.5 cm.

201. The system of any one of claims 186-196, wherein second hole of the second spacer array are separated by a second inter-hole distance is about 5 mm.

202. The system of any one of claims 186-201, wherein the second thickness of the second spacer array is from at least about 0.5 cm to at least about 5 cm.

203. The system of any one of claims 186-202, wherein a width of an outer side of the second spacer array is from about 1 cm to about 30 cm.

204. The system of any one of claims 186-202, wherein the second spacer array comprises at least about 2 second holes.

205. The system of any one of claims 186-202, wherein the second spacer array comprises from about 2 second holes to about 500 second holes.

206. The system of any one of claims 186-203, wherein the second spacer array comprises from about 2 second holes to about 100 second holes.

207. The system of any one of claims 186-203, wherein the second spacer array comprises from about 2 second holes to about 30 second holes.

208. The system of any one of claims 186-207, wherein the second thickness of the second spacer array is the same as the first thickness of the first spacer array.

209. The system of any one of claims 186-207, wherein the second thickness of the second spacer array is different than the first thickness of the first spacer array.

210. The system of any one of claims 186-207, wherein the second spacer array is identical to the first spacer array.

211. The system of any one of claims 186-208, wherein the second inter-hole distance of the second spacer array is different than the first inter-hole distance of the first spacer array.

212. The system of any one of claims 186-208, wherein the second inter-hole distance between two second holes of the second spacer array is different than at least one first interhole distance between two first holes of the first array of holes of the first spacer array.

213. The system of any one of claims 186-208, wherein the first hole width and the second hole width of the second hole of the second spacer array are the same as the first hole width and the second hole width of the first hole of the first spacer array.

214. The system of any one of claims 186-208, wherein the first hole width and the second hole width of the second hole of the second spacer array are different than the first hole width and the second hole width of the first hole of the first spacer array.

215. The system of any one of claims 98-214, further comprising a cathode.

216. The system of claim 215, wherein the cathode comprises a material that is electrically conductive and non-toxic to the at least one donor cell.

217. The system of claim 215 or 216, wherein the cathode comprises a three-dimensional (3D) cathode plate.

218. The system of any one of claims 215-217, wherein the cathode is configured to contact a surface of the first spacer array or the second spacer array.

219. The system of claim 215 or 216, wherein the cathode comprises a cathode coil to configured to be disposed within the first hole of the first spacer array or the second hole of the second spacer array.

220. The system of claim 215 or 216, wherein the cathode comprises an array of cathode coils.

221. The system of claim 220, wherein the array of cathode coils is configured to position individual cathode coils within the first hole of the first spacer array or the second hole of the second spacer array.

222. The system of claim 215, wherein the array of cathode coils is configured to position individual cathode coils within at least 2 holes of the first spacer array or within at least 2 second holes of the second spacer array.

223. The system of claim 221, wherein the array of cathode coils is configured to position individual cathode coils within at least 3 holes of the first spacer array or within at least 3 second holes of the second spacer array.

224. The system of claim 221, wherein the array of cathode coils is configured to position individual cathode coils within at least 5 holes of the first spacer array or within at least 5 second holes of the second spacer array.

225. The system of any one of claims 98-224, further comprising an anode.

226. The system of claim 225, wherein the anode comprises a material that is electrically conductive and non-toxic to the at least one donor cell.

227. The system of claim 225 or 226, wherein the anode comprises a three-dimensional (3D) anode plate.

228. The system of claim 225 or 226, wherein the anode is configured to contact a surface of the first spacer array or the second spacer array.

229. The system of claim 225 or 226, wherein the anode comprises a wire anode.

230. The system of claim 225 or 226, wherein the anode comprises an array of wire anodes configured to position individual wire anodes of the array of wire anodes within the individual holes of the first spacer array or second spacer array.

231. The system of claim 230, wherein the array of wire anodes is configured to position individual wire anodes of the array of wire anodes within the individual holes of the first spacer array or the second spacer array.

232. The system of any one of claims 98-231, further comprising an electric pulse generation system.

233. The system of claim 232, wherein the electric pulse generation system comprises: a) an electrical pulse generator; b) a shock pulse converter; and c) an electrical current output device.

234. The system of claim 232 or 233, wherein the electric pulse generation system is configured to adjust a voltage applied to the at least one donor cell in contact with the perforated membrane.

235. The system of any one of claims 232-234, wherein the electric pulse generation system is configured to sequentially deliver an electrical pulse to the anode wire or at least one of the individual wire anodes of the array of wire anodes.

236. The system of any one of claims 232-235, wherein the electric pulse generation system is configured to execute an automated routine to deliver an electrical pulse to the anode wire or at least one of the individual wire anodes of the array of wire anodes.

237. The system of any one of claims 232-236, further comprising an automated liquid handling or liquid distribution device configured to: a) dispose the at least one donor cell within at least one individual hole of the first spacer array; b) add or remove an electroporation buffer within at least one individual hole of the second spacer array; or c) add or remove a cell culture medium within at least one individual hole of the first spacer array.

238. A system for high-throughput cell electroporation comprising: a) a perforated membrane, wherein the perforated membrane comprises channels disposed through the perforated membrane; b) a first array spacer; c) a second array spacer; and d) at least one donor cell, wherein the first array spacer, the second array spacer, and the at least one donor cell are in contact with the perforated membrane.

239. The system of claim 238, wherein the perforated membrane comprises a silicon membrane, a track-etch membrane, a polymer membrane, a single-layer polymer membrane, or dual-layer polymer membrane.

240. The system of claim 238 or 239, wherein the channels are distributed throughout the perforated membrane in a patterned arrangement or a non-patterned arrangement.

241. The system of any of one claims 238-240, wherein the first spacer array comprises: a) a thickness greater than a diameter of a donor cell; and b) a plurality of first holes, wherein a first hole of the plurality of first holes comprises a first hole width and a second hole width orthogonal to the first width, wherein the first hole of the first spacer array has a cross-sectional area defined by a material of the first spacer array .

242. The system of claim 238-241, wherein the first hole width and the second hole width of the first hole of the first spacer array is less than a surface area of a surface of the perforated membrane having openings of a first plurality of channels.

243. The system of any one of claims 238-242, wherein the first hole of the first spacer array is covered by the perforated membrane.

244. The system of any one of claims 238-241, wherein the first hole width of the first hole is at least about 1 mm.

245. The system of any one of claims 238-241, wherein the first hole width of the first hole is from at least about 1 mm to about 10 cm.

246. The system of any one of claims 238-241, wherein the first hole width of the first hole is about 1 cm.

247. The system of any one of claims 238-241, wherein the second hole width of the first hole is at least about 5 mm.

248. The system of any one of claims 238-241, wherein the second hole width of the first hole is from at least about 1 mm to about 10 cm.

249. The system of any one of claims 238-241, wherein the second hole width of the first hole is about 1 cm.

250. The system of any one of claims 238-249, wherein the plurality of first holes of the first spacer array comprises: a) at least 2 rows of first holes of the plurality of first holes; and b) at least 2 columns of first holes of the plurality of first holes.

251. The system of any one of claims 238-249, wherein the first hole is separated from other first holes adjacent to the first hole of the first spacer array by a first inter-hole distance.

252. The system of claim 251, wherein the first hole of the first spacer array is separated from other first holes adjacent to the first hole of the first spacer array by a first inter -hole distance of at least about 1 mm.

253. The system of claim 251 , wherein the first hole of the first spacer array is separated from other first holes adj acent to the first hole of the first spacer array by a first inter-hole distance of from about 1 mm to about 1.5 cm or from about 1 mm to about 5 cm.

254. The system of claim 251, wherein the first hole of the first array spacer is separated from other first holes adjacent to the first hole of the first spacer array by a first inter -hole distance of about 5 mm.

255. The system of any one of claims 238-254, wherein the thickness of the first spacer array is from about 0.1 cm to about 5 cm.

256. The system of any one of claims 238-255, wherein a width of an outer side of the first spacer array is from about 1 cm to about 30 cm.

257. The system of any one of claims 238-256, wherein the first spacer array comprises at least about 2 first holes.

258. The system of any one of claims 238-256, wherein the first spacer array comprises from about 2 first holes to about 500 first holes.

259. The system of any one of claims 238-256, wherein the first spacer array comprises from about 2 first holes to about 100 first holes.

260. The system of any one of claims 238-256, wherein the first spacer array comprises from about 2 first holes to about 30 first holes.

261. The system of any one of claims 238-260, wherein the first spacer array comprises a polymer.

262. The system of claim 261, wherein the first spacer array comprises a thermoplastic polymer, a crosslinking polymer, a light-curing polymer, a thermal -curing polymer, a light- reactive polymer, a silicon polymer, a rubber, or a photoresist polymer.

263. The system of claim 261, wherein the first array spacer comprises polyethylene terephthalate (PET), polycarbonate, poly dimethylsiloxane (PDMS), polydimethylsiloxane (PDMA), polyvinyl alcohol (PVA), polyethylene, polypropylene, polystyrene, polyacrylamide, or polyacrylic acid.

264. The system of any one of claims 238-263, wherein the second spacer array comprises: a) a second thickness greater than a diameter of a donor cell; and b) a plurality of second holes, wherein a second hole of the second spacer array comprises a first hole width and a second hole width orthogonal to the first width, wherein the second hole of the second spacer array has a cross-sectional area defined by a material of the second spacer array .

265. The system of any one of claims 238-264, wherein the second spacer array is in contact with the perforated membrane.

266. The system of claim 238-265, wherein the second hole of the second spacer array does not exceed a surface area of a surface of the perforated membrane having openings of a second plurality of channels.

267. The system of any one of claims 238-266, wherein the second hole of the second spacer array is covered by the perforated membrane.

268. The system of any one of claims 238-267, wherein the first hole width of the second hole of the second spacer array is at least about 1 mm.

269. The system of any one of claims 238-267, wherein the first hole width of the second hole of the second spacer array is from at least about 1 mm to about 10 cm.

270. The system of any one of claims 238-267, wherein the first hole width of the second hole of the second spacer array is about 1 cm.

271. The system of any one of claims 238-267, wherein the second hole width of the second hole of the second spacer array is at least about 1 mm.

272. The system of any one of claims 238-267, wherein the second hole width of the second hole of the second spacer array is from about 1 mm to about 10 cm .

273. The system of any one of claims 238-267, wherein the second hole width of the second hole of the second spacer array is about 1 cm.

274. The system of any one of claims 238-273, wherein the second holesof the second spacer array comprise: a) at least 2 rows of second holes of the second spacer array; and b) at least 2 columns of second holes of the second spacer array .

275. The system of any one of claims 238-274, wherein the second hole of the second spacer array is separated from other second holes adjacent to the second hole of the second spacer array by a second inter-hole distance.

276. The system of any one of claims 238-274, wherein the second hole of the second spacer array is separated from other second holes adjacent to the second hole of the second spacer array by a second inter-hole distance of at least about 1 mm.

277. The system of any one of claims 238-274, wherein the second hole of the second spacer array is separated from other second holes adjacent to the second hole of the second spacer array by a second inter-hole distance of from about 1 mm to about 5 cm.

278. The system of any one of claims 238-274, wherein the second hole of the second spacer array is separated from other second holes adjacent to the second hole of the second spacer array by a second inter-hole distance of from about 1 mm to about 1.5 cm.

279. The system of any one of claims 238-274, wherein the second hole of the second spacer array is separated from other second holes adjacent to the second hole of the second spacer array by a second inter-hole distance is about 5 mm.

280. The system of any one of claims 238-279, wherein the second thickness of the second spacer array is from at least about 0. 1 cm to at least about 5 cm.

281. The system of any one of claims 238-280, wherein a width of an outer side of the second spacer array is from about 1 cm to about 30 cm.

282. The system of any one of claims 238-281, wherein the second spacer array comprises at least about 2 second holes.

283. The system of any one of claims 238-281, wherein the second spacer array comprises from about 2 second holes to about 500 second holes.

284. The system of any one of claims 238-281, wherein the second spacer array comprises from about 2 second holes to about 100 second holes.

285. The system of any one of claims 238-281, wherein the second spacer array comprises from about 2 second holes to about 30 second holes.

286. The system of any one of claims 238-285, wherein the second thickness of the second spacer array is the same as the first thickness of the first spacer array .

287. The system of any one of claims 238-286, wherein the second spacer array is identical to the first spacer array.

288. The system of any one of claims 238-285, wherein the second thickness of the second spacer array is different than the first thickness of the first spacer array.

289. The system of any one of claims 238-288, wherein the second inter-hole distance of the second spacer array is different than the first inter-hole distance of the first spacer array.

290. The system of any one of claims 238-288, wherein the second inter-hole distance separating the second hole from other second holes adjacent to the second hole of the second spacer array is different than the first inter-hole distance separating the first hole from the other first holes adj acent to the first hole of the first array of holes of the first spacer array .

291. The system of any one of claims 238-290, wherein the first hole width and the second hole width of the second hole of the second spacer array are the same as the first hole width and the second hole width of the first hole of the first spacer array .

292. The system of any one of claims 238-291, wherein the first hole width and the second hole width of the second hole of the second spacer array are different than the first hole width and the second hole width of the first hole of the first spacer array .

293. The system of any one of claims 238-292, wherein the second array spacer comprises a second thermoplastic polymer, a second crosslinking polymer, a second light-curing polymer, a second thermal -curing polymer, a second light-reactive polymer, a second silicon polymer, a second rubber, or a second photoresist polymer294. The system of any one of claims 238-293, further comprising a cathode.

295. The system of claim 294, wherein the cathode comprises a material that is electrically conductive and non-toxic to the at least one donor cell.

296. The system of claim 294 or 295, wherein the cathode comprises a three-dimensional (3D) cathode plate.

297. The system of any one of claims 294-296, wherein the cathode is configured to contact a surface of the first spacer array or the second spacer array .

298. The system of claim 294 or 295, wherein the cathode comprises a cathode coil to configured to be disposed within the first hole of the first spacer array or the second hole of the second spacer array.

299. The system of claim 294 or 295, wherein the cathode comprises an array of cathode coils.

300. The system of claim 299, wherein the array of cathode coils is configured to position individual cathode coils within the first hole of the first spacer array or the second hole of the second spacer array.

301. The system of claim 299, wherein the array of cathode coils is configured to position individual cathode coils within at least 2 holes of the first spacer array or within at least 2 second holes of the second spacer array.

302. The system of claim 300, wherein the array of cathode coils is configured to position individual cathode coils within at least 3 holes of the first spacer array or within at least 3 second holes of the second spacer array.

303. The system of claim 300, wherein the array of cathode coils is configured to position individual cathode coils within at least 5 holes of the first spacer array or within at least 5 second holes of the second spacer array.

304. The system of any one of claims 238-303, further comprising an anode.

305. The system of claim 304, wherein the anode comprises a material that is electrically conductive and non-toxic to the at least one donor cell.

306. The system of claim 304 or 305, wherein the anode comprises a three-dimensional (3D) anode plate.

307. The system of claim 304 or 305, wherein the anodeis configured to contact a surface of the first spacer array or the second spacer array .

308. The system of claim 304 or 305, wherein the anode comprises a wire anode.

309. The system of claim 304 or 305, wherein the anode comprises an array of wire anodes configured to position individual wire anodes of the array of wire anodes within the individual holes of the first spacer array or second spacer array.

310. The system of claim 309, wherein the array of wire anodes is configured to position individual wire anodes of the array of wire anodes within the individual holes of the first spacer array or the second spacer array .

311. The system of any one of claims 238-310, further comprising an electric pulse generation system.

312. The system of claim 311, wherein the electric pulse generation system comprises: a) an electrical pulse generator; b) a shock pulse converter; and c) an electrical current output device.

313. The system of claim 311 or 312, wherein the electric pulse generation system is configured to adjust a voltage applied to the at least one donor cell in contact with the perforated membrane.

314. The system of any one of claims 311-313, wherein the electric pulse generation system is configured to sequentially deliver an electrical pulse to the anode wire or at least one of the individual wire anodes of the array of wire anodes.

315. The system of any one of claims 311-314, wherein the electric pulse generation system is configured to execute an automated routine to deliver an electrical pulse to the anode wire or at least one of the individual wire anodes of the array of wire anodes.

316. The system of any one of claims 238-315, further comprising an automated liquid handling or liquid distribution device configured to: a) dispose the at least one donor cell within at least one individual hole of the first spacer array; b) add or remove an electroporation buffer within at least one individual hole of the second spacer array; or c) add or remove a cell culture medium within at least one individual hole of the first spacer array.

317. The system of any one of claims 238-316, wherein the first spacer array is in contact with a surface of the perforated membrane and defines an array of electroporation chambers; wherein individual cell electroporation chambers of the array of electroporation chambers are isolated from neighboring electroporation chambers by the first spacer array; and wherein the individual cell electroporation chambers are configured to contain at least one electroporation buffer, optionally wherein the individual cell electroporation chambers are configured to contain a different electroporation buffer, a different transfection reagent, or a different concentration of a transfection reagent than other individual cell electroporation chambers.

318. The system of any one of claims 238-317, wherein the second spacer array is in contact with a second surface of the perforated membrane opposite the surface of the perforated membrane in contact with the first spacer array; wherein the second spacer array defines an array of cell culture chambers; wherein individual cell culture chambers of the array of cell culture chambers are isolated from neighboring cell culture chambers by the second spacer array; wherein individual cell culture chambers are fluidically coupled to an individual electroporation chamber via the channels of the perforated membrane; and wherein the individual cell culture chambers are configured to contain at least one cell culture medium and the at least one donor cell, optionally wherein the individual cell culture chambers are configured to contain a different cell culture medium or a different donor cell type than other individual cell culture chambers.

319. The system of any one of claims 238-318, wherein the perforated membrane comprises:i. a first layer comprising a plurality of first channels disposed through the first layer; and ii. a second layer in contact with the first layer, the second layer comprising a plurality of second channels disposed through the second layer, wherein: a first average thickness of the first layer is different from a second average thickness of the second layer; a first channel of the plurality of first channels is in fluid communication of a second channel of the plurality of second channels; a first average diameter of the first channel is different from a second average diameter of the second channel.

320. The system of claim 319, wherein first average thickness is larger than the second average thickness.

321. The system of claim 320, wherein the first average thickness is from about 1 pm to about200 pm.

322. The system of claim 320 or 321, wherein the first average thickness is at least twice of the second average thickness.

323. The system of any one of claims 319-322, wherein the first average thickness is at least three times of the second average thickness.

324. The system of any one of claims 320-323, wherein the second average thickness is from about 100 nm to about 10 pm.

325. The system of any one of claims 319-324, wherein the first average diameter is larger than the second average diameter.

326. The system of any one of claims 319-325, wherein the first average diameter is from about 1 pm to about 20 pm.

327. The system of any one of claims 319-325, wherein the first average diameter is at least twice of the second average diameter.

328. The system of any one of claims 319-325, wherein the first average diameter is at least three times of the second average diameter.

329. The system of any one of claims 319-325, wherein the first average diameter is from about twice to about three times of the second average diameter.

330. The system of any one of claims 319-325, wherein the first average diameter is from about three times to about four times larger than the second average diameter.

331. The system of any one of claims 319-325, wherein the second average diameter is from about 100 nm to about 5 pm.

332. The system of any one of claims 319-325, wherein the firstaverage diameter is about half of the first average thickness.

333. The system of any one of claims 319-325, wherein the first average diameter is about one-third of the first average thickness.

334. The system of any one of claims 319-325, wherein the second average diameter is no more than half of the second average thickness.

335. The system of any one of claims 319-325, wherein the second average diameter is no more than one-third of the second average thickness.

336. The system of any one of claims 319-325, wherein the second average diameter is from about one-half to about one-third of the second average thickness.

337. The system of any one of claims 319-325, wherein the second average diameter is from about one-third to about one-fourth of the second average thickness.

338. The system of any one of claims 319-325, wherein the second average diameter is about half of the second average thickness.

339. The system of any one of claims 319-325, wherein the second average diameter is no more than half of the second average thickness.

340. The system of any one of claims 319-325, wherein the second average diameter is no more than half of the second average thickness.

341. The system of any one of claims 319-340, wherein the first layer comprises a first polymer material.

342. The system of claim 341, wherein the first polymer material comprises a first light- reactive polymer.

343. The system of claim 341, wherein the first polymer material comprises a first crosslinking polymer.

344. The system of claim 341, wherein the first polymer material comprises a first synthetic polymer.

345. The system of claim 341, wherein the first polymer material comprises a first thermosetting polymer.

346. The system of claim 341, wherein the first polymer material comprises a first light-curing polymer or a first thermal-curing polymer.

347. The system of claim 341, wherein the first polymer material comprises a first photoresist.

348. The system of claim 341, wherein the first polymer material comprises a first positive photoresist.

349. The system of claim 341, wherein the first polymer material comprises a first negative photoresist.

350. The system of claim 341, wherein the first polymer material comprises a first SU-8 photoresist.

351. The system of claim 341, wherein the first polymer material comprises a first SU-8 3000- series photoresist.

352. The system of claim 341, wherein the first polymer material comprises a SU-8 3010 photoresist.

353. The system of claim 341, wherein the first polymer material comprises a first SU-82000- series photoresist.

354. The system of claim 341, wherein the first polymer material comprises a first SU-8 2005 photoresist.

355. The system of any one of claims 319-354, wherein the second layer comprises a second polymer material.

356. The system of claim 355, wherein the second polymer material comprises a second light- reactive polymer.

357. The system of claim 355, wherein the second polymer material comprises a second crosslinking polymer.

358. The system of claim 355, wherein the second polymer material comprises a second synthetic polymer.

359. The system of claim 355, wherein the second polymer material comprises a second thermosetting polymer.

360. The system of claim 355, wherein the second polymer material comprises a second lightcuring polymer or a second thermal-curing polymer.

361. The system of claim 355, wherein the second polymer material comprises a second photoresist.

362. The system of claim 355, wherein the second polymer material comprises a second positive photoresist.

363. The system of claim 355, wherein the second polymer material comprises a second negative photoresist.

364. The system of claim 355, wherein the second polymer material comprises a second SU-8 photoresist.

365. The system of claim 355, wherein the second polymer material comprises a second SU-8 TF 6000-series photoresist.

366. The system of claim 355, wherein the second polymer material comprises a SU-8 TF 6002 photoresist.

367. The system of claim 355, wherein the second polymer material comprises a second SU-8 2000-series photoresist.

368. The system of claim 355, wherein the second polymer material comprises a SU-8 2002 photoresist.

369. The system of any one of claims 238-368, wherein first layer comprises at least one ingredient different from ingredients of the second materials.

370. The system of any one of claims 238-369, wherein the first layer is made of materials different from those of the second layer.

371. The system of claim 370, wherein the first layer comprises the first photoresist, and wherein the second layer comprises the second photoresist.

372. The system of claim 370, wherein the first layer comprises the first negative photoresist, and wherein the second layer comprises the second negative photoresist.

373. The system of claim 370, wherein the first layer comprises the first SU-8 photoresist, and wherein the second layer comprises the second SU-8 photoresist.

374. The system of claim 370, wherein the first layer comprises the SU-8 3010 photoresist or the SU-8 2005 photoresist, and wherein the second layer comprises the SU-8 TF 6002 photoresist or the SU-8 2002 photoresist.

375. The system of any one of claims 319-374, wherein a first tensile strength of the first layer is larger than a second tensile strength of the second layer.

376. The system of any one of claims 319-375, wherein the first layer and the second layer are made from photoresist polymers.

377. The system of any one of claims 238-376, wherein the first layer is made of the same materials as those of the second layer.

378. The system of claim 377, wherein the first layer and the second layer comprise the light- reactive polymer.

379. The system of claim 377, wherein the first layer and the second layer comprise the crosslinking polymer.

380. The system of claim 377, wherein the first layer and the second layer comprise the synthetic polymer.

381. The system of claim 377, wherein the first layer and the second layer comprise the lightcuring polymer.

382. The system of any one of claims 238-381, configured to electroporate a donor cell with a transfection reagent.

383. A method of producing and collecting extracellular vesicles comprising:a) providing an electroporation device comprising an array of cell electroporation wells, wherein the electroporation device comprises: i. a perforated membrane situated between a first spacer array and a second spacer array, wherein the perforated membrane comprises a plurality of channels that span a distance of a first surface of the perforated membrane and a second surface of the perforated membrane; ii. a first spacer array defining an array of cell culture chambers; iii. a second spacer array defining an array of electroporation reagent chambers, wherein a second arrangement of the array of electroporation reagent chambers of the second spacer array is complementary to a first arrangement of the array of cell culture chambers of the first spacer array; wherein the array of cell culture chambers of the first spacer array and the array of electroporation reagent chambers of the second spacer array create at least two isolated cell electroporation chambers when spatially aligned and when then first spacer array and the second spacer array are in contact with the perforated membrane, wherein a cell culture chamber of the array of cell culture chambers comprises a portion of the perforated membrane; and wherein an electroporation reagent chamber of the array of electroporation reagent chambers comprises a portion of the perforated membrane; wherein the cell culture chambers of the array of cell culture chambers are fluidically coupled to the electroporation reagent chambers of the array of electroporation reagent chambers via the channels of the portion of the perforated membrane; b) introducing donors cells into at least one cell culture chamber of the array of cell culture chambers; c) introducing a polynucleotide, DNA, RNA, vector, or plasmid into at least one electroporation buffer chamber of the array of electroporation buffer chambers. d) applying an electric field, a current, or a voltage through the electroporation device, thereby electroporating the donor cells in at least one cell electroporation well of the array of cell electroporation wells; and e) collecting extracellular vesicles (EVs) produced by the donors cells.

384. The method of claim 383, wherein the extracellular vesicles (EVs) are exosomes, apoptotic bodies or microvesicles.

385. The method of claim 383 or 384, wherein the polynucleotide, DNA, or RNA encodes a collagen or a dystrophin.

386. The method of claim 385, wherein polynucleotide, DNA, or RNA encodes Coll A.

387. The method of any one of claims 383-386, wherein the perforated membrane is a silicon membrane, a track-etch membrane, a single-layer polymer membrane, or dual -lay er polymer membrane.

388. The method of any one of claims 383-387, further comprising repeating (b) to (e).

389. The method of any one of claims 383-387, further comprising repeating (c) to (e).

390. The method of any one of claims 383-387, further comprising repeating (d) to (e).

391. The method of any one of claims 383 -390, wherein (d) comprises applying the electric field sequentially to at least two cell electroporation wells before (e).

392. The method of any one of claims 383 -391, wherein the extracellular vesicles (EVs) are collected up to 24 hours after (d).

393. The method of any one of claims 383-392, further comprising repeating (b) through (e).

394. The method of any one of claims 383-392, further comprising repeating (c) through (e).

395. The method of any one of claims 383-392, further comprising repeating (d) through (e).

396. The method of any one of claims 383 -392, further comprising repeating (d) before (e).

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