Porous electroporation device and method

Abstract

The present disclosure describes systems, devices, and methods for high-throughput electroporation of donor cells. The system can include a perforation membrane having one or more layers, each layer including a plurality of channels disposed through each layer. The system can also include an array of spacers in contact with the perforation membrane to create a plurality of cell electroporation chambers for high-throughput cell electroporation.

Description

[0001] Cross-references This application claims the benefits of U.S. Provisional Application No. 63 / 591,116, filed 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 Technology

[0002] Cell electroporation is primarily used to introduce biomolecules (such as DNA, RNA, or drugs, proteins, or other charged biomolecules) into cells by temporarily disrupting the cell membrane with an electric field. This allows for the efficient delivery of nucleic acids and / or other biomolecules that are not normally membrane-permeable. Cell electroporation is commonly used for gene delivery and drug delivery to treat diseases. Tools have been developed to allow for faster, more efficient, and scalable electroporation methods. Furthermore, high-throughput electroporation platforms capable of multiplexing electroporation can increase the power of screening and the development of therapeutics. While existing high-throughput electroporation platforms offer the ability to deliver many nucleic acids or biomolecules of interest to large and diverse cell populations, the electroporation conditions applied to the cells affect key aspects such as cell viability, the magnitude of extracellular vesicle secretion, and the uniformity of nucleic acid expression. Currently feasible cell electroporation systems typically employ electrical stimulation that disrupts the entire cell membrane. For example, developing a method for locally electroporating cells could increase electroporation efficiency, improve cell health, and accelerate the development of therapeutics. Summary of the Invention

[0003] This document describes systems, apparatus, and instruments for providing high-yield and high-throughput delivery of nucleic acids and other biomolecules into cells, as well as their uses or methods. This disclosure presents a solution to the problem of increasing the efficiency and bandwidth of current methods in cell electroporation by culturing cells on a perforated membrane having an array of channels such that the cells are in contact with the openings of the channels. The channels range from about 500 nm to about 20 µm to minimize the surface area of ​​the cell membrane disturbed during the application of an electric field to the cells via the openings of the channels within the perforated membrane. The system provided in this disclosure enables multiple high-throughput electroporation to deliver one or more nucleic acids or other biomolecules to one or more cell populations in contact with the channels of the perforated membrane.

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

[0005] In another aspect, this disclosure provides a method for fabricating a perforated film from a photoresist material, comprising: (a) spin-coating a first layer of a first photoresist material onto a substrate; (b) soften-baking the first layer obtained in (a); (c) exposing the softened first layer obtained in (b) to a first UV radiation using a first photomask; (d) after (c), spin-coating a second layer of a second photoresist material onto the first layer; (e) soften-baking the second layer obtained in (d); (f) exposing the softened second layer obtained in (e) to another UV radiation using a second photomask; (g) performing post-exposure baking; and (g) after (g), developing both the first layer and the second layer to fabricate a perforated film comprising two layers.

[0006] This document also provides 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 with a second channel of the plurality of second channels; a first average diameter of the first channels is different from a second average diameter of the second channels; and at least one donor cell is in contact with the perforated membrane.

[0007] Another aspect of this disclosure describes a system for high-throughput cell electroporation, comprising: (a) a perforated membrane, wherein the perforated membrane includes channels arranged 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 for generating and collecting extracellular vesicles, comprising: (a) providing an electroporation device comprising a plurality of pores, such device comprising a perforated membrane located between two spacer sheets, wherein: the spacer sheets comprise a complementary array of pores defining the cross-sectional area of ​​the pores; the pores 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 fluidly couple the cell culture chamber to the electroporation buffer chamber; (b) introducing donor cells into the cell culture chamber; introducing polynucleotides, DNA, RNA, vectors, or plasmids into the electroporation buffer chamber; (d) applying an electric field, current, or voltage through the electroporation device to electroporate the donor cells; and (e) collecting extracellular vesicles (EVs) generated by the donor cells.

[0009] Other aspects and advantages of this disclosure will readily become apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of this disclosure are shown and described. As will be appreciated, this disclosure is capable of other and different embodiments, and certain details thereof can be modified in various obvious respects, all without departing from this disclosure. Therefore, the drawings and descriptions should be considered illustrative in nature and not restrictive.

[0010] Incorporation All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is expressly and individually indicated to be incorporated by reference. Attached Figure Description

[0011] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description and accompanying drawings, which illustrate illustrative embodiments utilizing the principles of the invention, in which: Figure 1 A flowchart illustrating a method for manufacturing a bilayer perforated film according to some embodiments of the invention described herein is provided. A non-limiting example of a material used to manufacture the bilayer perforated film is SU-8 epoxy photoresist.

[0012] Figure 2Scanning electron micrographs with perspective views depicting a bilayer perforated film with a patterned array of "bottle"-shaped channels (e.g., channels having small-diameter and large-diameter portions) according to some embodiments of the invention described herein. A non-limiting example of a bilayer perforated film with a patterned SU-8 thin film shows a bilayer microstructure. The thin layer of the bilayer perforated film is made of SU-8 TF 6002 photoresist with channels (i.e., pores) having a diameter of approximately 1 µm and a depth / length of approximately 2 µm. The thick layer consists of SU-8 3010 photoresist with a patterned array of channels (i.e., pores) having a diameter of approximately 4 µm for the large pores, thereby aligning one opening of the large channel with a small 1 µm diameter pore. The total thickness of the SU-8 thin film is approximately 10 µm.

[0013] Figure 3 Depicting Figure 2 The image shows a scanning electron micrograph with a side view of a "bottle"-shaped channel (e.g., a nanochannel or microchannel) within a double-layered perforated membrane.

[0014] Figure 4A Scanning electron micrographs with perspective views depicting some embodiments of the double-layer perforated film according to the invention described herein. As a non-limiting example, the SU-8 double-layer structure is made of a thin layer composed of SU-8 2002 photoresist and a thick layer made of SU-8 2005 photoresist.

[0015] Figure 4B Depicting Figure 4A The image shows a scanning electron micrograph with a side view of the double-layer perforated membrane.

[0016] Figure 5 The dystrophin (DMD) mRNA copy number / quantification of electroporation donor cells derived from human skin fibroblasts via cell nanoporation (CNP) was characterized. A total of 24,000 cells were localized on a 0.9 cm x 0.9 cm surface area of ​​either a bilayer perforated membrane (SU-8 bilayer surface; SU-8) or a silicon perforated membrane (Si-CNP; Si). Electroporation conditions: 100 V, 4.5 mm electrode-to-electrode distance, and ten 10-ms pulses at 0.1 sec intervals. EV was collected in culture medium for 24 hours following cell nanoporation.

[0017] Figure 6 A schematic diagram of the workflow for manufacturing a polymer cell electroporation surface using sacrificial template imprinting (STI) technology according to some embodiments of the invention described herein is presented.

[0018] Figure 7A Describing as Figure 6Optical images of a PVA-based sacrificial template mold (male mold) shown in the image and according to some embodiments of the invention described herein. Figure 7B Depicting through Figure 6 Optical images of PDMS cell electroporation / nano-electroporation (CEP / CNP) surfaces prepared by the sacrificial template imprinting method provided in this paper are shown in the figure.

[0019] Figure 8 A schematic diagram of a high-throughput single-array electroporation device (e.g., a cell electroporation / nano-electroporation (CEP / CNP) device) according to one embodiment of the invention described herein is depicted. A partial enlarged view: a side view across the single-cell culture wells of the high-throughput single-array electroporation device. Donor cells adhere to the top surface of a Si CNP surface having channel openings (e.g., nanochannel openings) and are fluidly connected to an electroporation buffer solution (e.g., a buffer containing the DNA plasmid of interest) via channels on the Si CNP surface. Electrical pulses are applied across the cathode and anode structures of the high-throughput single-array cell electroporation device via an electrical pulse generator to mediate electroporation of the donor cells with genetic cargo (e.g., DNA plasmids) in the electroporation buffer.

[0020] Figure 9A A schematic diagram of a spacer array for a high-throughput single-array electroporation device according to some embodiments of the invention described herein is depicted. The spacer array creates isolated cell culture wells and / or isolated electroporation culture chambers for high-throughput electroporation, such as high-throughput cell nano-electroporation (HIT-CNP)). Figure 9B Images depict high-throughput single-array electroporation devices with spacer arrays according to some embodiments of the invention described herein.

[0021] Figure 10A Scanning electron micrographs (SEMs) depicting a patterned array of channels in a perforated electroporated film (Si CNP surface) made of silicon.

[0022] Figure 10B Scanning electron micrographs (SEMs) depict unpatterned channels in track-etched perforated electroporated films (TEM).

[0023] Figure 11AA schematic diagram of a high-throughput dual-array electroporation device (e.g., a cell nanoporation (CNP) device) with a perforated electroporation membrane (e.g., a tracked etched randomized channel or a silicon (Si) patterned array of channels) sandwiched between two PDMS spacer arrays is depicted according to one embodiment of the invention described herein. A partial enlarged view: a side view across the dual-layer high-throughput electroporation device. Donor cells are added to individual cell culture wells defined by the pores within the polymer spacer array, and the donor cells are adhered to the top surface and channel openings (e.g., nanochannel openings) of the tracked etched membrane, patterned perforated silicon membrane, or polymer dual-layer perforated membrane. Here, the sandwiched array forms individual cell culture wells, each of which is fluidly connected via a channel of the perforated membrane to a separate electroporation reagent well (e.g., a buffer containing a plasmid of interest). Any perforated membrane is compatible with this dual-array-based cell electroporation device configuration. Each cell culture well can be seeded with cells of a different type than those in neighboring cell culture wells, and each electroporation reagent well can be loaded with different types, concentrations, or mixtures of transfection reagents (e.g., DNA plasmids, RNA, or small molecules). Electrical pulses are applied across the cathode and anode structures of the high-throughput single-array electroporation device via an electrical pulse generator to mediate the electroporation of donor cells with genetic cargo (e.g., DNA plasmids) in the electroporation buffer.

[0024] Figure 11B A schematic diagram of a complete dual-well array chamber layer-based cell electroporation system for high-throughput cell electroporation is depicted, the system having an array of cathode coils inserted into each of the electroporation reagent wells.

[0025] Figure 12 A schematic diagram depicts the workflow of a dual-array cell electroporation system for high-throughput cell electroporation (e.g., cell electroporation / nano-electroporation (CEP / CNP)) according to some embodiments of the invention described herein. A perforated electroporation membrane is sandwiched between two arrays of spacers to create complementary arrays of isolated cell culture wells and electroporation reagent chambers. In this configuration, each individual cell culture well is fluidly coupled to one of the electroporation reagent chambers. Donor cells adhere to the surface of the perforated electroporation membrane within one or more of the isolated cell culture wells. Upon adhesion to the perforated membrane, the donor cells form contact with the openings of a channel (e.g., a nanochannel). The dual-array cell electroporation system is then inverted to load the electroporation reagent wells with an electroporation buffer containing genetic cargo of interest (e.g., DNA plasmids, RNA, etc.), and then an array of cathode coils (…) is applied. Figure 13 Insert it into the electroporation reagent well to complete the circuit for high-throughput cell electroporation / nano-electroporation (CEP / CNP).

[0026] Figure 13 The configuration for use with Figures 15A-15D A photograph of a 5 x 5 cathode coil used in an automated cell electroporation pulse generation system.

[0027] Figure 14 This study depicts the quantification of qPCR results for COL1A1 mRNA production in extracellular vesicles (EVs) (as enriched genetic material) from the same number of donor cells (e.g., nHDF cells) via cellular nanoporation using either a patterned silicon perforated electroporation membrane (CNP) or a track-etched perforated electroporation membrane (TEP) with unpatterned channels. All data are presented as mean ± SD; CTR: control for non-electroporated cells (i.e., cells not subjected to electrical stimulation).

[0028] Figure 15A An automated cell electroporation pulse generation system is described, configured to generate sequential electrical pulses for high-throughput single-array and dual-array electroporation devices with multiple cell culture wells. The pulse generation system includes an electrical pulse generator with adjustable voltage capability, an impulse pulse converter, and an impulse chamber. Figure 15B Depicting Figure 15A The diagram shows an electrical pulse generator for an automated cell electroporation electrical pulse generation system according to some embodiments of the invention described herein. Figure 15C Depicting Figure 15A The image shows a photograph of the impulse pulse generator of an automated cell electroporation pulse generation system according to some embodiments of the invention described herein. The impulse pulse generator sequentially sends electrical outputs to each electrode via a 25-pin connector. Figure 15D Depicting Figure 15A The image shows a photograph of the impact chamber of an automated cell electroporation pulse generation system according to some embodiments of the invention described herein. The impact chamber is configured to initiate high-throughput cell electroporation (e.g., cell electroporation / nano-electroporation (CEP / CNP)) in individual cell culture wells. Detailed Implementation

[0029] Overview This article describes systems, devices, and instruments that provide high-yield and high-throughput delivery of biomolecules into cells, as well as their uses or methods. In some cases, the systems, devices, and instruments achieve intracellular delivery of nucleic acids or other biomolecules into a high number of cells. In some cases, the systems, devices, and instruments achieve non-endocytic delivery of nucleic acids or other biomolecules into a high number of cells. In some cases, the systems, devices, and instruments achieve multiplexed high-throughput electroporation for at least one cell type or at least two different cell types. In some cases, the systems, devices, and instruments achieve multiplexed high-throughput electroporation to deliver at least one nucleic acid or other biomolecule into at least one cell type or at least two different cell types. In some cases, the systems, devices, and instruments achieve multiplexed high-throughput electroporation to deliver at least two nucleic acids or other biomolecules into at least one cell type or at least two different cell types. In some cases, the systems, devices, and instruments achieve non-endocytic delivery of at least one nucleic acid or other biomolecule into at least one cell type or at least two different cell types. In some cases, the systems, devices, and instruments achieve non-endocytic delivery of at least two nucleic acids or other biomolecules into at least one cell type or at least two different cell types. In some cases, systems, devices, and instruments allow for rapid cell electroporation, high expression of at least one nucleic acid or other biomolecule, and rapid post-transfection cell collection. In many cases, the systems, devices, instruments, and methods described herein enable consistent and accurate delivery of a wide range of transfection reagents, from small oligodeoxynucleotides to large plasmid DNA and nanoparticles, to at least one or more cell types. In some cases, delivery of at least one or more nucleic acids or other biomolecules to at least one or more cell types involves targeting cells to the pores of a perforated membrane (e.g., nano- or micro-sized pores), thereby performing cell electroporation or nanoelectroporation (CEP / CNP) when an electric field is applied across the perforated membrane to deliver at least one or more transfection reagents or other biomolecules to the cells.

[0030] I. Perforated membrane In one aspect, the present disclosure provides a perforated membrane comprising: a first layer including a plurality of first channels disposed through the first layer; and a second layer in contact with the first layer including 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; the first channels of the plurality of first channels are in fluid communication with the second channels of the plurality of second channels; and a first average diameter of the first channels is different from a second average diameter of the second channels.

[0031] In one aspect, this disclosure provides a perforated membrane with distinctive features. Typically, a perforated membrane is a flat structure having a first surface and a second surface facing each other. The first and second surfaces are generally substantially parallel to each other, and the planes of the first and second surfaces 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. Typically, a perforated membrane includes multiple channels that allow substances to pass from the first surface of the perforated membrane to the second surface (i.e., through the perforated membrane). For example, the multiple channels may allow liquid substances (e.g., cell culture media, electroporation buffers) or biomolecules (e.g., nucleic acids, DNA, DNA plasmids, RNA, proteins, or small molecule drugs) to pass from the first surface of the perforated membrane to the second surface (i.e., through the perforated membrane). Individual channels of the multiple channels may include cylindrical, cubic, or any other three-dimensional shape that allows substances, voltage, or current to pass from the first surface of the perforated membrane to the second surface.

[0032] In some embodiments, the perforated membrane is configured to withstand a voltage of at least about 1 millivolt (mV). In some cases, the perforated membrane is configured to withstand a voltage of no more than about 500 volts (V). The perforated membrane may also be configured to withstand a voltage from about 1 mV to about 300 V or from about 1 V to about 200 V. In some cases, the voltage applied to the perforated membrane channels allows the voltage to pass 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.

[0033] In some embodiments, the perforated membrane is configured to carry a current of at least about 0.001 amperes (Amp). In some cases, the perforated membrane is configured to carry a current of no more than about 20 amperes (Amp). The perforated membrane may also be configured to carry a current from about 0.01 Amp to about 15 Amp or from about 0.01 Amp to about 10 Amp. In some cases, the channels of the perforated membrane enable current to pass through the perforated membrane or to apply current to the device. In some cases, the channels can carry a current from at least about 0.01 amperes (Amp) to about 10 Amp. In some cases, the current is from at least about 0.01 amperes (Amp) to about 0.1 Amp. In some cases, the current is from at least about 0.01 amperes (Amp) to about 0.5 Amp. In some cases, the channels can carry a current from at least about 0.01 amperes (Amp) to about 1 Amp. In some cases, the current is from at least about 0.01 amperes (Amp) to about 2 Amp. In some cases, the current is at least about 0.01 amps (Amp) to about 3 Amp. In some cases, the current is at least about 0.01 amps (Amp) to about 4 Amp. In some cases, the channel can carry a current of at least about 0.01 amps (Amp) to about 5 Amp. In some cases, the current is at least about 0.01 amps (Amp) to about 6 Amp. In some cases, the channel can carry a current of at least about 0.01 amps (Amp) to about 7 Amp. In some cases, the current is at least about 0.01 amps (Amp) to about 8 Amp. In some cases, the current is at least about 0.01 amps (Amp) to about 9 Amp. In some cases, the current is at least about 0.01 amps (Amp) to about 10 Amp. In some cases, the channel can carry currents 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.

[0034] Accordingly, the individual channels of the multiple channels include diameters. In some cases, the channels have diameters 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, 1,500 nm, 1,600 nm, 1,700 nm, 1,800 nm, 1,900 nm, and 2,000 nm. In some cases, the channels have diameters of at least about 100 nanometers (nm) to about 2,000 nm. In some cases, the channels have diameters of at least about 200 nanometers (nm) to about 2,000 nm. In some cases, the channels have diameters of at least about 200 nanometers (nm) to about 1,000 nm. In some cases, the channels have diameters of at least about 200 nanometers (nm) to about 500 nm. In some cases, the channel has a diameter of at least about 200 nanometers (nm) to about 1,500 nm. In some cases, the channel has a diameter of at least about 1,000 nanometers (nm) to about 2,000 nm. In some cases, the channel has a variable diameter throughout the perforated membrane. In some cases, the channel has a uniform diameter throughout the perforated membrane. In some cases, the channel is a nanochannel comprising a diameter from about 100 nm to about 2,000 nm. In some cases, the channel comprises a diameter of no more than about 20 µm, no more than about 15 µm, about 10 µm, no more than about 8 µm, no more than about 5 µm, no more than about 4 µm, no more than about 3 µm, more than about 2 µm, or no more than about 1.5 µm.

[0035] In some cases, the channels comprise diameters from about 0.3 µm to about 50 µm, from about 0.3 µm to about 20 µm, from about 0.4 µm to about 10 µm, from about 0.4 µm to about 5 µm, from about 0.4 µm to about 2 µm, or preferably from about 0.4 µm to about 1.5 µm. In some cases, the channels are microchannels comprising diameters from about 0.8 µm to about 10 µm, from about 0.8 µm to about 5 µm, or from about 1 µm to about 5 µm. In some cases, the individual channels of the multiple channels comprise diameters that are substantially constant over the entire length of the individual channel of the multiple channels, spanning the distance between the first and second surfaces of the perforated membrane. It is also conceivable that the individual channels of the multiple channels can have complex three-dimensional shapes. In some cases, the individual channels of the multiple channels comprise multiple portions with different diameters, such as a first portion having a diameter different from that of a second portion.

[0036] In some cases, the multiple channels of the perforated membrane comprise a non-patterned arrangement or non-patterned distribution of channels penetrating the perforated membrane. In some cases, the multiple channels of the perforated membrane comprise a patterned distribution of channels penetrating the perforated membrane. In some cases, the multiple channels of the perforated membrane comprise a patterned distribution of not more than one type of channel penetrating 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 has a second distance between channels along the length and / or width of the perforated membrane, wherein the second distance between channels in the second cross-sectional portion of the perforated membrane is different from the first distance between channels in the first cross-sectional portion of the perforated membrane.

[0037] Multiple channels can be distributed throughout the perforated membrane at a uniform or non-uniform density (i.e., the number of individual channels per unit surface area of ​​the perforated membrane). In some cases, the multiple channels of the perforated membrane comprise at least about 1 × 10⁻⁶ channels. 5 Channels / cm 2 The channel density. In some cases, the multiple channels of the perforated membrane comprise up to approximately 1 × 10⁻⁶. 8 Channels / cm 2 The channel density. In some cases, the multiple channels of the perforated membrane include approximately 1 × 10⁻⁶. 5 Channels / cm 2 From approximately 1 × 10 8 Channels / cm 2 The channel density. In some cases, the multiple channels of the perforated membrane include approximately 5 × 10⁻⁶. 5 Channels / cm 2 To approximately 5 × 10 6 Channels / cm 2 Channel density.

[0038] The perforated membrane has a thickness (i.e., height or distance along the z-axis). In some cases, the thickness of the perforated membrane is at least about 10 µm and is easily handled using tweezers during cell electroporation (e.g., cell electroporation / nano-electroporation (CEP / CNP)) operations. In some cases, the perforated membrane has a thickness of at least about 5 micrometers (µm), at least about 10 µm, at least about 20 µm, at least about 30 µm, at least about 40 µm, or at least about 50 µm. In some cases, the thickness of the perforated membrane ranges from about 5 µm to about 50 µm. In some cases, the thickness of the perforated membrane ranges from about 5 µm to about 40 µm. In some cases, the thickness of the perforated membrane ranges from about 5 µm to about 30 µm. In some cases, the thickness of the perforated membrane ranges from about 5 µm to about 20 µm. In some cases, the thickness of the perforated membrane ranges from about 5 µm to about 10 µm.

[0039] The perforated membrane has a dimensional dimension defined by the width and length (i.e., the distance along the x-axis and y-axis of the first surface) of a 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 no more than about 100 cm. In some cases, the first surface of the perforated membrane has a length 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 no more than about 100 cm. In some cases, the first surface of the perforated membrane has a width 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.

[0040] In some cases, the distances along the x and y axes of the second surface of the perforated membrane are substantially equal to the distances along the x and y axes of the first surface.

[0041] In some cases, the perforated membrane comprises a single layer, such that the perforated membrane is made of a single material or a single chemical component. In some cases, the perforated membrane comprises a single layer, such that the perforated membrane is made of a single material or a single chemical component of uniform concentration. In some cases, 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 different cross-sectional portion along the z-axis (i.e., height) of the perforated membrane, having characteristics different from adjacent layers or a second layer along the z-axis of the perforated membrane. The different characteristics of the first layer and the second layer of the perforated membrane may include, for example, different channel diameters, different thicknesses (i.e., height or distance along the z-axis), or different materials or chemical components.

[0042] The perforated membrane described herein may comprise a polymer material, a silicon material, or an alumina material. More preferably, the perforated membrane comprises a polymer material. In some cases, the perforated membrane comprises polycarbonate, polyester, or polyimide.

[0043] Therefore, the perforated membrane is configured to electroporate donor cells with transfection reagents, including DNA, RNA, nucleic acids, synthetic nucleic acid molecules, other biomolecules, or small molecules.

[0044] First layer In some cases, the perforated membrane comprises a first layer, wherein the first layer contains a plurality of first channels arranged through the first layer.

[0045] 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 µm. In some cases, the first average thickness of the first layer of the perforated membrane is from about 1 µm to about 5000 µm. In some cases, the first average thickness of the first layer of the perforated membrane is from about 1 µm to about 1000 µm. In some cases, the first average thickness of the first layer of the perforated membrane is from about 1 µm to about 200 µm. In some cases, the first average thickness of the first layer of the perforated membrane is from about 1 µm to about 100 µm, 1 µm to about 50 µm, 1 µm to about 40 µm, 1 µm to about 30 µm, 1 µm to about 20 µm, 1 µm to about 10 µm, or 1 µm to about 5 µm. In some cases, the first average thickness of the first layer of the perforated membrane is about 20 µm, about 15 µm, about 12 µm, about 10 µm, about 8 µm, about 6 µm, about 5 µm, about 4 µm, about 3 µm, about 2 µm, or about 1 µm. In some cases, the first average thickness of the first layer of the perforated membrane is at most about 5,000 µm, about 1,000 µm, about 500 µm, about 200 µm, about 100 µm, about 50 µm, about 20 µm, about 10 µm, or about 5 µm.

[0046] In some cases, the plurality of first channels comprises individual channels having a first average diameter. In some cases, the plurality of first channels comprises individual channels having a first average diameter that is substantially consistent among the plurality of individual channels throughout the first layer of the perforated membrane. In some cases, the first average diameter is at least about 1 µm. In some cases, the first average diameter is no more than about 20 µm. In some cases, the first average diameter ranges from about 1 µm to about 20 µm, from about 3 µm to about 10 µm, from about 4 µm to about 8 µm, or from about 5 µm to about 7 µm.

[0047] The first channel also has an aspect ratio, which is defined as the ratio of the first average diameter to the first average thickness of the first channel. In some cases, the first average diameter is about half the first average thickness. In other cases, the first average diameter is about one-third the first average thickness of the first channel.

[0048] In some cases, the plurality of first channels arranged through the first layer comprise a first array of channels. In some cases, the first array of channels comprises individual channels having a first average diameter. In some cases, the first array of channels comprises individual channels 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.

[0049] The first layer of the perforated film is made of a material or chemical composition. In some cases, the first layer of the perforated film includes a material that provides a rigid structure to the first layer. Alternatively, in some cases, the first layer of the perforated film includes a material that provides a flexible structure to the first layer. In some cases, the first layer of the perforated film includes a first polymer material, a silicon material, an alumina material, a glass, a ceramic material, gold, copper, or other biomaterial. In some cases, the first layer includes a first polymer material. In some cases, the first layer includes a first thermoplastic material, a first photoreactive polymer, a first crosslinked polymer, a first synthetic polymer, a first thermosetting polymer, a first photocurable polymer, a first thermosetting polymer, or a first photoresist. The first layer preferably includes a first photoresist. In some cases, the first photoresist includes a positive photoresist. In some cases, the first photoresist includes a negative photoresist. In some cases, the first photoresist includes a first SU-8 photoresist, such as the first SU-83000 series photoresist. In some cases, the first layer includes SU-8 3010 photoresist or SU-8 3005 photoresist. In some cases, the first layer comprises a first SU-8 2000 series photoresist, such as SU-8 2005 photoresist. It is conceivable that the first layer of the perforated film may comprise any positive photoresist, or a negative photoresist may be suitably used to provide a first layer having multiple channels, thickness, length, and width.

[0050] Second floor In some cases, the perforated membrane includes a second layer, which contacts the first layer of the perforated membrane and includes a plurality of second channels arranged through the second layer.

[0051] The second layer of the perforated membrane has a second average thickness (i.e., height or distance along the z-axis). In some cases, the second average thickness of the second layer differs 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 greater than the second average thickness of the second layer. For example, the first average thickness of the first layer is at least two or at least three times greater than the second average thickness of the second layer. In some cases, the second average thickness of the second layer is at least about 100 nm. In some cases, the second average thickness of the second layer is no more than about 10 µm. In some cases, the second average thickness of the second layer is from about 300 nm to about 7 µm, from about 500 nm to about 5 µm, from about 600 nm to about 3 µm, from about 700 nm to about 2 µm, or from about 700 nm to about 1.5 µm. In some cases, the second average thickness of the second layer of the perforated membrane is about 700 nm, 800 nm, 1 µm, about 2 µm, about 3 µm, or about 4 µm.

[0052] In some cases, the plurality of second channels comprises individual channels having a second average diameter. In some cases, the plurality of second channels comprises individual channels 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 cases, the second average diameter of the second layer is at least about 100 nm. In some cases, the second average diameter of the second layer is no more than about 5 µm. In some cases, the second average diameter of the second layer ranges from about 100 nm to about 5 µm. In some cases, the second average diameter of the second layer ranges from about 300 nm to about 1.5 µm, from about 400 nm to about 1.2 µm, or preferably from about 600 nm to about 1 µm.

[0053] The second channel also has an aspect ratio, defined as the ratio of the second average diameter to the second average thickness of the second channel. In some cases, the second average diameter is no more than half the second average thickness of the second layer of the perforated membrane. In some cases, 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-quarter of the second average thickness. In some cases, the second average diameter is no more than half the second average thickness or no more than about 25% of the second average thickness.

[0054] In some cases, the plurality of second channels arranged through the second layer comprise a second array of channels. In some cases, the second array of channels comprises individual channels having a second average diameter. In some cases, the second array of channels comprises individual channels 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.

[0055] In some cases, the first average diameter of the first channel in the first layer of the perforated membrane differs from the second average diameter of the second channel in the second layer of the perforated membrane. In some cases, the first average diameter of the first channel in the first layer is larger than the second average diameter of the second channel in the second layer. For example, the first average diameter may be at least two, three, or four times larger than the second average diameter of the second channel in the second layer. Variations in the diameters of the first channel in the first layer and the second channel in the second layer are also possible. For example, the first average diameter of the first channel in the first layer may be approximately two to approximately three times, or approximately three to approximately four times, larger than the second average diameter of the second channel in the second layer of the perforated membrane.

[0056] The second layer of the perforated film is made of a material or chemical composition. In some cases, the second layer of the perforated film includes a material that provides a rigid structure to the second layer. Alternatively, in some cases, the second layer of the perforated film includes a material that provides a flexible structure to the second layer. In some cases, the second layer of the perforated film includes a second polymer material, a silicon material, an alumina material, a glass, a ceramic material, gold, copper, or other biomaterials. In some cases, the second layer includes a second polymer material. In some cases, the second layer includes a second thermoplastic material, a second photoreactive polymer, a second crosslinked polymer, a second synthetic polymer, a second thermosetting polymer, a second photocurable polymer, a second thermosetting polymer, or a second photoresist. The second layer preferably includes a second photoresist. In some cases, the second photoresist includes a positive photoresist. In some cases, the second photoresist includes a negative photoresist. In some cases, the second polymer material includes SU-8 TF 6000 series photoresists, such as SU-8 TF 6002 photoresist. In some cases, the second polymer material includes a second SU-8 2000 series photoresist, including but not limited to SU-8 2002 photoresist.

[0057] The difference between the first and second layers Some embodiments of the perforated membrane include a first layer of perforated membrane that is different from the second layer. For example, in some cases, the first layer contains at least one component that is different from the composition of the second layer material. In some cases, the first layer is made of a material different from the material of the second layer. Such differences give the first layer and the second layer different structural properties. For example, in some cases, the first tensile strength of the first layer is greater than the second tensile strength of the second layer.

[0058] For example, in some cases, the first layer includes a first photoresist, and the second layer includes a second photoresist. More specifically, the first layer includes a first negative photoresist, and the second layer includes a second negative photoresist. For example, the first layer includes a first SU-8 photoresist, and the second layer includes a second SU-8 photoresist. Various combinations of the first SU-8 photoresist of the first layer and the second SU-8 photoresist of the second layer are compatible with embodiments of the perforated film according to this disclosure. In some cases, the first layer includes SU-8 3010 photoresist, SU-8 3005 photoresist, or SU-8 2005 photoresist, and the second layer includes SU-8 TF 6002 photoresist or SU-8 2002 photoresist. It is also conceivable that the first layer includes a first positive photoresist, and the second layer includes a second positive photoresist.

[0059] Similarity between the first and second layers Alternatively, according to some embodiments, the first and second layers of the perforated film have similar characteristics. In some cases, the first and second layers are made of a photoresist polymer. In some cases, the first layer is made of the same material as the second layer. For example, in some embodiments, the first and second layers comprise a first photoreactive polymer, a first crosslinked polymer, a first synthetic polymer, or a first photocurable polymer.

[0060] Typically, the first channels of a plurality of first channels are in fluid communication with the second channels of a plurality of second channels. In the case where the first plurality of channels of the first layer and the second plurality of second layers form an adjoining channel, the adjoining channel spans the distance (i.e., thickness) between the first and second surfaces of the perforated membrane.

[0061] II. Methods for manufacturing perforated membranes This document also describes methods for fabricating the perforated membrane of this disclosure, which includes 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 includes coating a first material onto a substrate. In some cases, spin coating, blade coating, push coating, drop casting, or spray coating methods may be used to coat the first layer of the first material onto the substrate. In some cases, the method of fabricating the perforated membrane includes coating the first material onto the 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 may be used to coat the second layer of the second material onto the substrate.

[0062] Another aspect of this disclosure provides a method for fabricating a perforated film using photoresist materials, including a perforated film having a first layer and a perforated film having a first layer and a second layer, as previously described. According to some embodiments, the method described herein for fabricating a perforated film using photoresist materials includes coating a first layer of a first photoresist material onto a substrate; and coating a second layer of a second photoresist material onto the substrate. In some cases, spin coating, blade coating, push coating, drop casting, or spray coating methods can be used to coat 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 to coat the second layer of the second material onto the substrate. In some cases, the first layer has the characteristics of a first layer of a perforated film as described above.

[0063] The first material can be any of the aforementioned materials used to fabricate the first layer of the perforated membrane (see also Section I: Perforated Membranes, which describes the second layer of the perforated membrane). In some cases, the first material used to fabricate the first layer of the perforated membrane includes a material that provides a rigid structure to the first layer of the perforated membrane. Alternatively, in some cases, the first layer of the perforated membrane includes a material that provides a flexible structure to the first layer. In some cases, the first material used to fabricate the first layer of the perforated membrane includes a first polymer material, silicon material, alumina material, glass, ceramic material, gold, copper, track-etched film, or other biomaterial. In some cases, the first material includes a first polymer material. In some cases, the first material includes a first thermoplastic material, a first photoreactive polymer, a first crosslinked polymer, a first synthetic polymer, a first thermosetting polymer, a first photocurable polymer, a first thermosetting polymer, or a first photoresist. The first material preferably includes a first photoresist. In some cases, the first photoresist includes a first positive photoresist. In some cases, the first material includes a negative photoresist. In some cases, the first polymer material includes SU-8 TF 6000 series photoresist, such as SU-8 TF 6002 photoresist. In some cases, the first polymer material includes a first SU-8 2000 series photoresist, including but not limited to SU-8 2002 photoresist. In some cases, the first polymer material includes a negative photoresist, including a first SU-8 TF 6000 series photoresist (e.g., SU-8 TF 6002 photoresist, etc.) or a first SU-8 2000 series photoresist (e.g., SU-8 2002 photoresist).

[0064] The second material can be any of the aforementioned materials used to fabricate the second layer of the perforated membrane (see also Section I: Perforated Membranes, which describes the first layer of the perforated membrane). In some cases, the second material used to fabricate the second layer of the perforated membrane includes a material that imparts a rigid structure to the second layer. Alternatively, in some cases, the second layer of the perforated membrane includes a material that imparts a flexible structure to the second layer. In some cases, the second material used to fabricate the second layer of the perforated membrane includes a second polymer material, a track-etched film, a silicon material, an alumina material, a glass, a ceramic material, gold, copper, or other biomaterials. In some cases, the second material includes a second polymer material. In some cases, the second material includes a second thermoplastic material, a second photoreactive polymer, a second crosslinked polymer, a second synthetic polymer, a second thermosetting polymer, a second photocurable polymer, a second thermosetting polymer, or a second photoresist. The second material preferably includes a second photoresist. In some cases, the second photoresist includes a second positive photoresist. In some cases, the second material includes a negative photoresist. In some cases, the second material includes a second SU-8 photoresist, such as a second SU-8 3000 series photoresist. In some cases, the second material includes SU-8 3010 photoresist or SU-8 3005 photoresist. In some cases, the second material includes a second SU-8 2000 series photoresist, such as SU-8 2005 photoresist. In some cases, the second negative photoresist includes a second SU-8 3000 series photoresist (e.g., SU-8 3010 photoresist, SU-8 3005 photoresist, etc.) or a second SU-8 2000 series photoresist (e.g., SU-8 2005 photoresist, etc.).

[0065] 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 and the second photoresist material are different. For example, in some cases, the first photoresist material has a higher resolution than the second photoresist material.

[0066] In some cases, coating a first layer of the first photoresist material onto the substrate includes (a) spin-coating the first layer of the first photoresist material onto the substrate. For example, the substrate is a silicon film, preferably a silicon film coated with a release layer that allows the first photoresist material to separate from the substrate. It is conceivable that any substrate with a rigid structure can be used. The first photoresist material may include any of the aforementioned photoresist materials used for the first layer of a perforated film (see also Section I: Perforated Films, which describes the second layer of a perforated film).

[0067] In some cases, the release layer includes a photoresist release layer. In other cases, the release layer includes 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 a polymer (including polystyrene, PDMS (polydimethylsiloxane), SAM (self-assembled molecules), OmniCoat, PMGI (polydimethylglutarimide)).

[0068] In some cases, spin coating in (a) applies a first layer onto the release layer. In some cases, spin coating involves spin coating a first photoresist material at a rotation speed from about 1,000 RPM to about 10,000 RPM. In some cases, spin coating involves spin coating a first photoresist material at a rotation speed of about 4,000 RPM.

[0069] In some cases, the method further includes: (b) soft baking the first layer obtained in (a), whereby (a) is a step of spin-coating the first layer of the first photoresist material onto the substrate. In some cases, the first layer obtained in (a) comprises a layer of perforated film having a plurality of channels, whereby the channels have an average diameter from about 300 nm to about 1.5 µm, from about 400 nm to about 1.2 µm, or preferably from about 600 nm to about 1 µm (e.g., Figure 3 (The smaller diameter channel portion). In some cases, softening includes softening 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, softening includes softening 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, softening includes softening the first layer for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, or about 5 minutes. In some cases, softening includes softening the first layer for about 3 minutes.

[0070] In some cases, softening includes softening the first layer at a temperature of at least about 90°C. In some cases, softening includes softening the first layer at a temperature of no more than about 200°C. In some cases, softening includes softening the first layer at a temperature from about 90°C to about 140°C or from about 100°C to about 120°C. Preferably, softening includes softening the first layer at a temperature of about 110°C.

[0071] In some cases, the method further includes: (c) exposing the softened first layer obtained in (b) to first UV radiation using a first photomask.

[0072] In some cases, the first photomask includes fused silica, chromium, borosilicate, glass, or polyethylene terephthalate (PET).

[0073] First optical mask The first photomask includes a width and a length (i.e., the distance 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 no more than about 100 cm. In some cases, the first photomask has a length 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 no more than about 100 cm. In some cases, the first photomask has a width 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.

[0074] A first photomask includes a first pattern. In some cases, the first pattern of the first photomask includes a plurality of first holes. The first holes may include, for example, a circle, a square, a rectangle, a triangle, or any geometric shape. Thus, each individual hole of the plurality of first holes includes a diameter. In some cases, the plurality of first holes includes individual holes having a first average diameter. In some cases, the plurality of first holes includes individual holes 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 cases, the first average diameter of the first photomask is at least about 100 nm. In some cases, the first diameter of the first photomask is no more than about 5 µm. In some cases, the first average diameter of the first photomask is from about 100 nm to about 5 µm. In some cases, the first average diameter of the first photomask is from about 300 nm to about 1.5 µm, from about 400 nm to about 1.2 µm, or preferably from about 600 nm to about 1 µm.

[0075] In some cases, the individual first aperture of the first photomask has a diameter substantially equivalent to that of the other individual first apertures of the first photomask.

[0076] 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 penetrating the first photomask. In some cases, the plurality of first holes of the first photomask comprises a patterned distribution of the first holes penetrating the first photomask. In some cases, the plurality of first holes of the first photomask comprises a patterned distribution of no more than one type of first hole penetrating the first photomask, such that a first cross-sectional portion of the first photomask has a first distance between the first holes along the length and / or width of the first photomask, and a second cross-sectional portion of the first photomask has a second distance between the first holes along the length and / or width of the first photomask, wherein the second distance between the first holes in the second cross-sectional portion of the first photomask is different from the first distance between the first holes in the first cross-sectional portion of the first photomask.

[0077] Multiple first apertures can be distributed at a uniform or non-uniform density (i.e., the number of individual first apertures per unit surface area of ​​the first photomask) throughout the first photomask. In some cases, the multiple first apertures of the first photomask comprise at least about 1 × 10⁻⁶. 5 First hole / cm 2 The aperture density. In some cases, the multiple first apertures of the first photomask comprise at most about 1 × 10⁻⁶. 8 First hole / cm 2 The aperture density. In some cases, the multiple first apertures of the first photomask include those ranging from approximately 1 × 10⁻⁶. 5 First hole / cm 2 From approximately 1 × 10 8 First hole / cm 2 The aperture density. In some cases, the multiple first apertures of the first photomask include those ranging from approximately 5 × 10⁻⁶. 5 First hole / cm 2 To approximately 5 × 10 6 First hole / cm 2 pore density.

[0078] In some cases, the method of fabricating a perforated film further includes: (d) after (c), spin-coating a second layer of a second photoresist material onto the first layer. The second photoresist material may include any of the aforementioned photoresist materials used for the second layer of the perforated film (see also Section I: Perforated Film, which describes the first layer of the perforated film).

[0079] In some cases, spin coating in (d) coats the second layer onto the first layer. In some cases, spin coating in (d) involves spin coating the second photoresist material at a rotation speed from about 1,000 RPM to about 10,000 RPM. In some cases, spin coating involves spin coating the second photoresist material at a rotation speed of about 4,000 RPM.

[0080] In some cases, the method of producing a perforated membrane further includes performing another exposure followed by baking after (c) but before (d).

[0081] In some cases, performing a second post-exposure bake includes 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 minute to about 4 minutes. In some cases, the second post-exposure bake includes baking the first layer for no more than 60 minutes, no more than 20 minutes, no more than 10 minutes, or no more than 5 minutes. In some cases, the post-exposure bake includes 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 bake includes baking the first layer for about 3 minutes.

[0082] In some cases, post-exposure baking includes exposing the first layer to a temperature of at least about 90°C and then baking it. In some cases, post-exposure baking includes exposing the first layer to a temperature of no more than about 200°C and then baking it. In some cases, post-exposure baking includes exposing the first layer to a temperature from about 90°C to about 140°C or from about 100°C to about 120°C and then baking it. Preferably, post-exposure baking includes exposing the first layer to a temperature of about 110°C and then baking it.

[0083] In some cases, the method of fabricating a perforated membrane further includes: applying a barrier layer on top of the first layer after (c) but before (d). The barrier layer may, for example, include silicon oxide, gold, silver, or any other barrier layer known in the art.

[0084] In some cases, the method of making a perforated membrane further includes: (e) softening the second layer obtained in (d).

[0085] In some cases, the softening in (e) includes softening 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 softening in (e) includes softening the second layer for no more than 60 minutes, no more than 20 minutes, no more than 10 minutes, or no more than 8 minutes. Preferably, the softening in (e) includes softening the second layer for about 7 minutes.

[0086] In some cases, the softening in (e) includes softening the first layer at a temperature of at least about 70°C. In some cases, the softening in (e) includes softening the first layer at a temperature of no more than about 200°C. In some cases, the softening in (e) includes softening the first layer at a temperature from about 85°C to about 105°C. Preferably, the softening in (e) includes softening the first layer at a temperature of about 95°C.

[0087] In some cases, the method of fabricating a perforated membrane further includes: (f) exposing the softened second layer obtained in (e) to another UV radiation using a second photomask.

[0088] Second light mask The second photomask includes a width and a length (i.e., the distance 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 no more than about 100 cm. In some cases, the second photomask has a length 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 no more than about 100 cm. In some cases, the second photomask has a width 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 photomask includes a width and length substantially the same as the second photomask's width and length.

[0089] The second photomask includes a second pattern. In some cases, the second pattern of the second photomask includes a plurality of second holes. Therefore, each individual hole of the plurality of second holes includes a diameter. In some cases, the plurality of second holes includes individual holes having a second average diameter. In some cases, the plurality of second holes includes individual holes 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 may, for example, include circles, squares, rectangles, triangles, or any geometric shape. Preferably, the second holes of the second photomask have a shape substantially similar to the first holes of the first photomask.

[0090] In some cases, the plurality of second pores comprises individual pores having a second average diameter. In some cases, the second average diameter is substantially consistent among the plurality of individual pores in the second layer of the perforated membrane. In some cases, the second average diameter is at least about 1 µm. In some cases, the second average diameter is no more than about 20 µm. In some cases, the second average diameter ranges from about 1 µm to about 20 µm, from about 3 µm to about 10 µm, from about 4 µm to about 8 µm, or from about 5 µm to about 7 µm.

[0091] In some cases, the plurality of second holes in the second photomask comprises a non-patterned arrangement or non-patterned distribution of second holes penetrating the second photomask. In some cases, the plurality of second holes in the second photomask comprises a patterned distribution of second holes penetrating the second photomask. In some cases, the plurality of second holes in the second photomask comprises a patterned distribution of no more than one type of second hole penetrating the second photomask, whereby a second cross-sectional portion of the second photomask has a second distance between the second holes along the length and / or width of the second photomask, and the second cross-sectional portion of the second photomask has a second distance between the second holes along the length and / or width of the second photomask, wherein the second distance between the second holes in the second cross-sectional portion of the second photomask is different from the second distance between the second holes in the second cross-sectional portion of the second photomask. Preferably, the individual second holes of the plurality of second holes comprise a diameter substantially equivalent to the other individual second holes of the second photomask.

[0092] Multiple second apertures can be distributed at a uniform or non-uniform density (i.e., the number of individual second apertures per unit surface area of ​​the second photomask) throughout the second photomask. In some cases, the multiple second apertures of the second photomask comprise at least about 1 × 10⁻⁶. 5 Second hole / cm 2 The aperture density. In some cases, the multiple second apertures of the second photomask comprise up to approximately 1 × 10⁻⁶. 8 Second hole / cm 2 The aperture density. In some cases, the multiple second apertures of the second photomask include those ranging from approximately 1 × 10⁻⁶. 5 Second hole / cm 2 From approximately 1 × 10 8 Second hole / cm 2 The aperture density. In some cases, the multiple second apertures of the second photomask include those ranging from approximately 5 × 10⁻⁶. 5 Second hole / cm 2 To approximately 5 × 10 6 Second hole / cm 2 The aperture density. Preferably, the distribution and density of the individual second apertures of the second photomask are substantially equivalent to the distribution and density of the individual first apertures of the first photomask.

[0093] In some cases, the method of fabricating the perforated film further includes: (g) performing a post-exposure baking of the second layer. In some cases, performing the post-exposure baking in (g) includes 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 minute to about 3 minutes. In some cases, the post-exposure baking includes baking the second layer for no more than 60 minutes, no more than 20 minutes, no more than 10 minutes, or no more than 5 minutes. In some cases, the post-exposure baking includes 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 includes baking the second layer for about 3 minutes.

[0094] In some cases, post-exposure baking includes exposing the second layer to a temperature of at least about 50°C and then baking it. In some cases, the post-exposure baking in (g) includes exposing the second layer to a temperature of no more than about 200°C and then baking it. In some cases, the post-exposure baking in (g) includes a first step of post-exposure baking the second layer, which includes post-exposure baking for about 0.5 minutes to about 2 minutes at a temperature from about 50°C to about 90°C or from about 60°C to about 80°C; and a second step of post-exposure baking the second layer, which includes post-exposure baking for about 1 minute to about 5 minutes at a temperature from about 70°C to about 100°C or from about 85°C to about 105°C, or preferably post-exposure baking for about 2 minutes at about 95°C.

[0095] In some cases, the method for fabricating the perforated film further includes (h) developing both the first and second layers after (g). In some cases, development includes simultaneously developing the first and second photoresist materials. The developer solution may contain alkaline substances, promoters, or inhibitors.

[0096] In some cases, the first pattern of the first photomask is configured to generate a plurality of first channels arranged in the first layer after (h), and the second pattern of the first photomask is configured to generate a plurality of second channels arranged in the second layer after (h).

[0097] In some cases, the method of making a perforated membrane further includes removing the release layer after (h).

[0098] In some cases, the method of fabricating a perforated membrane results in a perforated membrane comprising a top layer having a plurality of top channels arranged through the top layer; and a bottom layer in contact with the top layer having a plurality of bottom channels arranged 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 with a second channel of the plurality of bottom channels; and a first average diameter of the first channel is different from a second average diameter of the second channel.

[0099] In some cases, the method of fabricating a perforated film from a photoresist material further includes: (a) spin-coating a first layer of a first photoresist material onto a substrate; (b) softening the first layer obtained in (a); (c) exposing the softened first layer obtained in (b) to a first UV radiation using a first photomask; (d) after (c), spin-coating a second layer of a second photoresist material onto the first layer; (e) softening the second layer obtained in (d); (f) exposing the softened second layer obtained in (e) to another UV radiation using a second photomask; (g) performing post-exposure baking; and (h) after (g), developing both the first and second layers to fabricate a perforated film comprising two layers.

[0100] Polymer film imprinted by a sacrificial template This disclosure further provides a method for producing a perforated membrane from a sacrificial template. The method for producing a perforated membrane from a sacrificial template includes: (a) providing a female mold comprising a recessed 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.

[0101] The method can produce perforated membranes (such as polymer membranes) with channels passing through the perforated membrane arrangement and of average thickness.

[0102] A female mold containing a recessed pattern has the features required for a perforated film, such as those described above for perforated films. In some cases, the pattern of the female mold is substantially the same as the pattern of the perforated film.

[0103] Typically, a female mold is a flat structure having a first surface for contacting the sacrificial male mold material and a second surface opposite the first surface. The first and second surfaces are generally substantially parallel to each other, and the planes of the first and second surfaces 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. Typically, the female mold contains multiple channels that can generate a desired perforated membrane, allowing material to pass from the first surface of the perforated membrane to the second surface (i.e., through the perforated membrane). The individual channels of the female mold can include cylindrical, cubic, or any other three-dimensional shape that can generate a perforated membrane with channels, allowing material, voltage, or current to pass from the first surface of the perforated membrane to the second surface.

[0104] Therefore, individual channels of multiple channels include diameters. In some cases, the channels have diameters 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, 1,500 nm, 1,600 nm, 1,700 nm, 1,800 nm, 1,900 nm, and 2,000 nm. In some cases, the channels have diameters of at least about 100 nanometers (nm) to about 2,000 nm. In some cases, the channels have diameters of at least about 200 nanometers (nm) to about 2,000 nm. In some cases, the channels have diameters of at least about 200 nanometers (nm) to about 1,000 nm. In some cases, the channels have diameters of at least about 200 nanometers (nm) to about 500 nm. In some cases, the channel has a diameter of at least about 200 nanometers (nm) to about 1,500 nm. In some cases, the channel has a diameter of at least about 1,000 nanometers (nm) to about 2,000 nm. In some cases, the channel has a variable diameter throughout the negative mold. In some cases, the channel has a uniform diameter throughout the negative mold. In some cases, the channel is a nanochannel comprising a diameter from about 100 nm to about 2,000 nm. In some cases, the channel comprises a diameter of no more than about 20 µm, no more than about 15 µm, about 10 µm, no more than about 8 µm, no more than about 5 µm, no more than about 4 µm, no more than about 3 µm, more than about 2 µm, or no more than about 1.5 µm.

[0105] In some cases, the channel comprises a diameter from about 0.3 µm to about 50 µm, from about 0.3 µm to about 20 µm, from about 0.4 µm to about 10 µm, from about 0.4 µm to about 5 µm, from about 0.4 µm to about 2 µm, or preferably from about 0.4 µm to about 1.5 µm. In some cases, the channel is a microchannel comprising a diameter from about 0.8 µm to about 10 µm, from about 0.8 µm to about 5 µm, or from about 1 µm to about 5 µm. In some cases, the individual channels of the multiple channels comprise a diameter that is substantially constant over the entire length of the individual channel of the multiple channels. It is also conceivable that the individual channels of the multiple channels can have complex three-dimensional shapes. In some cases, the individual channels of the multiple channels comprise multiple portions with different diameters, such as a first portion having a diameter different from that of a second portion.

[0106] In some cases, the multiple channels of the female mold comprise a non-patterned arrangement or non-patterned distribution of channels penetrating the mold. In some cases, the multiple channels of the female mold comprise a patterned distribution of channels penetrating the mold. In some cases, the multiple channels of the female mold comprise a patterned distribution of not more than one type of channel penetrating the mold, such that 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 has a second distance between channels along the length and / or width of the female mold, wherein the second distance between channels in the second cross-sectional portion of the female mold is different from the first distance between channels in the first cross-sectional portion of the female mold.

[0107] Multiple channels can be distributed throughout the mold at a uniform or non-uniform density (i.e., the number of individual channels per unit surface area of ​​the mold). In some cases, the multiple channels of the mold comprise at least approximately 1 × 10⁻⁶ channels. 5 Channels / cm 2 The channel density. In some cases, the multiple channels of the female mold include up to approximately 1 × 10⁻⁶. 8 Channels / cm 2 The channel density. In some cases, the multiple channels of the negative mold include approximately 1 × 10⁻⁶. 5 Channels / cm 2 From approximately 1 × 10 8 Channels / cm 2 The channel density. In some cases, the multiple channels of the female mold include approximately 5 × 10⁻⁶. 5 Channels / cm 2 To approximately 5 × 10 6 Channels / cm 2 Channel density.

[0108] The female mold has dimensional dimensions defined by the width and length of the distance between its first surfaces (i.e., the distance 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 no more than about 100 cm. In some cases, the first surface of the female mold has a length 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 no more than about 100 cm. In some cases, the first surface of the female mold has a width 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.

[0109] In some cases, the distances along the x and y axes of the second surface of the female mold are substantially equal to the distances along the x and y axes of the first surface.

[0110] In some cases, the female mold comprises a single layer, such that the female mold is made of a single material or a single chemical component. In other cases, the female mold comprises a single layer, such that the female mold is made of a single material or a single chemical component of uniform concentration.

[0111] In some cases, preparing a sacrificial male mold based on the female mold in (b) involves casting a male mold material onto the female mold. In some cases, the male mold material includes a polymer. In some cases, the male mold material includes a water-soluble polymer. For example, the male mold material may include water-soluble polymers such as poly(vinyl alcohol) (PVA), cellulose, polyacrylamide (PAM), polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), carrageenan, chitosan derivatives, guar gum, or poly(acrylic acid). In some cases, the water-soluble polymer is polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or a polyvinylpyrrolidone / vinyl alcohol copolymer (PVP / VA copolymer). In some cases, the male mold material may include a water-soluble polymer such as poly(vinyl alcohol) (PVA).

[0112] Preparing a perforated membrane based on the sacrificial male mold in (c) involves applying a polymeric resin (e.g., a perforated membrane material) to the sacrificial male mold. In some cases, the polymeric resin is a thermosetting resin, such as an epoxy resin, polyester resin, polyimide resin, polyurethane resin, bisimide, or benzoxazine.

[0113] In some cases, the preparation of the perforated membrane based on the sacrificial male mold in (c) further includes curing a polymer resin. In some cases, the preparation of the perforated membrane based on the sacrificial male mold in (c) further includes thermosetting a polymer resin.

[0114] In some cases, the preparation of the perforated membrane based on the sacrificial anode in (c) further includes photocuring a polymeric resin. For example, photocuring the polymeric resin involves exposing the polymeric resin to ultraviolet (UV) light. In some cases, the polymeric resin is a photocurable resin. For example, the photocurable resin may be BAPR-α, BAPR-β, BAPR-γ, BAPR-δ, poly(ethylene glycol) diacrylate (PEGDA), poly(ethylene glycol) dimethacrylate (PEGDMA), or poly(propylene fumarate) / diethyl fumarate (PPF / DEF).

[0115] Preparing a perforated membrane based on the sacrificial mold in (c) may include imprinting. In some cases, imprinting includes contacting the perforated membrane material with the sacrificial mold. Optionally, imprinting may include placing a weight over the perforated membrane material while it is in contact with the sacrificial mold to form a perforated membrane having a clear array of channels through the perforated membrane arrangement. In some cases, imprinting includes providing a substrate and a weight.

[0116] Imprinting may further include applying a release layer to a flexible substrate. In some cases, the substrate may be a rubber layer. In some cases, imprinting includes coating the substrate (e.g., a rubber layer, etc.) with a release layer. The release layer may include a releaseable polymer. In some cases, the release layer includes silicone-based polymers, fluoropolymers (e.g., polytetrafluoroethylene (PTFE)), polyolefins, polymers with long alkyl chains, and poly(vinyl alcohol). In some cases, polyolefins include polyethylene (low-density polyethylene, high-density polyethylene, linear low-density polyethylene), polypropylene, and polybutene (polybutene-1 and polyisobutylene). In some cases, polymers with long alkyl chains may be polyethylene (PE), polypropylene (PP), poly(vinyl chloride) (PVC), poly(methyl methacrylate) (PMMA), polyisoprene (natural rubber), polybutadiene, or synthetic rubbers with long alkyl side chains. In some cases, the release polymer may also include water-soluble polymers such as poly(vinyl alcohol) (PVA), cellulose, polyacrylamide (PAM), polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), carrageenan, chitosan derivatives, guar gum, or poly(acrylic acid). In some cases, the water-soluble polymer is polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or polyvinylpyrrolidone / vinyl alcohol copolymer (PVP / VA copolymer).

[0117] In some cases, (c) preparing a perforated membrane based on a sacrificial male mold includes casting a perforated membrane material onto the sacrificial male mold. In some cases, the perforated membrane material includes a polymer, such as a polymer resin. In some cases, the polymer resin may include polydimethylsiloxane (PDMS) resin or an acrylic resin. In some cases, the perforated membrane material includes a silicone polymer, such as polycarbonate (PC), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), polyethylene (PE), or polydimethylsiloxane (PDMS).

[0118] In some cases, contacting the perforated membrane material with the sacrificial male mold may include spin-coating, scraping, pushing, or spraying the perforated membrane material onto the sacrificial male mold. In some cases, (c) preparing the perforated membrane based on the sacrificial male mold may include spin-coating from about 1,000 RPM to about 10,000 RPM for at least 1 minute or from about 1 minute to about 10 minutes.

[0119] (c) The preparation of a perforated membrane based on a sacrificial mold further includes: removing the sacrificial mold. Removing the sacrificial mold includes exposing a release layer on the cured polymer resin to water vapor at a release temperature of at least about 50°C, so that the release layer dissolves from the cured polymer resin, thereby forming a sacrificial template for the perforated membrane. 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.

[0120] The method may further include, before (a) providing a negative mold containing a recessed pattern, (d) preparing the negative mold. In some cases, (d) may include performing soft lithography. In some cases, the negative mold is complementary to the master mold. In some cases, the negative mold is made of a negative mold material, such as a polymer. In some cases, the negative mold material includes a silicone polymer, such as polycarbonate (PC), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), polyethylene (PE), or polydimethylsiloxane (PDMS).

[0121] The method may further include, prior to (d) fabricating the negative mold, (e) fabricating the master mold. In some cases, (e) includes fabricating the master mold, such as a silicon master mold. In some cases, (e) may include deep reactive ion etching (DRIE) of the master mold.

[0122] III. A high-throughput cell electroporation system with a double-layered perforated membrane and spacer array This document describes a system for high-throughput cell electroporation. In some cases, the system for high-throughput cell electroporation includes: (a) a perforated membrane, wherein the perforated membrane includes: (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 differs from a second average thickness of the second layer; the first channels of the plurality of first channels are in fluid communication with the second channels of the plurality of second channels; the first average diameter of the first channels differs from the second average diameter of the second channels; and (b) at least one donor cell is in contact with the perforated membrane.

[0123] In some cases, the perforated membrane includes any perforated membrane and its embodiments previously described in this disclosure. For example, a system may include a perforated membrane comprising a first layer and a second layer, according to the embodiments provided in Section I: Perforated Membranes of this disclosure.

[0124] 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 µm to about 200 µm.

[0125] The second average thickness of the second layer can be any of the previously described embodiments, such as those in Section I: Perforated Membranes. In some cases, the second average thickness is from about 100 nm to about 10 µm.

[0126] In some cases, the first average thickness of the first layer of the perforated membrane is greater than the second average thickness of the second layer of the perforated membrane. In some cases, the first average thickness is at least two or at least three times the second average thickness.

[0127] 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 ranges from about 1 µm to about 20 µm.

[0128] The first channel of the first layer has an aspect ratio, wherein the first average diameter is about half or about one-third of the first average thickness of the first layer.

[0129] 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 ranges from about 100 nm to about 5 µm.

[0130] The second channels of the second layer have an aspect ratio in which 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 or from about one-third to about one-quarter of the second average thickness. The second average diameter may be about half the second average thickness of the second layer of the perforated membrane. In some cases, the second average diameter is no more than half the second average thickness.

[0131] In some cases, the first average diameter is larger than the second average diameter. In some cases, the first average diameter is at least two or at least three times the second average diameter. In some embodiments, the first average diameter is from about two to about three times the second average diameter. The first average diameter may, for example, be about three to about four times the second average diameter.

[0132] The first layer may include a first polymeric material according to any of the embodiments described above, such as those provided in Section I: Perforated Film. In some cases, the first layer includes a first polymeric material. In some cases, the first polymeric material includes a first photoreactive polymer, a first crosslinked polymer, a first synthetic polymer, a first thermosetting polymer, a first photocurable polymer, a first thermosetting polymer, or a first photoresist. In some cases, the first polymeric material includes a first positive photoresist or a first negative photoresist. In some cases, the first polymeric material includes 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.).

[0133] The second layer may include a second polymeric material according to any of the embodiments described above, such as those provided in Section I: Perforated Film. In some cases, the second layer includes a second polymeric material. In some cases, the second polymeric material includes a second photoreactive polymer, a second crosslinked polymer, a second synthetic polymer, a second thermosetting polymer, a second photocurable polymer, a second thermosetting polymer, or a second photoresist. In some cases, the second polymeric material includes a second positive photoresist or a second negative photoresist. In some cases, the second polymeric material includes a second SU-8 photoresist, such as a second SU-8 TF 6000 series photoresist (e.g., SU-8 TF 6002 photoresist, etc.) or a second SU-8 2000 series photoresist (e.g., SU-8 2002 photoresist).

[0134] In some embodiments, the first layer contains at least one component that is different from the composition of the second material. In some cases, the first layer is made of a material different from the material of the second layer. According to some embodiments, the first layer includes a first photoresist, and the second layer includes a second photoresist. In some cases, the first layer includes a first negative photoresist, and the second layer includes a second negative photoresist. In some cases, the first layer includes a first SU-8 photoresist, and the second layer includes a second SU-8 photoresist. Preferably, the first layer includes SU-8 3010 photoresist, SU-8 3005 photoresist, or SU-8 2005 photoresist, and the second layer includes SU-8 TF 6002 photoresist or SU-8 2002 photoresist. According to some embodiments, the first tensile strength of the first layer is greater than the second tensile strength of the second layer.

[0135] The first and second layers can be made of photoresist polymers. In some cases, the first layer is made of the same material as the second layer, such as when the first and second layers comprise a first photoreactive polymer, a first crosslinked polymer, a first synthetic polymer, or a first photocurable polymer.

[0136] donor cells Systems for high-throughput cell electroporation also include donor cells. The donor cells of the system are in contact with a perforated membrane. In some cases, the donor cells adhere to a first surface of the perforated membrane. In some cases, multiple donor cells are in contact with the first surface of the perforated membrane. In some cases, multiple donor cells adhere to the first surface of the perforated membrane as a monolayer of donor cells. For example, the donor cells may be in contact with the first surface of the perforated membrane or a portion thereof. In some cases, the donor cells adhere to a perforated membrane, such as the first surface of the perforated membrane. In some cases, the donor cells are in contact with the opening of at least one channel of the perforated membrane. Preferably, the donor cell contacts the opening of at least one channel on the first surface of the perforated membrane, wherein the at least one channel has an average diameter from about 0.3 µm to about 50 µm, from about 0.3 µm to about 20 µm, from about 0.4 µm to about 10 µm, from about 0.4 µm to about 5 µm, from about 0.4 µm to about 2 µm, or from about 0.4 µm to about 1.5 µm. Preferably, the donor cell contacts the opening of at least one channel on the first surface of the perforated membrane, wherein the at least one channel has an average diameter from about 0.4 µm to about 1.5 µm.

[0137] Donor cells can be any type of cell. In some cases, donor cells are cells that secrete or are capable of secreting extracellular vesicles. In some cases, donor cells are eukaryotic cells (e.g., mammalian cells, human cells, non-human mammalian cells, rodent cells, mouse cells, etc.). In some cases, donor cells are derived from cell lines, stem cells, primary cells, or differentiated cells. In some embodiments, donor cells are primary cells. In some cases, donor cells are mouse embryonic fibroblasts (MEF), human embryonic fibroblasts (HEF), human dermal fibroblasts (HDF), dendritic cells, mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipose-derived stromal cells, enucleated cells, neural stem cells, immature dendritic cells, or immune cells. Donor cells can be adherent cells. In some cases, donor cells are adherent cells. In some cases, donor cells are suspension cells. In some cases, donor cells are cells from suspension cell lines. In some cases, donor cells are suspension primary cells. In some cases, donor cells are human cells.

[0138] In some cases, systems used for high-throughput cell electroporation are configured to culture donor cells.

[0139] In some cases, systems used for high-throughput cell electroporation are configured to electroporate donor cells with transfection reagents.

[0140] Transfection reagent Transfection reagents can be any type of biomolecule. In some cases, the transfection reagent is at least one heteropolynucleotide, such as a vector (e.g., plasmid, DNA). In certain cases, at least one heteropolynucleotide encodes at least one polypeptide. In some cases, at least one polypeptide is therapeutic. In some cases, at least one polypeptide is used for targeted delivery of extracellular vesicles. In some cases, at least one polypeptide is both therapeutic and used for targeted delivery of extracellular vesicles. In other cases, the transfection reagent can be a therapeutic compound (e.g., 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, lncRNA, piRNA, snoRNA, snRNA, exRNA, or scaRNA), a drug, or a combination thereof. In other cases, the transfection reagent can be a non-therapeutic compound (e.g., a non-therapeutic polynucleotide). In some cases, the transfection reagent is loaded in an electroporation chamber, as further described below.

[0141] First spacer array The system for high-throughput cell electroporation further includes at least one array of spacers (e.g., a first spacer array). When the system is assembled, the first spacer array has pores that can be used to form an array with multiple cell culture wells or chambers, thus providing a porous electroporation device for realizing electroporation of one or more types of donor cells. Typically, a perforated membrane extends across the cell culture chamber, such that a chamber suitable for a buffer (e.g., an electroporation buffer) is located below the membrane (or on one side of the perforated membrane), and a chamber is located above the membrane (such as a cell culture chamber), allowing donor cells to be seeded and cultured on top of the perforated membrane (or on one side of the perforated membrane opposite the side facing the buffer chamber). Spacers can also be used to separate chambers. For example, chambers can be separated by a length of 5 mm, such as... Figure 8 , Figure 9A and Figure 9BAs shown in the diagram. Typically, at least a portion of the perforated membrane is located within one or more cell culture chambers. In some cases, the first spacer array is in contact with a first surface of the perforated membrane. In some cases, the first spacer array is adhered to the perforated membrane via van der Waals interactions. In some cases, the first spacer array is adhered to the perforated membrane using an adhesive material. In some cases, the first spacer array is adhered to the perforated membrane using a bioadhesive material. In some cases, the first spacer array is in contact with a first surface of the perforated membrane, which has a first average diameter of a first channel that is larger than the second average diameter of a second channel in the second layer of the perforated membrane.

[0142] Typically, the first spacer array is a plate having a specific thickness (i.e., the height or the distance between the first surface of the first spacer array and the second surface of the first spacer array opposite the first surface). In some cases, the thickness of the first spacer array is greater than the diameter of the 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.

[0143] 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, or 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, from about 1 mm to about 40 mm, or from about 1 mm to about 50 mm.

[0144] The first spacer array also includes a plurality of first holes, wherein the cross-sectional area of ​​each individual first hole is defined by the absence of a first material in the first spacer array. In some cases, at least one hole of the first spacer array is covered by a perforated membrane. In some cases, a plurality of first holes of the first spacer array are covered by a 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 the first holes of the plurality of first holes is uniform throughout the first spacer array. In some cases, the distribution of the first holes of the plurality of first holes is non-uniform throughout the first spacer array.

[0145] The first aperture of a plurality of first apertures is defined by the absence of a first material of a first spacer array, and has walls defined by the first material surrounding each first aperture of the first spacer array. Therefore, the first aperture of the first spacer array includes a first aperture width and a second aperture width perpendicular to the first aperture width, wherein the first aperture has a cross-sectional area defined by the first aperture width and the second aperture width of the first aperture (i.e., individual aperture) of the first spacer array. In some cases, the first aperture width and the second aperture width of the aperture of the array of apertures of the first spacer array do not exceed the surface area of ​​the perforated membrane surface having a first plurality of channel openings.

[0146] In some cases, the width of the first hole is at least about 1 mm. In some cases, the 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 width of the first hole ranges from at least about 1 mm to about 10 cm. In some cases, the width of the first hole is about 1 cm, about 9 mm, or about 5 mm. In some cases, the width of the first hole 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 width of the first hole can range from at least about 1 mm to about 50 mm.

[0147] In some cases, the width of the second hole of the first hole is at least about 1 mm. In some cases, the width of the second hole 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 width of the second hole of the first hole ranges from at least about 1 mm to about 10 cm. In some cases, the width of the second hole of the first hole is about 1 cm, about 9 mm, or about 5 mm. In some cases, the width of the second hole 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 width of the second hole can range from at least about 1 mm to about 50 mm. In some cases, the width of the first hole of the first hole is substantially the same as the width of the second hole of the first hole. In some cases, the width of the first hole of the first hole is different from the width of the second hole of the first hole.

[0148] In some cases, the cross-sectional area of ​​the first hole in the first spacer array (e.g., the first hole formed through holes in the first spacer array) can be, for example, 0.1 cm². 2 0.5 cm 2 1 cm 2 5 cm 2 10 cm2 Values ​​that are greater than or less than these values. In some cases, the cross-sectional area of ​​the first hole in the first spacer array is from approximately 0.1 cm². 2 approximately 5cm 2 From 1 cm 2 approximately 2 cm 2 Or from about 0.5 cm 2 Approximately 4 cm 2 In some cases, the cross-sectional area of ​​the first hole is no more than about 10 cm². 2 No more than about 5 cm 2 No more than about 3 cm 2 No more than about 2 cm 2 or no more than about 1 cm 2 .

[0149] In some cases, the cross-sectional area of ​​the first hole is less than, greater than, or equal to about 10 mm × about 10 mm, and other dimensions such as these. In some cases, the cross-sectional area of ​​the first hole is about 2 mm × 2 mm or about 50 mm × 50 mm.

[0150] The first spacer array also includes a plurality of first holes, whereby the first holes of the plurality of first holes are separated from other first holes of the adjacent first spacer array by a distance between the first holes. In some cases, the first holes of the first spacer array are separated from other first holes of the adjacent first spacer array by a distance between the first holes of the first holes of the first spacer array of at least about 1 mm. In some cases, the first holes of the first spacer array are separated from other first holes of the adjacent first spacer array by a distance between the first holes of the first holes of the first spacer array of no more than about 10 cm. In some cases, the first holes of the first spacer array are separated from other first holes of the adjacent first spacer array of the first holes by a distance between the first holes of the first spacer array of from about 1 mm to about 5 cm or from about 1 mm to about 1.5 cm. In some cases, the first holes of the first spacer array are separated from other first holes of the adjacent first spacer array of the first holes by a distance between the first holes of the first spacer array of about 5 mm. In some cases, the first aperture of the first spacer array is separated from the other first apertures of the adjacent first spacer array by a first aperture 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.

[0151] In some cases, the distance between the first holes of each first hole in the first spacer array is substantially the same. In other cases, the distance between the first holes differs from the distance between the first holes of another first hole in the first spacer array.

[0152] The first spacer array also includes an outer perimeter surrounding a plurality of first holes. The outer perimeter of the first spacer array is defined by an outer width (e.g., the length of a first outer side of the second spacer array measured in a plane parallel to the surface of the second spacer array) and an outer length (e.g., a second length of a second outer side of the second spacer array perpendicular to the first outer side of the second spacer array, and measured in a plane parallel to the surface of the second spacer array). In some cases, the outer width of the outer side of the first spacer array is at least about 1 cm. In some cases, the outer width of the outer side of the first spacer array is no more than about 30 cm. In some cases, the outer width of the outer side of the first spacer array ranges 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 width of the 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 outermost width of the outermost part is at least about 1 mm to about 5 mm. In some cases, the outermost width of the outermost part is at least about 1 mm to about 10 mm. In some cases, the length is at least about 1 mm to about 20 mm. In some cases, the first hole of the first spacer array does not exceed the surface area of ​​the perforated membrane having openings with a first plurality of channels.

[0153] The first spacer array includes a number of first holes. In some cases, the first spacer array includes at least one first hole or at least two first holes. In some cases, the first spacer array includes no more than about 2,500 first holes. In some cases, the first spacer array includes 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 includes 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 in any desired number of rows and columns.

[0154] In some cases, the plurality of first holes in the first spacer array have a specific shape. The shape can be, for example, a square, a circle, a triangle, or any geometric shape. In some cases, the shape is a square, rectangle, rhombus, polygon, circle, or ellipse. In some cases, the shape is a shape having at least one angle less than or greater than 90 degrees. In some cases, the shape of the first hole may differ from the shape of another first hole in the plurality of first holes. In some cases, the shape of the first hole may be substantially equivalent to the shape of another first hole in the plurality of first holes.

[0155] In some cases, the first spacer array comprises a plurality of first holes arranged in an array having rows and columns. In some cases, the array format of the first holes comprises an array of rows and columns of the first holes. In some cases, the first spacer array includes rows and columns of first holes of 1 × 1, 2 × 2, 3 × 2, 6 × 4, 12 × 8, 2 × 2, 3 × 3, 4 × 3, 4 × 4, 5 × 5, 6 × 6, 7 × 7, 8 × 8, 8 × 12, 9 × 9, 10 × 10, 11 × 11, 12 × 12, 16 × 16, 16 × 24, 24 × The first spacer array can have 24 rows and columns of first holes or 48 × 60 rows and columns of first holes. The first spacer array can have any first rows and columns of first holes, arranged in any desired number of rows and columns of first holes.

[0156] In some cases, the first spacer array contains at least one row or more than one row. In some cases, the second spacer array contains at least two rows. In some cases, the first spacer array contains no more than 100 rows. In some cases, the first spacer array contains 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 contains about 5 rows.

[0157] In some cases, the first spacer array comprises at least one column or more columns. In some cases, the first spacer array comprises at least two columns. In some cases, the first spacer array 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 aperture of the first spacer array comprises: (a) at least two rows of the first aperture of the first spacer array; and (b) at least two columns of the first aperture of the first spacer array.

[0158] 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 bond well with another smooth surface, such as a perforated film (e.g., a track-etched film, a silicon perforated film, a photoresist perforated film, or any polymer perforated film). In some cases, the spacers comprise or are made of rubber. For example, the spacers can be polymers, such as silicone rubber. In some cases, the spacers are made of polymers. In some cases, the first spacer array includes thermoplastic polymers, crosslinked polymers, photocurable polymers, thermosetting polymers, photoreactive polymers, silicone polymers, rubber, thermoplastic elastomers, or photoresist polymers. In some cases, the first spacer array can include water-soluble polymers (e.g., polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), etc.) or curable polymers, such as curable polymer resins (e.g., acrylic acid, polydimethylsiloxane (PDMS), etc.). In some cases, the first spacer array includes polyethylene terephthalate (PET), polycarbonate, polydimethylsiloxane (PDMS), polydimethylsiloxane (PDMA), polyvinyl alcohol (PVA), polyethylene, polypropylene, polystyrene, polyacrylamide, or polyacrylic acid.

[0159] As described above, the pores 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. Therefore, the cell culture chamber is configured for culturing donor cells. The number of donor cells that can be cultured in the cell culture chamber depends on the cross-sectional area of ​​the cell culture chamber, as defined by the first pore width and the second pore width of the first spacer array in any embodiment. In some cases, the average cell culture chamber may contain at least about 10,000 channels / cm² of perforated membrane. 2 In some cases, the average cell culture chamber can contain from about 10,000 to about 1 x 10⁻⁶ cells. 8 Channels per perforated membrane / cm 2 In some cases, the average cell culture chamber can contain from approximately 1 x 10⁻⁶ cells. 5 To approximately 1x10 8Channels per perforated membrane / cm 2 Preferably, the average cell culture chamber contains cells ranging from about 5 x 10⁻⁶. 5 Approximately 5x10 6 Channels per perforated membrane / cm 2 .

[0160] In some implementations, the multi-well cell culture chamber has a size matching or approximating that of a standard 6, 12, 24, 48, or 96-well cell culture plate or any of the aforementioned dimensions described in the first well of the first spacer array. The multi-well cell culture chamber can facilitate liquid handling for purposes such as cell loading, washing, EV collection, etc., using automated robotic multichannel pipettes and dispensers. In some cases, the multi-well cell culture chamber can 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 well and prevent well-to-well cross-contamination. The multi-well cell culture chamber can efficiently determine multiple conditions or replicate equivalent conditions.

[0161] Second spacer array A system for high-throughput cell electroporation can further include a second array of spacers having pores that can be used to form an array of multiple electroporation reagent wells or chambers. In this configuration, the system can be assembled such that the second spacer array contacts a second surface of a perforated membrane, the second surface of which opposes a first surface of a perforated membrane that contacts the first spacer array. This forms a "sandwich" structure, whereby the perforated membrane is positioned between the first and second spacer arrays, as... Figure 11A and Figure 11B As shown in the diagram. In some cases, the first spacer array contacts a first surface of the perforated membrane, and the second spacer array contacts a 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. However, the first spacer array is used to form an array with multiple cell culture wells, and the wells 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 be used to separate second wells to form multiple electroporation reagent chambers. Typically, the first and second spacer arrays are aligned to form an array of “miniature” cell culture / electroporation chambers, whereby the individual culture / electroporation chambers are fluidly isolated from the other culture / electroporation chambers of the porous electroporation device described herein. Furthermore, each individual chamber of the porous 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 fluidly coupled via channels of the perforated membrane positioned between the first and second spacer arrays.

[0162] Typically, a perforated membrane extends across at least one cell culture chamber, such that a buffer-suitable chamber is located below the membrane (or on one side of the perforated membrane), and a chamber is located above the membrane (such as a cell culture chamber), allowing cells to be seeded and cultured on top of the perforated membrane (or on one side of the perforated membrane opposite the side facing the buffer chamber). In some cases, particularly when an electric field is applied to the electroporation device, reagents present in the buffer chamber (e.g., DNA, RNA, plasmids, vectors, polynucleotides) are transferred to the cell chamber via pores in the perforated membrane. In some embodiments, a porous electroporation / nanoporosis (CEP / CNP) device comprises cell culture chambers typically spaced apart by spacers. Thus, a system partially comprises a porous 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 any combination of transfection reagents described herein). In some cases, particularly when an electric field is applied to the electroporation device, reagents (e.g., DNA, RNA, plasmids, vectors, polynucleotides) present in the buffer compartment are transferred to the cell compartment via pores in the perforated membrane. In some cases, the porous electroporation device includes a reservoir or buffer well or buffer compartment containing a buffer solution and polynucleotides (e.g., DNA, RNA, DNA plasmids, microRNAs) to be electroporated into donor cells in cell culture wells. In some cases, the electroporation reagent compartment has a depth considered to be approximately the distance between the front of the non-perforated membrane and the surface of the spacer (e.g., the thickness of the spacer). In some cases, the depth of the electroporation reagent compartment is at least about 1 mm to about 5 mm. In some cases, the depth of the electroporation reagent compartment has a thickness of at least about 1 mm to about 10 mm. In some cases, the depth of the electroporation reagent compartment 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 as the depth of the cell culture well.

[0163] In some cases, 12-well Transwell plates (cell growth area 1.12 cm²) 2 Transwell perforated membranes larger than 6-well plates (cell growth area 4.67 cm²) 2 Or a Transwell perforated membrane much larger than a 100 mm culture dish (cell growth area 44 cm²) 2 (This is used in the apparatus provided herein.)

[0164] In some cases, the second spacer array is in contact with a second surface of the perforated membrane. In some cases, the second spacer array is adhered to the perforated membrane via van der Waals interactions. In some cases, the second spacer array is adhered to the perforated membrane using an adhesive material. In some cases, the second spacer array is adhered to the perforated membrane using a bio-adhesive material. In some cases, the second spacer array is in contact with a second surface of the perforated membrane, which has a second average diameter of a second channel that is larger than the second average diameter of a second channel in the second layer of the perforated membrane.

[0165] Typically, the second spacer array is a plate with a specific thickness (i.e., the height or the distance between the first surface of the second spacer array and the second surface of the second spacer array opposite to the first surface). In some cases, the thickness of the second spacer array is greater than the diameter of the 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 ranges from at least about 0.5 cm to at least about 5 cm.

[0166] 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, or 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, from about 1 mm to about 40 mm, or from about 1 mm to about 50 mm.

[0167] The second spacer array also includes a plurality of second holes, wherein the cross-sectional area of ​​each individual second hole is defined by the absence of a second material in the second spacer array. In some cases, at least one hole of the second spacer array is covered by a perforated membrane. In some cases, a plurality of second holes of the second spacer array are covered by a perforated membrane. In some cases, the plurality of second holes are arranged in a non-patterned arrangement or distribution throughout the second spacer array. In some cases, the plurality of second holes are arranged in a patterned arrangement throughout the second spacer array. In some cases, the distribution of the second holes of the plurality of second holes is uniform throughout the second spacer array. In some cases, the distribution of the second holes of the plurality of second holes is non-uniform throughout the second spacer array.

[0168] The second holes of the plurality of second holes are defined by the absence of a second material in the second spacer array, and have walls defined by the second material surrounding each second hole in the second spacer array. Therefore, the second holes of the second spacer array include a first hole width and a second hole width perpendicular 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 the second hole (i.e., individual hole) in the second spacer array. In some cases, the first hole width and the second hole width of the array of holes in the second spacer array do not exceed the surface area of ​​the perforated membrane surface having a second plurality of channel openings.

[0169] In some cases, the width of the first aperture of the second aperture is at least about 1 mm. In some cases, the width of the first aperture of the second aperture 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 width of the first aperture of the second aperture ranges from at least about 1 mm to about 10 cm. In some cases, the width of the first aperture of the second aperture is about 1 cm, about 9 mm, or about 5 mm. In some cases, the width of the first aperture 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 width of the first aperture can range from at least about 1 mm to about 50 mm. In some cases, the width of the first aperture of the second aperture of the second spacer array is about 1 cm.

[0170] In some cases, the width of the second hole is at least about 1 mm. In some cases, the 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 width of the second hole ranges from at least about 1 mm to about 10 cm. In some cases, the width of the second hole is about 1 cm, about 9 mm, or about 5 mm. In some cases, the width of the second hole 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 width of the second hole can range from at least about 1 mm to about 50 mm. In some cases, the width of the second hole in the second spacer array is about 1 cm. In some cases, the width of the first hole of the second hole is substantially the same as the width of the second hole. In some cases, the width of the first hole of the second hole differs from the width of the second hole.

[0171] In some cases, the cross-sectional area of ​​the second hole in the second spacer array (e.g., a second hole formed through holes in the second spacer array) can be, for example, 0.1 cm². 20.5 cm 2 1 cm 2 5 cm 2 10 cm 2 The value may be greater than or less than these values. In some cases, the cross-sectional area of ​​the second hole in the second spacer array is from approximately 0.1 cm². 2 approximately 5cm 2 From 1 cm 2 approximately 2 cm 2 Or from about 0.5 cm 2 Approximately 4 cm 2 In some cases, the cross-sectional area of ​​the second hole is no more than about 10 cm². 2 No more than about 5 cm 2 No more than about 3 cm 2 No more than about 2 cm 2 or no more than about 1 cm 2 .

[0172] In some cases, the cross-sectional area of ​​the second hole is less than, greater than, or equal to about 10 mm × about 10 mm, and other dimensions such as these. In some cases, the cross-sectional area of ​​the second hole is about 2 mm × 2 mm or about 50 mm × 50 mm.

[0173] The second spacer array also includes a plurality of second holes, whereby the second holes of the plurality of second holes are separated from other second holes of the adjacent second spacer array by a distance between the second holes. In some cases, the second holes of the second spacer array are separated from other second holes of the adjacent second spacer array by a distance between the second holes of at least about 1 mm. In some cases, the second holes of the second spacer array are separated from other second holes of the adjacent second spacer array by a distance between the second holes of no more than about 10 cm. In some cases, the second holes of the second spacer array are separated from other second holes of the adjacent second spacer array by a distance between the second holes of about 1 mm and about 5 cm, or from about 1 mm and about 1.5 cm. In some cases, the second holes of the second spacer array are separated from other second holes of the adjacent second spacer array by a distance between the second holes of about 5 mm. In some cases, the second aperture of the second spacer array is separated from the other second apertures of the adjacent second spacer array by a second aperture 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.

[0174] In some cases, the distance between the second holes of each second hole in the second spacer array is substantially the same. In other cases, the distance between the second holes of the second holes differs from the distance between the second holes of another second hole in the second spacer array.

[0175] The second spacer array also includes an outer perimeter surrounding the plurality of second holes. The outer perimeter of the second spacer array is defined by its outer width and outer length. In some cases, the outer width of the outer side of the second spacer array is at least about 1 cm. In some cases, the outer width of the outer side of the second spacer array is no more than about 30 cm. In some cases, the outer width of the outer side of the second spacer array ranges 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 width of the 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 width of the outer side is at least about 1 mm to about 5 mm. In some cases, the outer width of the outer side is at least about 1 mm to about 10 mm. In some cases, the length is at least about 1 mm to about 20 mm. In some cases, the second aperture of the second spacer array does not exceed the surface area of ​​the perforated membrane with a second plurality of channels.

[0176] The second spacer array includes a number of second holes. In some cases, the second spacer array includes at least one or at least two second holes. In some cases, the second spacer array includes no more than about 2,500 second holes. In some cases, the second spacer array includes from about 2 to about 500 second holes, from about 2 to about 100 second holes, or from about 2 to about 30 second holes. In some cases, the number of second holes includes 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 second holes. The second spacer array can have any number of second holes arranged in any desired number of rows and columns.

[0177] In some cases, the multiple second holes of the second spacer array have a specific shape. The shape can be, for example, a square, circle, triangle, or any geometric shape. In some cases, the shape is a square, rectangle, rhombus, polygon, circle, or ellipse. In some cases, the shape has at least one angle less than or greater than 90 degrees. In some cases, the shape of the second hole may differ from the shape of another second hole in the array. In some cases, the shape of the second hole may be substantially identical to the shape of another second hole in the array.

[0178] In some cases, the multiple second holes of the second spacer array have a specific shape. The shape can be, for example, a square, circle, triangle, or any geometric shape. In some cases, the shape is a square, rectangle, rhombus, polygon, circle, or ellipse. In some cases, the shape has at least one angle less than or greater than 90 degrees. In some cases, the shape of the second hole may differ from the shape of another second hole in the array. In some cases, the shape of the second hole may be substantially identical to the shape of another second hole in the array.

[0179] In some cases, the second spacer array comprises a plurality of second holes arranged in an array having rows and columns. In some cases, the array format of the second holes comprises an array of rows and columns of second holes. In some cases, the second spacer array includes rows and columns of second holes of 1 × 1, 2 × 2, 3 × 2, 6 × 4, 12 × 8, 2 × 2, 3 × 3, 4 × 3, 4 × 4, 5 × 5, 6 × 6, 7 × 7, 8 × 8, 8 × 12, 9 × 9, 10 × 10, 11 × 11, 12 × 12, 16 × 16, 16 × 24, 24 × The second spacer array can have 24 rows and columns of second holes or 48 × 60 rows and columns of second holes. The second spacer array can have any second rows and columns of second holes, arranged in any desired number of rows and columns of second holes.

[0180] In some cases, the second spacer array comprises at least one column or more columns. In some cases, the second spacer array comprises at least two 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 to about 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 aperture of the second spacer array comprises: (a) at least two rows of the second aperture of the second spacer array; and (b) at least two columns of the second aperture of the second spacer array.

[0181] 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 bond well with another smooth surface, such as a perforated film (e.g., a track-etched film, a silicon perforated film, a photoresist perforated film, or any polymer perforated film). In some cases, the spacers comprise or are made of rubber. For example, the spacers can be polymers, such as silicone rubber. In some cases, the spacers are made of polymers. In some cases, the second spacer array includes thermoplastic polymers, crosslinked polymers, photocurable polymers, thermosetting polymers, photoreactive polymers, silicone polymers, rubber, thermoplastic elastomers, or photoresist polymers. In some cases, the second spacer array can include water-soluble polymers (e.g., polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), etc.) or curable polymers, such as curable polymer resins (e.g., acrylic acid, polydimethylsiloxane (PDMS), etc.). In some cases, the second spacer array includes polyethylene terephthalate (PET), polycarbonate, polydimethylsiloxane (PDMS), polydimethylsiloxane (PDMA), polyvinyl alcohol (PVA), polyethylene, polypropylene, polystyrene, polyacrylamide, or polyacrylic acid.

[0182] As described above, the pores of the second spacer array, when in contact with the second surface of the perforated membrane, can be used to form an array of electroporation reagent chambers. Therefore, the electroporation reagent chambers are configured for electroporating donor cells. The number of donor cells that can be electroporated within the cell culture chamber also depends on the cross-sectional area of ​​the electroporation reagent chamber fluidly coupled to the cell culture chamber, as defined by the first and second pore widths of the second pores of the second spacer array in any embodiment. In some cases, the average electroporation reagent chamber may contain at least about 10,000 channels / cm² of perforated membrane. 2 In some cases, the average electroporation reagent chamber can contain from about 10,000 to about 1 x 10⁻⁶. 8 Channels per perforated membrane / cm 2 In some cases, the average electroporation reagent chamber can contain from approximately 1 x 10⁻⁶.5 To approximately 1x10 8 Channels per perforated membrane / cm 2 Preferably, the average electroporation reagent chamber contains from about 5 x 10 5 Approximately 5x10 6 Channels per perforated membrane / cm 2 .

[0183] In some implementations, the porous electroporation reagent chamber has a size matching or approximating that of a standard 6, 12, 24, 48, or 96-well electroporation reagent plate or any of the aforementioned dimensions described in the first well of the first spacer array. The porous electroporation reagent chamber can facilitate liquid handling using automated robotic multichannel pipettes and dispensers for purposes such as cell loading, washing, EV collection, loading transfection reagents, etc. In some cases, the porous electroporation reagent chamber can increase the mechanical strength of the perforated membrane. The porous electroporation reagent chamber can ensure that each well is physically isolated from the next well and prevent well-to-well cross-contamination. The porous electroporation chamber can efficiently determine multiple conditions or replicate equivalent conditions.

[0184] Comparison of the first spacer array and the second spacer array Some embodiments of a system for high-throughput electroporation include a first spacer array that differs from the second spacer array. In some cases, the thickness of the second spacer array differs from the thickness of the first spacer array. In some embodiments, the distance between the second holes in the second spacer array differs from the distance between the first holes in the first spacer array. For example, the distance between two second holes in the second spacer array may differ from the distance between two first holes in the first array of holes in the first spacer array. In some embodiments, the width of the first hole in the second hole of the second spacer array may differ from the width of the first hole in the first spacer array. It is also conceivable that the width of the second hole in the second spacer array may differ from the width of the first hole in the first spacer array. In some cases, the number of first holes in the first spacer array differs from the number of second holes in the second spacer array. Furthermore, it is conceivable that the first spacer array contains at least one component that differs from the composition of the material of the second spacer array. In some cases, the first spacer array is made of a material different from the material of the second spacer array.

[0185] Alternatively, according to some embodiments of a system for high-throughput electroporation, a first spacer array and a second spacer array are used. 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 may, for example, be equivalent to the first spacer array. For example, the distance between the second holes of the second spacer array may be substantially the same as or equivalent to the distance between the first holes of the first array of holes in the first spacer array. In some embodiments, the width of the first hole of the second hole of the second spacer array may be substantially the same as or equivalent to the width of the first hole of the first hole of the first spacer array. It is also conceivable that the width of the second hole of the second hole of the second spacer array may be substantially the same as or equivalent to the width of the second hole of the first hole of the first spacer array. In some cases, the number of first holes in the first spacer array is substantially the same as or equivalent to the number of second holes in the second spacer array. Furthermore, it is conceivable that the first spacer array is made of at least one material that is the same as the material of the second spacer array. In some cases, the first spacer array is made of a material equivalent to the second spacer array.

[0186] Cathode Array Systems for high-throughput electroporation may also include a cathode. The cathode can be made of any conductive material that is non-toxic to 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 aperture (e.g., a cell culture chamber) of the first spacer array. In some cases, the cathode is configured to contact the surface of either the first or second spacer array. Thus, the cathode plate can have a length and width that are substantially the outer length and width of the first spacer array or substantially the outer length and width of the second spacer array. The cathode plate can have a length and width that covers only a portion of the first 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. The placement of the anode within the first or second spacer array will depend on the charge of the material disposed within it. Therefore, in some cases, contact between the cathode plate and the first or second spacer array is appropriate. The cathode plate can also be electrically connected to the first or second spacer array via a solution.

[0187] In other cases, the cathode may be rod-shaped or linear, including wires forming a coil (e.g., a cathode coil). For example, the cathode may be a cathode coil configured to be disposed within a first hole of a first spacer array or a second hole of a second spacer array.

[0188] Furthermore, to accommodate the multi-hole configuration of the high-throughput electroporation system and apparatus provided herein, the cathode may include an array of cathode coils. For example, the cathode coil array is configured to position cathode coils within a first hole of a first spacer array or within a second hole of a second spacer array. In some cases, the cathode coil array is configured to position individual cathode coils within at least two, at least three, or at least five first holes of the first spacer array. In some cases, the cathode coil array is configured to position individual cathode coils within a portion of a first hole of the first spacer array. In some cases, the cathode coil array is configured to position individual cathode coils within all first holes of the first spacer array.

[0189] In some cases, the cathode coil array is configured to position individual cathode coils within at least two, at least three, or at least five second holes of the second spacer array. In some cases, the cathode coil array is configured to position individual cathode coils within a portion of the second holes of the second spacer array. In some cases, the cathode coil array is configured to position individual cathode coils within all the second holes of the second spacer array.

[0190] The cathode wire may have a diameter smaller than the cross-sectional area of ​​the first aperture of the first spacer array or smaller than the cross-sectional area of ​​the second aperture 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.

[0191] A cathode array contains a number of cathodes. In some cases, a cathode array contains at least one cathode or at least two cathodes. In some cases, a cathode array contains no more than about 2,500 cathodes. In some cases, a cathode array contains from about two cathodes to about 500 cathodes, from about two cathodes to about 100 cathodes, or from about two cathodes to about 30 cathodes. In some cases, the number of cathodes includes 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 cathodes. A cathode array can have any desired number of cathodes.

[0192] In some cases, the cathodes of the cathode array have a specific shape. The shape can be, for example, a square, circle, triangle, or any geometric shape. In some cases, the shape is a square, rectangle, rhombus, polygon, circle, or ellipse. In some cases, the shape has at least one angle less than or greater than 90 degrees. In some cases, the shape of the first aperture may differ from the shape of another first aperture of the plurality of cathodes. In some cases, the shape of the first aperture may be substantially equivalent to the shape of another first aperture of the plurality of cathodes.

[0193] In some cases, the cathode array comprises multiple cathodes arranged in an array of rows and columns. In other cases, the cathode array format includes an array of rows and columns of cathodes. In some cases, the cathode array includes rows and columns of 1 × 1 cathodes, 2 × 2 cathodes, 3 × 2 cathodes, 6 × 4 cathodes, 12 × 8 cathodes, 2 × 2 cathodes, 3 × 3 cathodes, 4 × 3 cathodes, 4 × 4 cathodes, 5 × 5 cathodes, 6 × 6 cathodes, 7 × 7 cathodes, 8 × 8 cathodes, 8 × 12 cathodes, 9 × 9 cathodes, 10 × 10 cathodes, 11 × 11 cathodes, 12 × 12 cathodes, 16 × 16 cathodes, 16 × 24 cathodes, 24 × 16 cathodes, and so on. A cathode array can have 24 rows and columns of cathodes or 48 × 60 rows and columns of cathodes. The cathode array can have any number of cathodes arranged in any desired number of rows and columns. The cathodes of the cathode array can be cathode wires, cathode coils, or have any shape. The cathodes of the cathode array can have a different shape than the other cathodes in the array.

[0194] In some cases, the cathode array comprises at least one row or more than one row. In some cases, the second spacer array comprises at least two 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.

[0195] In some cases, the cathode array comprises at least one or more columns of cathodes. In some cases, the cathode array comprises at least two 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 cathodes of the cathode array include: (a) cathodes of at least two rows of cathode arrays; and (b) cathodes of at least two columns.

[0196] In some cases, the cathode wires are structurally fixed to a common sleeve. In other cases, the cathode wires are reversibly connected to a cathode wire array. In still other cases, the cathode wire array can be customized by adding, removing, or repositioning cathode wires to, for example, adapt to any of the aforementioned embodiments of a porous system used for high-throughput electroporation.

[0197] Anode array Systems for high-throughput electroporation may also include an anode. The anode can be made of any conductive material that is non-toxic to 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 aperture (e.g., an electroporation reagent chamber) of the second spacer array. In some cases, the anode is configured to contact the surface of either the first or second spacer array. Thus, the anode plate can have a length and width that are substantially the outer length and width of the first or second spacer array. The anode plate can have a length and width that covers only a portion of the first 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. The placement of the anode within the first or second spacer array will depend on the charge of the material disposed within the first or second spacer array. Therefore, in some cases, contact between the cathode plate and the first or second spacer array is appropriate. The cathode plate can also be electrically connected to the first or second spacer array via a solution.

[0198] In other cases, the anode may be rod-shaped or linear, including wires forming a coil (e.g., an anode coil). For example, the anode may be an anode coil configured to be disposed within a first hole of a first spacer array or a second hole of a second spacer array.

[0199] Furthermore, to accommodate the porous configuration of the high-throughput electroporation system and apparatus provided herein, the anode may include an array of anode coils. For example, the anode coil array is configured to position anode coils within a first hole of a first spacer array or within a second hole of a second spacer array. In some cases, the anode coil array is configured to position individual anode coils within at least two, at least three, or at least five first holes of the first spacer array. In some cases, the anode coil array is configured to position individual anode coils within a portion of a first hole of the first spacer array. In some cases, the anode coil array is configured to position individual anode coils within all first holes of the first spacer array.

[0200] In some cases, the anode coil array is configured to position individual anode coils within at least two, at least three, or at least five second holes of the second spacer array. In some cases, the anode coil array is configured to position individual anode coils within a portion of the second holes of the second spacer array. In some cases, the anode coil array is configured to position individual anode coils within all the second holes of the second spacer array.

[0201] The anode wire may have a diameter smaller than the cross-sectional area of ​​the first hole in the first spacer array or smaller than the cross-sectional area of ​​the second hole in 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.

[0202] An anode array comprises a certain number of anodes. In some cases, an anode array comprises at least one anode or at least two anodes. In some cases, an anode array comprises no more than about 2,500 anodes. In some cases, an anode array comprises from about two anodes to about 500 anodes, from about two anodes to about 100 anodes, or from about two anodes to about 30 anodes. In some cases, the number of anodes includes 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 anodes. An anode array can have any desired number of anodes.

[0203] In some cases, the anodes of the anode array have a specific shape. The shape can be, for example, a square, circle, triangle, or any geometric shape. In some cases, the shape is a square, rectangle, rhombus, polygon, circle, or ellipse. In some cases, the shape is one with at least one angle less than or greater than 90 degrees. In some cases, the shape of the first aperture may differ from the shape of another first aperture among the multiple anodes. In some cases, the shape of the first aperture may be substantially equivalent to the shape of another first aperture among the multiple anodes.

[0204] In some cases, the anode array comprises multiple anodes arranged in an array having rows and columns. In other cases, the anode array format includes an array of rows and columns of anodes. In some cases, the anode array includes rows and columns of 1 × 1 anodes, 2 × 2 anodes, 3 × 2 anodes, 6 × 4 anodes, 12 × 8 anodes, 2 × 2 anodes, 3 × 3 anodes, 4 × 3 anodes, 4 × 4 anodes, 5 × 5 anodes, 6 × 6 anodes, 7 × 7 anodes, 8 × 8 anodes, 8 × 12 anodes, 9 × 9 anodes, 10 × 10 anodes, 11 × 11 anodes, 12 × 12 anodes, 16 × 16 anodes, 16 × 24 anodes, 24 × Anode arrays can have 24 rows and columns or 48 × 60 rows and columns. An anode array can have any number of anodes arranged in any desired number of rows and columns. The anodes in an anode array can be anode wires, anode coils, or have any shape. The anodes in an anode array can have shapes different from those of the anodes in the array.

[0205] In some cases, the anode array comprises at least one row or more than one row. In some cases, the second spacer array comprises at least two 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 to about 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.

[0206] In some cases, the anode array comprises at least one column or more of anodes. In some cases, the anode array comprises at least two 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 anodes of the anode array include: (a) anodes of at least two rows of anode arrays; and (b) anodes of at least two columns.

[0207] In some cases, the anode wires are structurally fixed to a common sleeve. In other cases, the anode wires are reversibly connected to an anode wire array. In still other cases, the anode wire array can be customized by adding, removing, or repositioning anode wires to, for example, adapt to any of the aforementioned embodiments of a porous system used for high-throughput electroporation.

[0208] Electrical pulse generation system Systems for high-throughput electroporation may also include an electrical pulse generation system configured to supply voltage or current to donor cells (e.g., configured to electroporate donor cells) via the cathode and anode described above. Here, the electrical pulse generation system includes: (a) an electrical pulse generator; (b) an impulse pulse converter; and (c) a current output device.

[0209] The electrical pulse generation system can be automated or manually operated. Preferably, the electrical pulse generation system is configured to adjust the characteristics of the electrical pulses applied to at least one donor cell in contact with the perforated membrane. Adjustable characteristics of the electrical pulses may include voltage, current, pulse duration, pulse interval, pulse frequency, or pulse delay. Any one or a combination of pulse characteristics can be adjusted to optimize the system for high-throughput cell electroporation of donor cells. In some cases, the electrical pulse generation system is configured to adjust the voltage applied to at least one donor cell in contact with the perforated membrane.

[0210] Pulse characteristics In some cases, the electrical pulse generation system delivers electrical pulses to the cathode and anode of the system to generate an electric field within at least one or more of the porous cell culture chamber / electroporation chamber described above. In some cases, the voltage generated by the cathode and anode driven by the electrical pulse generation system includes voltages between about 10 V and about 500 V.

[0211] In some cases, the voltage generated by the cathode and anode driven by the electrical pulse generation system includes approximately 10 V to approximately 25 V, approximately 10 V to approximately 50 V, approximately 10 V to approximately 100 V, approximately 10 V to approximately 125 V, approximately 10 V to approximately 150 V, approximately 10 V to approximately 175 V, approximately 10 V to approximately 200 V, approximately 10 V to approximately 225 V, approximately 10 V to approximately 250 V, approximately 10 V to approximately 300 V, approximately 10 V to approximately 500 V, approximately 25 V to approximately 50 V, approximately 25 V to approximately 100 V, approximately 25 V to approximately 125 V, approximately 25 V to approximately 150 V, approximately 25 V to approximately 175 V, approximately 25 V to approximately 200 V, approximately 25 V to approximately 225 V, approximately 25 V to approximately 250 V, approximately 25 V to approximately 300 V, approximately 25 V to approximately 500 V, approximately 50 V to approximately 100 V, and approximately 50 V to approximately 125 V. V, approximately 50 V to approximately 150 V, approximately 50 V to approximately 175 V, approximately 50 V to approximately 200 V, approximately 50 V to approximately 225 V, approximately 50 V to approximately 250 V, approximately 50 V to approximately 300 V, approximately 50 V to approximately 500 V, approximately 100 V to approximately 125 V, approximately 100 V to approximately 150 V, approximately 100 V to approximately 175 V, approximately 100 V to approximately 200 V, approximately 100 V to approximately 225 V, approximately 100 V to approximately 250 V, approximately 100 V to approximately 300 V, approximately 100 V to approximately 500 V, approximately 125 V to approximately 150 V, approximately 125 V to approximately 175 V, approximately 125 V to approximately 200 V, approximately 125 V to approximately 225 V, approximately 125 V to approximately 250 V, approximately 125 V to approximately 300 V, approximately 125 V to approximately 500 V, approximately 150 V to approximately 175 V. Voltages between approximately 150 V and approximately 200 V, approximately 150 V and approximately 225 V, approximately 150 V and approximately 250 V, approximately 150 V and approximately 300 V, approximately 150 V and approximately 500 V, approximately 175 V and approximately 200 V, approximately 175 V and approximately 225 V, approximately 175 V and approximately 250 V, approximately 175 V and approximately 300 V, approximately 175 V and approximately 500 V, approximately 200 V and approximately 225 V, approximately 200 V and approximately 250 V, approximately 200 V and approximately 300 V, approximately 200 V and approximately 500 V, approximately 225 V and approximately 250 V, approximately 225 V and approximately 300 V, approximately 225 V and approximately 500 V, approximately 250 V and approximately 300 V, approximately 250 V and approximately 500 V, or approximately 300 V and approximately 500 V.In some cases, the voltage generated by the cathode and anode driven by the electrical pulse generation system includes voltages between approximately 10 V, approximately 25 V, approximately 50 V, approximately 100 V, approximately 125 V, approximately 150 V, approximately 175 V, approximately 200 V, approximately 225 V, approximately 250 V, approximately 300 V, or approximately 500 V. In some cases, the voltage generated by the cathode and anode driven by the electrical pulse generation system includes voltages ranging from 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.

[0212] In some cases, the electric field generated by the cathode and anode driven by the electric pulse generation system includes an electrical strength ranging from about 0.1 volts / mm to about 50,000 volts / mm. In other cases, the electric field generated by the cathode and anode driven by the electric pulse generation system includes strengths ranging from about 0.1 volts / mm to about 0.5 volts / mm, about 0.1 volts / mm to about 1 volt / mm, about 0.1 volts / mm to about 5 volts / mm, about 0.1 volts / mm to about 10 volts / mm, about 0.1 volts / mm to about 50 volts / mm, about 0.1 volts / mm to about 1,000 volts / mm, about 0.1 volts / mm to about 5,000 volts / mm, and about 0.1 volts / mm to... Approximately 10,000 volts / mm, approximately 0.1 volts / mm to approximately 50,000 volts / mm, approximately 0.5 volts / mm to approximately 1 volt / mm, approximately 0.5 volts / mm to approximately 5 volts / mm, approximately 0.5 volts / mm to approximately 10 volts / mm, approximately 0.5 volts / mm to approximately 50 volts / mm, approximately 0.5 volts / mm to approximately 100 volts / mm, approximately 0.5 volts / mm to approximately 500 volts / mm, approximately 0.5 volts / mm to approximately 1,000 volts / mm, approximately 0.5 volts / mm to approximately 5,000 volts / mm, approximately 0.5 volts / mm to approximately 10,000 volts / mm, approximately 0.5 volts / mm to about 50,000 volts / mm, about 1 volt / mm to about 5 volts / mm, about 1 volt / mm to about 10 volts / mm, about 1 volt / mm to about 50 volts / mm, about 1 volt / mm to about 100 volts / mm, about 1 volt / mm to about 500 volts / mm, about 1 volt / mm to about 1,000 volts / mm, about 1 volt / mm to about 5,000 volts / mm, about 1 volt / mm to about 10,000 volts / mm, about 1 volt / mm to about 50,000 volts / mm, about 5 volts / mm to about 10 volts / mm, about 5 volts / mm to about 50 volts / mm, about 5 volts / mm to about 100 volts / mm Approximately 5 volts / mm to approximately 500 volts / mm, approximately 5 volts / mm to approximately 1,000 volts / mm, approximately 5 volts / mm to approximately 5,000 volts / mm, approximately 5 volts / mm to approximately 10,000 volts / mm, approximately 5 volts / mm to approximately 50,000 volts / mm, approximately 10 volts / mm to approximately 50 volts / mm, approximately 10 volts / mm to approximately 100 volts / mm, approximately 10 volts / mm to approximately 500 volts / mm, approximately 10 volts / mm to approximately 1,000 volts / mm, approximately 10 volts / mm to approximately 5,000 volts / mm, approximately 10 volts / mm to approximately 10,000 volts / mm, approximately 10 volts / mm to approximately 50,000 volts / mm m, approximately 50 volts / mm to approximately 100 volts / mm, approximately 50 volts / mm to approximately 500 volts / mm, approximately 50 volts / mm to approximately 1,000 volts / mm, approximately 50 volts / mm to approximately 5,000 volts / mm, approximately 50 volts / mm to approximately 10,000 volts / mm, approximately 50 volts / mm to approximately 50,000 volts / mm, approximately 100 volts / mm to approximately 500 volts / mm, approximately 100 volts / mm to approximately 1,000 volts / mm, approximately 100 volts / mm to approximately 5,000 volts / mm, approximately 500 volts / mm to approximately 10,000 volts / mm, approximately 100 volts / mm to approximately 50,000 volts / mm, approximately 500 volts / mm to approximately 1 ... Electric field strengths ranging from approximately 0.5 volts / mm to about 1,000 volts / mm, approximately 500 volts / mm to about 5,000 volts / mm, approximately 500 volts / mm to about 10,000 volts / mm, approximately 500 volts / mm to about 50,000 volts / mm, approximately 1,000 volts / mm to about 5,000 volts / mm, approximately 1,000 volts / mm to about 50,000 volts / mm, approximately 5,000 volts / mm to about 10,000 volts / mm, approximately 5,000 volts / mm to about 50,000 volts / mm, or approximately 10,000 volts / mm to about 50,000 volts / mm. In some cases, the electric field generated by the cathode and anode driven by the electric pulse generation system includes values ​​from approximately 0.5 volts / mm.Electric field strengths of 1 volt / mm, approximately 0.5 volts / mm, approximately 1 volt / mm, approximately 5 volts / mm, approximately 10 volts / mm, approximately 50 volts / mm, approximately 100 volts / mm, approximately 500 volts / mm, approximately 1,000 volts / mm, approximately 5,000 volts / mm, approximately 10,000 volts / mm, or approximately 50,000 volts / mm. In some cases, the electric fields generated by the cathode and anode driven by the electric pulse generation system include electric field strengths of at least approximately 0.1 volts / mm, approximately 0.5 volts / mm, approximately 1 volt / mm, approximately 5 volts / mm, approximately 10 volts / mm, approximately 50 volts / mm, approximately 100 volts / mm, approximately 500 volts / mm, approximately 1,000 volts / mm, approximately 5,000 volts / mm, or approximately 10,000 volts / mm. In some cases, the electric fields generated by the cathode and anode driven by the electric pulse generation system include field strengths ranging from up to about 0.5 V / mm, about 1 V / mm, about 5 V / mm, about 10 V / mm, about 50 V / mm, about 100 V / mm, about 500 V / mm, about 1,000 V / mm, about 5,000 V / mm, about 10,000 V / mm, or about 50,000 V / mm.

[0213] In some cases, the electric field generated by the cathode and anode driven by the electric pulse generation system comprises multiple pulses having pulse durations ranging from about 0.01 ms / pulse to about 5,000 ms / pulse. In other cases, the electric field generated by the cathode and anode driven by the electric pulse generation system comprises pulses having durations ranging from about 0.01 ms / pulse to about 0.05 ms / pulse, about 0.01 ms / pulse to about 0.1 ms / pulse, about 0.01 ms / pulse to about 0.5 ms / pulse, about 0.01 ms / pulse to about 1 ms / pulse, about 0.01 ms / pulse to about 5 ms / pulse, about 0.01 ms / pulse to about 10 ms / pulse, about 0.01 ms / pulse to about 50 ms / pulse, about 0.01 ms / pulse to about 100 ms / pulse, and about 0.01 ms / pulse to about 100 ms / pulse. 0.01 ms / pulse to about 500 ms / pulse, about 0.01 ms / pulse to about 1,000 ms / pulse, about 0.01 ms / pulse to about 5,000 ms / pulse, about 0.05 ms / pulse to about 0.1 ms / pulse, about 0.05 ms / pulse to about 0.5 ms / pulse, about 0.05 ms / pulse to about 1 ms / pulse, about 0.05 ms / pulse to about 5 ms / pulse, about 0.05 ms / pulse to about 10 ms / pulse, about 0.05 ms / pulse to about 50 ms / pulse, about 0.05 ms / pulse to Approximately 100 milliseconds / pulse, approximately 0.05 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 0.05 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 0.05 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 0.1 milliseconds / pulse to approximately 0.5 milliseconds / pulse, approximately 0.1 milliseconds / pulse to approximately 1 millisecond / pulse, approximately 0.1 milliseconds / pulse to approximately 50 milliseconds / pulse, approximately 0.1 milliseconds / pulse to approximately 100 milliseconds / pulse, approximately 0.1 milliseconds / pulse / pulse to approximately 500 ms / pulse, approximately 0.1 ms / pulse to approximately 1,000 ms / pulse, approximately 0.1 ms / pulse to approximately 5,000 ms / pulse, approximately 0.5 ms / pulse to approximately 1 ms / pulse, approximately 0.5 ms / pulse to approximately 5 ms / pulse, approximately 0.5 ms / pulse to approximately 10 ms / pulse, approximately 0.5 ms / pulse to approximately 500 ms / pulse, approximately 0.5 ms / pulse to approximately 1,000 ms / pulse, approximately 0.5 ms / pulse to about 5,000 ms / pulse, about 1 ms / pulse to about 5 ms / pulse, about 1 ms / pulse to about 10 ms / pulse, about 1 ms / pulse to about 50 ms / pulse, about 1 ms / pulse to about 100 ms / pulse, about 1 ms / pulse to about 500 ms / pulse, about 1 ms / pulse to about 1,000 ms / pulse, about 1 ms / pulse to about 5,000 ms / pulse, about 5 ms / pulse to about 10 ms / pulse, about 5 ms / pulse to about 50 ms / pulse, about 5 ms / pulse to about 100 ms / pulse, about 5 ms / pulse to about 500 ms / pulse, about 5 ms / pulse to about 1,000 ms / pulse, about 5 ms / pulse to about 5,000 ms / pulse, about 10 ms / pulse to about 50 ms / pulse, about 10 ms / pulse to about 100 ms A series of pulses with a duration of approximately 10 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 10 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 10 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 50 milliseconds / pulse to approximately 100 milliseconds / pulse, approximately 50 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 50 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 50 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 100 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 100 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 500 milliseconds / pulse to approximately 1,000 milliseconds / pulse, or approximately 1,000 milliseconds / pulse to approximately 5,000 milliseconds / pulse. In some cases, the electric field generated by the cathode and anode driven by the electric pulse generation system comprises multiple pulses having pulse durations ranging from about 0.01 ms / pulse, about 0.05 ms / pulse, about 0.1 ms / pulse, about 0.5 ms / pulse, about 1 ms / pulse, about 5 ms / pulse, about 10 ms / pulse, about 50 ms / pulse, about 100 ms / pulse, about 500 ms / pulse, about 1,000 ms / pulse, or about 5,000 ms / pulse. In some cases, the electric field generated by the cathode and anode driven by the electric pulse generation system comprises multiple pulses having pulse durations ranging from at least about 0.01 ms / pulse, about 0.05 ms / pulse, about 0.1 ms / pulse, about 0.5 ms / pulse, about 1 ms / pulse, about 5 ms / pulse, about 10 ms / pulse, about 50 ms / pulse, about 100 ms / pulse, about 500 ms / pulse, or about 1,000 ms / pulse. In some cases, the electric fields generated by the cathode and anode driven by the electric pulse generation system include those with pulse durations ranging from up to about 0.05 ms / pulse, about 0.1 ms / pulse, and about 0.Multiple pulses with durations of approximately 5 milliseconds / pulse, approximately 1 millisecond / pulse, approximately 5 milliseconds / pulse, approximately 10 milliseconds / pulse, approximately 50 milliseconds / pulse, approximately 100 milliseconds / pulse, approximately 500 milliseconds / pulse, approximately 1,000 milliseconds / pulse, or approximately 5,000 milliseconds / pulse. In some cases, the cathode and anode, driven by the electrical pulse generation system, generate pulses including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more pulses to each porous cell culture chamber / electroporation chamber of multiple electroporation devices or systems for high-throughput electroporation of donor cells.

[0214] Preferably, the electrical pulse generation system is configured to sequentially deliver electrical pulses 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 the electrical pulses are delivered to each of the culture wells / electroporation wells of the porous electroporation apparatus or system. In some cases, the sequential delivery of electrical pulses may include delivering one or more electrical pulses sequentially to each culture well / electroporation well. In some cases, the sequential delivery of electrical pulses or multiple pulses may include electroporating the culture wells / electroporation wells along rows or columns of the porous electroporation apparatus or system, optionally wherein electrical pulses are simultaneously delivered to all culture wells / electroporation wells in a first row of wells, and then simultaneously delivered to all culture wells / electroporation wells in a second row of wells, until the electrical pulses or multiple electrical pulses are delivered to a desired portion or all of all culture wells / electroporation wells. As another example, the sequential delivery of electrical pulses may include simultaneously delivering one or more electrical pulses to more than one culture well / electroporation well that is not adjacent to each other (e.g., a partial checkerboard pattern). In yet another non-limiting example, the sequential delivery of electrical pulses may include simultaneously delivering one or more electrical pulses to a first group of more than one culture well / electroporation well that is not adjacent to each other, then simultaneously delivering one or more electrical pulses to a second group of culture wells / electroporation wells that are not adjacent to each other after the electrical pulses in the previous group have been delivered, and optionally sequentially delivering one or more electrical pulses to a third and / or fourth group of culture wells / electroporation wells after the electrical pulses in the previous group have been delivered. In some cases, the electrical pulse generation system is configured to execute a user-defined design grating pattern to deliver electrical pulses or multiple electrical pulses to at least one culture well / electroporation well, more than one culture well / electroporation well, a first portion of a culture well / electroporation well, a second portion of a culture well / electroporation well, a third portion of a culture well / electroporation well, a fourth portion of a culture well / electroporation well, any desired combination of culture well / electroporation wells, or all of the culture well / electroporation wells in a multi-well electroporation device or system.

[0215] In some cases, the electrical pulse generation system is configured to perform automated routines to deliver electrical pulses to at least one of the individual line anodes of an anode line or line anode array.

[0216] Accessories for automation Systems for high-throughput electroporation may also include liquid handling or liquid dispensing devices configured to: (A) arrange at least one donor cell in at least one individual well of a first spacer array; (b) add or remove electroporation buffer in at least one individual well of a second spacer array; and (c) add or remove cell culture medium in at least one individual well of the first spacer array.

[0217] In some cases, the liquid handling or dispensing device is configured to perform (a), (b), and (c) automatically. Alternatively, the liquid handling or dispensing device may be manually operated.

[0218] In some cases, the liquid handling or dispensing device is configured to add cell culture medium to at least one, more than one, a portion of, or all of the cell culture chambers of a porous electroporation device or system. The liquid handling or dispensing device may also be configured to remove cell culture medium from at least one, more than one, a portion of, or all of the cell culture chambers of a porous electroporation device or system. The liquid handling or dispensing device may also be configured to replace the cell culture medium in at least one, more than one, a portion of, or all of the cell culture chambers of a porous electroporation device or system.

[0219] In some cases, the liquid handling or liquid dispensing device is configured to arrange at least one or more donor cells into at least one cell culture chamber, more than one cell culture chamber, a portion of or all of the cell culture chambers of a porous electroporation device or system.

[0220] The porous electroporation apparatus or system is also configured to culture more than one type of donor cell, including at least two, three, four, five, eight, ten, or more types of donor cells. For example, a first type of donor cell may be arranged and cultured in a first cell culture chamber, a second type of donor cell in a second cell culture chamber, a third type of donor cell in a third cell culture chamber, a fourth type of donor cell in a fourth cell culture chamber, and so on. In some cases, the porous electroporation apparatus or system is also configured to culture from one type of donor cell to approximately one hundred types of donor cells, from one type of donor cell to 500 types of donor cells, from one type of donor cell to approximately 50 types of donor cells, or from one type of donor cell to approximately 25 types of donor cells.

[0221] In some cases, the liquid handling or dispensing device is configured to add electroporation buffer to at least one, more than one, a portion of, or all of the electroporation reagent chambers of a porous electroporation apparatus or system. The liquid handling or dispensing device may also be configured to remove the electroporation buffer from at least one, more than one, a portion of, or all of the electroporation reagent chambers of a porous electroporation apparatus or system. The liquid handling or dispensing device may also be configured to replace the electroporation buffer in at least one, more than one, a portion of, or all of the electroporation reagent chambers of a porous electroporation apparatus or system.

[0222] Systems for high-throughput electroporation can also be configured to perform multiplex cell electroporation. For example, each electroporation reagent chamber of a multi-well electroporation device or system can be loaded with a different electroporation buffer containing one or any combination of the transfection reagents described above. As another example, the electroporation buffer in the first electroporation reagent chamber can contain a concentration of transfection reagent different from the second concentration of the second electroporation reagent buffer in the second electroporation reagent chamber, a third concentration of the third electroporation reagent buffer in the third electroporation reagent chamber, and so on. Therefore, a multi-well electroporation device or system is configured to titrate transfection reagents across multiple cell culture wells / electroporation wells of the multi-well electroporation device or system.

[0223] In some cases, if necessary, the electroporation buffer in the first electroporation reagent chamber may be the same as the second electroporation buffer in the second electroporation reagent chamber and the third electroporation buffer in the third electroporation reagent chamber. In some cases, the first electroporation buffer may be added to all electroporation reagent chambers of the porous electroporation device or system.

[0224] Systems for high-throughput electroporation may also include a power supply. The power supply for the system or porous electroporation apparatus may be configured to have a voltage output of at least about 1 V, from about 1 V 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 deliver a current of 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 a current of about 10 A to the porous electroporation apparatus or system.

[0225] In some cases, a second spacer array contacts the surface of a perforated membrane and defines an electroporation chamber array; wherein individual cell electroporation chambers of the electroporation chamber array are isolated from adjacent electroporation chambers by the second spacer array; and wherein each individual cell electroporation chamber is configured to contain at least one electroporation buffer, optionally wherein each individual cell electroporation chamber is configured to contain a different electroporation buffer, a different transfection reagent, or a different concentration of transfection reagent than other individual cell electroporation chambers. In some cases, a first spacer array contacts a first surface of a perforated membrane relative to the surface of the perforated membrane in contact with the first spacer array; wherein the first spacer array defines a cell culture chamber array; wherein individual cell culture chambers of the cell culture chamber array are isolated from adjacent cell culture chambers by the first spacer array; wherein each individual cell culture chamber is fluidly coupled to an individual electroporation chamber via a channel of the perforated membrane; and wherein each individual cell culture chamber is configured to contain at least one cell culture medium and at least one donor cell, optionally wherein each individual cell culture chamber is configured to contain a different cell culture medium or a different donor cell type than other individual cell culture chambers.

[0226] Devices used in EVs The system for high-throughput electroporation provided herein can be used to generate extracellular vesicles (EVs) containing one or more transfection reagents described above. The system can also be used to collect electroporated extracellular vesicles (e.g., extracellular vesicles (EVs) containing any one or more transfection reagents such as DNA, DNA plasmids, RNA, proteins, etc.) secreted from donor cells, which are cultured and electroporated in an isolated cell culture / electroporation chamber of a porous electroporation apparatus. Any perforated membrane described herein and known in the art can be used.

[0227] In some cases, porous electroporation devices can stimulate the production of extracellular vesicles (EVs) from donor cells within a cell culture chamber. In some cases, porous electroporation devices can lead to an increase in EV production. In some cases, porous electroporation devices can lead to an increase of at least about 1, 2, 3, 4, 5, 10, 100, 1000, 2000, 3000, 4000, or 5000 times in EV production from donor cells within a cell culture chamber. In some cases, compared to Transwell electroporation devices, porous electroporation devices can increase the production of extracellular vesicles (EVs) from donor cells within a cell culture chamber. In some cases, compared to a 6-well Transwell electroporation device, porous electroporation devices can increase the production of extracellular vesicles (EVs) from donor cells within a cell culture chamber. In some cases, compared to a 12-well Transwell electroporation device, porous electroporation devices can increase the production of extracellular vesicles (EVs) from donor cells within a cell culture chamber. In some cases, multi-well electroporation devices can increase the production of extracellular vesicles (EVs) from donor cells in the cell culture chamber by at least approximately 50-fold, compared to 6-well Transwell electroporation devices. In some cases, multi-well electroporation devices can increase the production of extracellular vesicles (EVs) from donor cells in the cell culture chamber by at least approximately 2,000-fold, compared to 12-well Transwell electroporation devices.

[0228] III. High-throughput cell electroporation system with dual spacer array configuration This 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 includes 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 at least one donor cell are in contact with the perforated membrane.

[0229] Perforated membranes may include polymer membranes, silicon membranes, alumina membranes, or track-etched membranes. In some cases, perforated membranes include polymer microporous membranes, micromachined silicon membranes, alumina membranes, water-soluble polymer membranes, or track-etched membranes. In some cases, perforated membranes include any perforated membrane and embodiments thereof previously described in this disclosure. For example, a system may include a perforated membrane comprising a first layer and a second layer, according to the embodiments provided in Section I: Perforated Membranes of this disclosure. As another example, a system may include a perforated membrane manufactured according to the methods and embodiments provided in Section II: Methods of Manufacturing Perforated Membranes, such as a perforated membrane manufactured by sacrificial template imprinting.

[0230] In some cases, perforated membranes comprise microporous patterned silicon membranes, nanoporous patterned silicon membranes, ceramic microporous membranes, ceramic nanoporous membranes, other porous materials, or combinations thereof. In some cases, perforated membranes are made of rigid materials. Alternatively, perforated membranes may be made of flexible materials.

[0231] The perforated track-etched film can be made of a polymer, such as a thermoplastic polymer (e.g., polyethylene terephthalate (PET), polycarbonate, etc.). The perforated track-etched film has a thickness. In some cases, the thickness of the track-etched film is at least about 10 µm. In some cases, the thickness of the track-etched film is no more than about 100 µm. In some cases, the thickness of the track-etched film ranges from about 1 µm to about 100 µm, from about 1 µm to about 50 µm, from about 1 µm to about 30 µm, from about 5 µm to about 30 µm, or from about 5 µm to about 15 µm. In some cases, the thickness of the track-etched film is about 5 µm, about 10 µm, about 15 µm, about 20 µm, about 25 µm, or about 30 µm. The perforated track-etched film can have channels having the features previously described in Section I: Perforated Films of this disclosure.

[0232] In some cases, a perforated membrane comprises: (i) a first layer including a plurality of first channels disposed through the first layer; and (ii) a second layer in contact with the first layer, the second layer including a plurality of second channels disposed through the second layer, wherein: a first average thickness of the first layer differs from a second average thickness of the second layer; the first channels of the plurality of first channels are in fluid communication with the second channels of the plurality of second channels; and a first average diameter of the first channels differs from a second average diameter of the second channels. Features of such perforated membranes, as described above, are found in Section I of this disclosure: Perforated Membranes.

[0233] The embodiments of channels arranged through the perforated membrane can be any of the features of the channels previously discussed in the perforated membranes of this disclosure. Such features of channels for perforated membranes are provided, for example, in the channels of perforated membranes (such as perforated membranes made by sacrificial template imprinting) produced according to Section I: Perforated Membranes of this disclosure and according to Section II: Methods of Manufacturing Perforated Membranes. In some cases, the channels are distributed throughout the perforated membrane in a patterned or unpatterned arrangement.

[0234] The first spacer array includes a first thickness, a plurality of pores defined by a 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 of this disclosure: High-throughput Cell Electroporation Systems with Bilayer Perforated Membranes and Spacer Arrays. In some cases, the first spacer array includes: (a) a thickness greater than the diameter of the donor cell; and (b) a plurality of first pores, wherein the first pores of the plurality of first pores include a first pore width and a second pore width perpendicular to the first width, wherein the first pores of the first spacer array have a cross-sectional area defined by the material of the first spacer array. In some cases, the first pore width and the second pore width of the first pores of the first spacer array are smaller than the surface area of ​​the perforated membrane surface having a first plurality of channel openings. As previously described, the first spacer array includes a plurality of first pores. In some cases, the first pores of the first spacer array are covered by a perforated membrane.

[0235] The first aperture of the first spacer array has a first aperture width and a second aperture width, as previously described. In some cases, the first aperture width is at least about 1 mm. In some cases, the first aperture width ranges from at least about 1 mm to about 10 cm. In some cases, the first aperture width is about 1 cm. In some cases, the second aperture width is at least about 5 mm. In some cases, the second aperture width ranges from at least about 1 mm to about 10 cm. In some cases, the second aperture width is about 1 cm.

[0236] The first aperture of the first spacer array also has an inter-aperture distance defined by the presence of material in the first spacer array, which separates the first aperture from another first aperture adjacent to it. In some cases, the first aperture is separated by the inter-aperture distance and another first aperture adjacent to the first aperture of the first spacer array. In some cases, the first aperture of the first spacer array is separated by an inter-aperture distance of at least about 1 mm and another first aperture adjacent to the first aperture of the first spacer array. In some cases, the first aperture of the first spacer array is separated by an inter-aperture distance from about 1 mm to about 1.5 cm or from about 1 mm to about 5 cm and another first aperture adjacent to the first aperture of the first spacer array. In some cases, the first aperture of the first array spacer is separated by an inter-aperture distance of about 5 mm and another first aperture adjacent to the first aperture of the first spacer array.

[0237] As previously provided, the first spacer array also has a first thickness. In some cases, the thickness of the first spacer array ranges from about 0.1 cm to about 5 cm.

[0238] The first spacer array also has an outer perimeter defined by its outer length and width, and lies in a plane parallel to the surface of the first spacer array in contact with the perforated membrane. In some cases, the width of the outer side of the first spacer array ranges from about 1 cm to about 30 cm.

[0239] The first spacer array also has a number of first holes. In some cases, the first spacer array contains at least about 2 first holes. In some cases, the first spacer array contains 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.

[0240] As outlined above, the plurality of first holes in the first spacer array can be arranged in an array having rows and columns. In some cases, the plurality of first holes in the first spacer array comprises: (a) at least two rows of first holes; and (b) at least two columns of first holes. The first spacer array can have any number of first holes arranged in any desired number of rows and columns.

[0241] As described above, the first spacer array is made of a material such as any material described in the spacer array of this disclosure. For example, the first spacer array may include a polymer. In some cases, the first spacer array includes a thermoplastic polymer, a crosslinked polymer, a photocurable polymer, a thermosetting polymer, a photoreactive polymer, a silicone polymer, a rubber, or a photoresist polymer. In some cases, the first array spacers include polyethylene terephthalate (PET), polycarbonate, polydimethylsiloxane (PDMS), polydimethylsiloxane (PDMA), polyvinyl alcohol (PVA), polyethylene, polypropylene, polystyrene, polyacrylamide, or polyacrylic acid.

[0242] The second spacer array includes a second thickness, a plurality of pores defined by a 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 of this disclosure: High-throughput Cell Electroporation System with a Double-Layer Perforated Membrane and Spacer Array. The second spacer array of the system includes: (a) a second thickness greater than the diameter of the donor cell; and (b) a plurality of second pores, wherein the second pores of the second spacer array include a first pore width and a second pore width perpendicular to the first width; wherein the second pores of the second spacer array have a cross-sectional area defined by the 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 pores of the second spacer array do not exceed the surface area of ​​the perforated membrane surface having a second plurality of channel openings. In some cases, the second pore width and the second pore width of the second spacer array are smaller than the surface area of ​​the perforated membrane surface having a second plurality of channel openings. As previously described, the second spacer array includes a plurality of second pores. In some cases, the second pores of the second spacer array are covered by the perforated membrane.

[0243] The second aperture of the second spacer array has a first aperture width and a second aperture width, as previously described. In some cases, the first aperture width of the second aperture is at least about 1 mm. In some cases, the first aperture width of the second aperture ranges from at least about 1 mm to about 10 cm. In some cases, the first aperture width of the second aperture is about 1 cm. In some cases, the second aperture width of the second aperture is at least about 5 mm. In some cases, the second aperture width of the second aperture ranges from at least about 1 mm to about 10 cm. In some cases, the second aperture width of the second aperture is about 1 cm. In some cases, the first aperture width and the second aperture width of the second aperture of the second spacer array are the same as the first aperture width and the second aperture width of the first aperture of the first spacer array. In some cases, the first aperture width and the second aperture width of the second aperture of the second spacer array are different from the first aperture width and the second aperture width of the first aperture of the first spacer array.

[0244] The second aperture of the second spacer array also has an inter-aperture distance defined by the presence of material in the second spacer array, which separates the second aperture from another adjacent second aperture. In some cases, the second aperture is separated by the inter-aperture distance and another adjacent second aperture of the second spacer array. In some cases, the second aperture of the second spacer array is separated by an inter-aperture distance of at least about 1 mm and another adjacent second aperture of the second spacer array. In some cases, the second aperture of the second spacer array is separated by an inter-aperture distance from about 1 mm to about 1.5 cm or from about 1 mm to about 5 cm and another adjacent second aperture of the second spacer array. In some cases, the second aperture of the second array spacer is separated by an inter-aperture distance of about 5 mm and another adjacent second aperture of the second spacer array. In some cases, the inter-aperture distance of the second spacer array differs from the inter-aperture distance of the first spacer array. In some cases, the inter-aperture distance of the second spacer array is substantially the same as the inter-aperture distance of the first spacer array. In some cases, the inter-aperture distance of the second spacer array is equivalent to the inter-aperture distance of the first spacer array. In some cases, the distance between a second hole that separates from other second holes adjacent to the second holes of the second spacer array is different from the distance between a first hole that separates from other first holes adjacent to the first holes of the first array of holes of the first spacer array. In some cases, the distance between a second hole that separates from other second holes adjacent to the second holes of the second spacer array is substantially the same as the distance between a first hole that separates from other first holes adjacent to the first holes of the first array of holes of the first spacer array. In some cases, the distance between a second hole that separates from other second holes adjacent to the second holes of the second spacer array is equivalent to the distance between a first hole that separates from other first holes adjacent to the first holes of the first array of holes of the first spacer array.

[0245] 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 first thickness of the first spacer array. In some cases, the second thickness of the second spacer array is different from the first thickness of the first spacer array.

[0246] The second spacer array also has an outer perimeter defined by its outer length and width, and lies in a plane parallel to the surface of the second spacer array in contact with the perforated membrane. In some cases, the width of the outer side of the second spacer array ranges from about 1 cm to about 30 cm.

[0247] The second spacer array also has a number of second holes. In some cases, the second spacer array contains at least about 2 second holes. In some cases, the second spacer array contains 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.

[0248] As outlined above, the plurality of second holes in the second spacer array can be arranged in an array having rows and columns. In some cases, the plurality of second holes in the second spacer array comprises: (a) at least two rows of second holes; and (b) at least two columns of second holes. The second spacer array can have any number of second holes arranged in any desired number of rows and columns.

[0249] In some cases, the second spacer array is equivalent to the first spacer array. In some cases, the second spacer array is substantially the same as the first spacer array. In some cases, the second spacer array differs from the first spacer array in at least one respect.

[0250] As described above, the second spacer array is made of a material such as any material described in the spacer array of this disclosure. For example, the second spacer array may include a polymer. In some cases, the second spacer array includes a thermoplastic polymer, a crosslinked polymer, a photocurable polymer, a thermosetting polymer, a photoreactive polymer, a silicone polymer, a rubber, or a photoresist polymer. In some cases, the second array spacers include polyethylene terephthalate (PET), polycarbonate, polydimethylsiloxane (PDMS), polydimethylsiloxane (PDMA), polyvinyl alcohol (PVA), polyethylene, polypropylene, polystyrene, polyacrylamide, or polyacrylic acid.

[0251] Systems for high-throughput electroporation may also include a cathode. The cathode is made of a conductive material that is non-toxic to at least one donor cell and has a three-dimensional shape, other features described in this disclosure, such as those in Section III: High-throughput Cell Electroporation Systems with Double-Layer Perforated Membranes and Spacer Arrays. The cathode may have any one or a combination of the embodiments provided throughout the disclosure. As previously outlined, the cathode may be configured to be disposed within a first aperture (e.g., a cell culture chamber) of the first spacer array. In some cases, the cathode includes shapes such as three-dimensional (3D) cathode plates or rods. In some cases, the cathode is configured to contact the surface of the first or second spacer array. In some embodiments, the cathode includes a cathode coil configured to be disposed within a first aperture of the first spacer array or a second aperture of the second spacer array. The cathode may include an array of cathode coils having any of the features described above.

[0252] Systems for high-throughput electroporation may also include an anode. The anode is made of a conductive material that is non-toxic to at least one donor cell and has a three-dimensional shape, other features described in this disclosure, such as those in Section III: High-throughput Cell Electroporation Systems with Double-Layer Perforated Membranes and Spacer Arrays. The anode may have any one or a combination of the embodiments provided throughout the disclosure. As previously outlined, the anode may be configured to be disposed within a first well (e.g., a cell culture chamber) of a first spacer array. In some cases, the anode includes shapes such as three-dimensional (3D) anode plates or rods. In some cases, the anode includes a wire anode. In some cases, the anode is configured to contact the surface of a first spacer array or a second spacer array. In some embodiments, the anode includes an anode coil configured to be disposed within a first well of a first spacer array or a second well of a second spacer array. The anode may include a wire anode array configured to position individual wire anodes of the wire anode array within individual wells of a first spacer array or a second spacer array. The anode array may have any of the features of the anode array described above.

[0253] Systems for high-throughput electroporation may also include an electrical pulse generation system configured to supply voltage or current to donor cells (e.g., configured to electroporate donor cells) via the cathode and anode described above. As described above in Section III: High-throughput Cell Electroporation Systems with a Double-Layer Perforated Membrane and Spacer Array, the electrical pulse generation system includes: (a) an electrical pulse generator; (b) an impulse pulse transducer; and (c) a current output device. The electrical pulse generation system may be automated or manually operated. Preferably, the electrical pulse generation system is configured to adjust the characteristics of the electrical pulse applied to at least one donor cell in contact with the perforated membrane. Such electrical pulse generation systems for high-throughput cell electroporation may have any of the characteristics of the electrical pulse generation systems described throughout this disclosure.

[0254] As outlined above, the systems for high-throughput electroporation provided herein may also include liquid handling or dispensing devices configured to: (a) arrange at least one donor cell in at least one individual well of a first spacer array; (b) add or remove electroporation buffer in at least one individual well of a second spacer array; and (c) add or remove cell culture medium in at least one individual well of the first spacer array. Liquid handling or dispensing devices suitable for such systems for high-throughput electroporation may have any one or a combination of the features and embodiments of the previously described liquid handling or dispensing devices.

[0255] Furthermore, the donor cells for the high-throughput electroporation system are in contact with a perforated membrane, such as with the opening of at least one channel of the perforated membrane, and have other characteristics, including cell type. The donor cells of the system may have any of the characteristics and embodiments described in this disclosure, such as those in Section III: High-throughput Cell Electroporation Systems with a Double-Layer Perforated Membrane and Spacer Array.

[0256] IV. High-throughput cell electroporation methods This document provides a method for generating and collecting extracellular vesicles. The method includes: (a) providing an electroporation device comprising a plurality of pores, such device comprising a perforated membrane located between a first array of spacers and optionally a second array of spacers, wherein: the spacer array comprises a complementary array of pores defining the cross-sectional area of ​​the pores; the pores 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 fluidly couple the cell culture chamber to the electroporation buffer chamber; (b) introducing donor cells into at least one cell culture chamber; (c) introducing polynucleotides, DNA, RNA, vectors, or plasmids into at least one electroporation buffer chamber; (d) applying an electric field, current, or voltage to one or more electroporation reagent chambers of the porous electroporation device to electroporate the donor cells; and (e) collecting extracellular vesicles (EVs) generated by the donor cells. In some cases, the method includes sequentially electroporating 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 donor cells may include simultaneously introducing at least two transfection reagents into the donor cells. In some cases, delivering more than one transfection reagent to donor cells may include repeating steps (b) through (e), steps (c) through (d), or only repeating step (d) of the method, thereby introducing one or a portion of the transfection reagent to be electroporated into the donor cells into the electroporation reagent chamber, and repeating the method until all transfection reagents are electroporated into the donor cells. A method for generating extracellular vesicles is provided in U.S. Patent No. 11,674,130, the details of which are incorporated herein by reference. In some cases, steps (a) through (e) or steps (a) through (d) of the method may be repeated at least once, twice, three times, or five times.

[0257] Extracellular vesicles (EVs) generated by this method can be extracellular vesicles or any membrane-bound granules (e.g., vesicles with a lipid bilayer). Typically, the extracellular vesicles presented herein are secreted by cells. In some cases, extracellular vesicles are membrane-bound granules generated in vitro. In some cases, extracellular vesicles are produced and secreted by extracellular vesicle donor cells transfected with at least one heterologous polynucleotide. In some cases, extracellular vesicles are exosomes, microvesicles, retrovirus-like granules, apoptotic bodies, oncosomes, exophers, enveloped viruses, exomere, or other very large extracellular vesicles (such as large oncosomes). In some cases, extracellular vesicles are exosomes.

[0258] In some cases, the method produces at least about 3 times more extracellular vesicles (EVs) secreted by electroporated donor cells compared to the number of EVs secreted by a non-electroplated donor cell population. In some cases, the method produces at least about 5, 10, 100, 1,000, 5,000, 10,000, 20,000, 50,000, or 100,000 times more extracellular vesicles (EVs) secreted by electroporated donor cells compared to the number of EVs secreted by a non-electroplated donor cell population. In some cases, the method produces an increase in the number of extracellular vesicles (EVs) secreted by electroporated donor cells by about 3 to about 100,000 times, about 10 to about 10,000 times, about 10 to about 1,000 times, about 10 to about 1,000 times, about 10 to about 100 times, about 10 to about 100 times, about 100 to about 100,000 times, about 100 to about 100,000 times, about 100 to about 10,000 times, about 100 to about 100,000 times, about 1,000 to about 5,000 times, about 5,000 to about 10,000 times, or about 100 to about 20,000 times compared to the number of EVs secreted by a non-electroplated donor cell population.

[0259] In some cases, the method produces at least about 1.1 times more extracellular vesicles (EVs) secreted by electroporated donor cells than by a donor cell population electroporated on a track-etched membrane, compared to a baseline amount of secreted EVs. In some cases, the method produces at least about 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 1,000 times, 10,000 times, 20,000 times, 50,000 times, or 100,000 times more extracellular vesicles (EVs) secreted by electroporated donor cells than by a donor cell population electroporated on a track-etched membrane, compared to a baseline amount of secreted EVs. In some cases, the method produces an increase in the number of extracellular vesicles (EVs) secreted by electroporated donor cells by approximately 1.2-fold to approximately 50-fold, approximately 1.2-fold to approximately 20-fold, approximately 1.2-fold to approximately 10-fold, approximately 1.2-fold to approximately 20-fold, approximately 1.2-fold to approximately 10-fold, and approximately 2-fold to approximately 100-fold, compared to a baseline amount of secreted EVs. In some cases, the method produces an increase in the number of extracellular vesicles (EVs) secreted by electroporated donor cells by at least approximately 1.5-fold, compared to a baseline amount of secreted EVs.

[0260] In some cases, the method produces at least about 1.1 times more extracellular vesicles (EVs) secreted by electroporated donor cells than by a population of donor cells electroporated on an electroporated membrane with a patterned array lacking channels, compared to a baseline amount of secreted EVs. In some cases, the method produces at least about 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 1,000 times, 10,000 times, 20,000 times, 50,000 times, or 100,000 times more extracellular vesicles (EVs) secreted by electroporated donor cells than by a population of donor cells electroporated on an electroporated membrane with a patterned array lacking channels, compared to a baseline amount of secreted EVs. In some cases, the method produces an increase in the number of extracellular vesicles (EVs) secreted by electroporated donor cells by approximately 1.2-fold to approximately 50-fold, approximately 1.2-fold to approximately 20-fold, approximately 1.2-fold to approximately 10-fold, approximately 1.2-fold to approximately 20-fold, approximately 1.2-fold to approximately 10-fold, and approximately 2-fold to approximately 100-fold, compared to a baseline amount of secreted EVs. In some cases, the method produces an increase in the number of extracellular vesicles (EVs) secreted by electroporated donor cells by at least approximately 1.5-fold, compared to a baseline amount of secreted EVs.

[0261] In some cases, the method produces at least approximately 3-fold more extracellular vesicles (EVs) secreted by electroporated donor cells compared to the baseline amount of secreted EVs from donor cell populations transfected with lipofectamine, compared to the baseline amount of secreted EVs. In some cases, the method produces at least approximately 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 more extracellular vesicles (EVs) secreted by electroporated donor cells compared to the baseline amount of secreted EVs from donor cell populations transfected with lipofectamine, compared to the baseline amount of secreted EVs. In some cases, the method produces an increase in the number of extracellular vesicles (EVs) secreted by donor cells from electroporation compared to the baseline amount of secreted EVs from donor cell populations transfected with lipofectamine, ranging from about 3 to about 100,000 times, from about 10 to about 10,000 times, from about 10 to about 1,000 times, from about 10 to about 1,000 times, from about 10 to about 100 times, from about 100 to about 100,000 times, from about 100 to about 100,000 times, from about 100 to about 10,000 times, from about 100 to about 100,000 times, from about 1,000 to about 5,000 times, from about 5,000 to about 10,000 times, or from about 100 to about 20,000 times.

[0262] In some cases, at least about 50% of the extracellular vesicles (EVs) secreted by electroporated donor cells contain polynucleotides. In some cases, at least about 70% of the extracellular vesicles (EVs) secreted by electroporated donor cells contain polynucleotides. In some cases, at least about 75%, 80%, 85%, 90%, 95%, 97.5%, or 100% of the extracellular vesicles (EVs) secreted by electroporated donor cells contain polynucleotides. In some cases, about 50% to about 95% of the extracellular vesicles (EVs) secreted by electroporated donor cells contain polynucleotides. In some cases, about 70% to about 95% of the extracellular vesicles (EVs) secreted by electroporated donor cells contain polynucleotides. In some cases, about 80% to about 95% of the extracellular vesicles (EVs) secreted by electroporated donor cells contain polynucleotides. In some cases, approximately 85% to 95% of extracellular vesicles (EVs) secreted by donor cells from electroporation contain polynucleotides.

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

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

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

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

[0267] In some cases, the introduction of polynucleotides includes introducing at least two, at least three, at least four, at least five, or more polynucleotides into at least one electroporation buffer chamber.

[0268] In some cases, extracellular vesicles can have a diameter of about 10 nm to about 50,000 nm. In some cases, extracellular vesicles can have a diameter of about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 50 nm, about 10 nm to about 100 nm, about 10 nm to about 200 nm, about 10 nm to 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 nm to 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 nm to 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 nm to about 5,000 nm, about 30 nm to about 50 nm. nm, about 30 nm to about 100 nm, about 30 nm to about 200 nm, about 30 nm to about 500 nm, about 30 nm to 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 nm to about 2,000 nm, about 100 nm to about 5,000 nm nm, about 100 nm to about 10,000 nm, about 100 nm to 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 nm to about 50,000 nm, about 500 nm to about 1,000 nm, about 500 nm to about 2,000 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,The diameters range from about 1,000 nm to about 5,000 nm, from about 1,000 nm to about 10,000 nm, from about 1,000 nm to about 50,000 nm, from about 2,000 nm to about 5,000 nm, from about 2,000 nm to about 10,000 nm, from about 2,000 nm to about 50,000 nm, from about 5,000 nm to about 10,000 nm, from about 5,000 nm to about 50,000 nm, or from about 10,000 nm to about 50,000 nm. In some cases, extracellular vesicles have diameters of about 10 nm, about 20 nm, about 30 nm, about 50 nm, about 100 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, extracellular vesicles may have a diameter of at least about 10 nm, about 20 nm, about 30 nm, about 50 nm, about 100 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 other cases, extracellular vesicles may have a diameter of at most about 20 nm, about 30 nm, about 50 nm, about 100 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.

[0269] In some cases, extracellular vesicles (EVs) are exosomes, apoptotic bodies, or microvesicles.

[0270] Providing a porous electroporation apparatus may include providing a porous electroporation apparatus or system as previously described throughout this disclosure.

[0271] The perforated membrane of an electroporation device containing multiple pores can be a silicon membrane, a track-etched membrane, a single-layer polymer membrane, a double-layer polymer membrane, or any perforated membrane having the characteristics of a perforated membrane discussed above.

[0272] Introducing donor cells into one or more cell culture chambers may include adding, positioning, or arranging the donor cells within at least one or more of a plurality of cell culture chambers in the porous electroporation apparatus and system for high-throughput cell electroporation described above. In some cases, introducing donor cells into the cell culture chamber includes contacting or adhering the donor cells to a first surface of a perforated membrane provided throughout this disclosure.

[0273] Introducing polynucleotides, DNA, RNA, vectors, or plasmids into an electroporation buffer chamber may include introducing any one or a combination of the transfection reagents provided herein into at least one or more of the electroporation reagent chambers (e.g., electroporation buffer chambers) of the porous electroporation apparatus and system of this disclosure.

[0274] Applying an electric field to donor cells may include applying an electric field or current of any voltage, electric field strength, or as described in, for example, Section III: High-throughput Cell Electroporation Systems with an Array of Perforated Membranes and Spacers. Applying an electric field to donor cells may include applying any described electrical pulse generation routine, such as the number of cell culture chambers / electroporation chambers to be stimulated, the stimulation pattern (e.g., chamber-to-chamber sequence, checkerboard pattern, simultaneous stimulation of non-adjacent cell culture chambers / electroporation chambers, etc.), and any other parameters provided in this disclosure, according to any embodiment of the electrical pulse generation system utilizing a porous electroporation device. In some cases, applying an electric field to donor cells includes sequentially applying an electric field to at least 1, 2, 3, 4, 5, 10, 12, 16, 25, or more cell culture chambers / electroporation chambers of the porous electroporation device. In some cases, applying an electric field to donor cells includes sequentially applying an electric field to the cell culture chambers / electroporation chambers of the porous electroporation device until all cell culture chambers / electroporation chambers have been applied. In some cases, the electric field applied to one cell culture chamber / electroporation chamber may be different from the electric field applied to another cell culture chamber / electroporation chamber of the porous electroporation apparatus.

[0275] Once an electric field is applied, the donor cell is an electroporated donor cell capable of expressing a transfection reagent or a combination of transfection reagents. In some cases, the electroporated donor cell may generate extracellular vesicles (EVs) that encapsulate the expression product (e.g., mRNA, protein, or other biomolecule) of one or more transfection reagents that have been electroporated into the donor cell. In some cases, the transfection reagent may include a polynucleotide, a protein encoded by DNA or RNA, or a biomolecule of interest such as collagen (e.g., Col1A1) or dystrophin. In the case of a repetitive method, the electric field applied in each iteration may differ from the electric field applied during another iteration of the method.

[0276] Collecting extracellular vesicles (EVs) generated from donor cells may include removing EVs by manual or automated liquid handling devices. EVs can then be further processed by any method to concentrate, isolate, and quantify parameters or characteristics of the EVs.

[0277] Extracellular vesicles (EVs) are collected some time after an electric field is applied to the donor cells. In some cases, EVs are collected within one hour of applying the electric field to the donor cells. In some cases, EVs are collected approximately 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 16, 20, 24, 30, 40, 45, 50, 72, 84, or 96 hours after applying the electric field to the donor cells. In some cases, EVs are collected at least approximately 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 after applying the electric field to the donor cells. In some cases, extracellular vesicles (EVs) are collected within approximately 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 16, 20, 24, 30, 40, 45, 50, 72, 84, or 96 hours after an electric field is applied to the donor cells.

[0278] When the method is repeated sequentially iteratively to add additional electroporation reagents to donor cells, extracellular vesicles (EVs) are collected approximately 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 16, 20, 24, 30, 40, 45, 50, 72, 84, or 96 hours after the last iteration of the method is applied to the donor cells. When the method is repeated sequentially iteratively, EVs can be collected after the application of the electric field of the method's iteration and before the next iteration after introducing one or more types of donor cells or adding one or more electroporation reagents to one or more electroporation reagent chambers of the porous electroporation apparatus. When the method is repeated sequentially iteratively, extracellular vesicles (EVs) are collected approximately 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 16, 20, 24, 30, 40, 45, 50, 72, 84, or 96 hours after the last iteration of the method is applied to the donor cells. When the method is repeated iteratively in sequence, extracellular vesicles (EVs) are collected 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 after the electric field of the last iteration of the method is applied to the donor cells.

[0279] definition Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or specialized expressions used herein are intended to have the same meaning as 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 convenience of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a material difference from what is commonly understood in the art.

[0280] Throughout this application, various cases may be presented in a range format. It should be understood that the range format is merely for convenience and brevity and should not be construed as an immutable limitation on the scope of this disclosure. Therefore, a description of a range should be considered as specifically disclosing all possible subranges and individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be considered as specifically disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0281] As used in the specification and claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly specifies otherwise. For example, the terms “channel,” “cell culture well,” “electroporation reagent well,” and “well distance” include multiple channels, multiple cell culture wells, or multiple electroporation reagent wells.

[0282] As used herein, open terms such as “include,” “containing,” “including,” “have,” etc., mean “include,” unless otherwise indicated.

[0283] "Optional" or "optionally" means that the event or situation described below may or may not occur, and such description includes instances where the event or situation occurs and instances where it does not occur.

[0284] Throughout the description and claims of this specification, the word “comprising” and variations thereof, such as “including” and “comprising”, mean “including but not limited to” and are not intended to exclude, for example, other additives, components, integers, or steps. “Exemplary” means “an example of…” and is not intended to convey indications of preferred or desirable aspects. “Like” is not used in a limiting sense but for interpretive purposes.

[0285] As used herein, the term "about (approximately / approximately)" refers to a number plus or minus 10% of that number. The term "about (approximately / approximately)" refers to a range minus 10% of its lowest value and plus 10% of its highest value. Similarly, when the term "about (approximately / approximately)" is used before a non-numerical term (e.g., horizontal, vertical, alignment) as an alternative to a numerical value, the term "about (approximately / approximately)" refers to the value of the non-numerical term (e.g., 90 degrees, 180 degrees) plus or minus 10% of that value.

[0286] As used herein, the term “substantially” when describing a relative value, relative quantity, or relative degree between two objects typically means 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 terms of value, quantity, or degree.

[0287] Unless otherwise specified, whenever the terms “at least,” “greater than,” “greater than or equal to,” “not exceeding,” “less than,” or “less than or equal to” precede the first value in a series of two or more values, the term applies to each value in that series. 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.

[0288] When the terms "not exceeding," "less than," or "less than or equal to" precede the first value in a series of two or more values, the terms "not exceeding," "less than," or "less than or equal to" apply to each value in the series. 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.

[0289] As used herein, the term “about (approximately / approximately)” means within 10% of a given value, either above or below. For example, “about 10” would include values ​​from 9 to 11, unless the context in which the term is used indicates otherwise.

[0290] As used herein, “or” can mean “and,” “or,” or “and / or,” and can be used individually or inclusively. For example, the term “A or B” can mean “A or B,” “A without B,” “B without A,” and “A and B.” In some cases, the context may prescribe a specific meaning.

[0291] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0292] The term “diameter” is used in this document to refer to the distance between the first wall of the pore and the other wall opposite the first wall of the pore.

[0293] The terms “array,” “patterned arrangement,” and “patterned distribution” are used interchangeably herein to refer to the consistent distance between adjacent or adjacent individual channels of a perforated membrane with respect to individual channels. For example, an “array” of individual channels may have consistent distances between adjacent individual channels in a first dimension (e.g., the width of the perforated membrane) and consistent distances between adjacent individual channels in a second dimension (e.g., the length of the perforated membrane). As a non-limiting illustrative example, where the distance between individual channels in the channel array is constant across the first and second surfaces of the perforated membrane, individual channels can be separated by a distance of approximately 5 µm along the length of the perforated membrane and approximately 5 µm along the width of the perforated membrane. Furthermore, where the rows or columns of individual channels in the channel array are staggered, individual channels can be separated by a distance of approximately 5 µm along the length of the perforated membrane and approximately 10 µm along the width of the perforated membrane.

[0294] The term “inter-aperture width” is used herein to refer to the distance between adjacent individual apertures of an array of spacers, defined by the presence of the spacer array material between the wall of the first individual aperture and the wall of another individual aperture adjacent to the first individual aperture.

[0295] The term “first hole width” is used herein to refer to the distance between the first wall of a single hole along a dimension parallel to the first surface of the spacer array and the second wall of a single hole opposite to the first wall of the single hole in the spacer array, and this distance is defined by the absence of material in the spacer array.

[0296] The term "second aperture width" is used herein to refer to the wall-to-wall distance between individual apertures perpendicular to the first aperture width of the individual channels of the spacer array, whereby the wall-to-wall distance is defined by the absence of material from the spacer array. As a non-limiting example, an individual square aperture of the spacer array has four walls defined by the material of the spacer array. The "first aperture width" is the distance between the first pair of opposing walls of the individual aperture, defined by the absence of spacer array material. The "second aperture width" is a second distance between a second pair of opposing walls, which are perpendicular to the first pair of opposing walls of the same individual aperture.

[0297] As used herein, the term "substrate" or "solid substrate" generally refers to a substance, structure, surface, material, method, or composition that includes non-biological, synthetic, inanimate, planar, spherical, or flat surfaces. Substrates can include, for example, but not limited to, semiconductors, synthetic metals, synthetic semiconductors, insulators, and dopants; metals, alloys, elements, compounds, and minerals; synthetic, cut, etched, photolithographic, printed, machined, and microfabricated slides, devices, structures, and surfaces; industrial polymers, plastics, and films; silicon, silicates, glass, metals, and ceramics; wood, paper, cardboard, cotton, wool, cloth, woven and non-woven fibers, materials, and fabrics; nanostructures and microstructures. Substrates can include immobilized matrices, such as, but not limited to, insoluble substances, solid phases, surfaces, layers, coatings, woven and non-woven fibers, matrices, crystals, films, insoluble polymers, plastics, glass, biological or biocompatible or biodegradable polymers or matrices, microparticles, or nanoparticles. Other examples may include, for example, but not limited to, monolayers, bilayers, commercial membranes, resins, matrices, fibers, separation media, chromatographic supports, polymers, plastics, glass, mica, gold, beads, microspheres, nanospheres, silicon, gallium arsenide, organic and inorganic metals, semiconductors, insulators, microstructures, and nanostructures. Microstructures and nanostructures may include, but are not limited to, miniaturized, nanoscale, and supramolecular probes, tips, rods, plugs, stoppers, rods, sleeves, wires, filaments, and tubes. For example, the substrate may exist as one or more particles, chains, precipitates, gels, sheets, tubes, spheres, containers, capillaries, pads, sheets, films, plates, glass slides, or semiconductor integrated surfaces. The substrate may be flat or may present alternative surface configurations. For example, the substrate may contain raised or recessed regions where synthesis or deposition occurs. In some examples, the substrate may contain raised or recessed regions present in different 3-D shapes and / or heights. In some examples, the substrate may include multiple features. In some examples, the substrate may include a topographic pattern, and the topography may include a set of grooves, a set of rods, a set of pillars, a set of holes, or combinations thereof. In some cases, topographic patterns can be formed using at least two different materials, such as a silicon surface covered with a layer of photoresist or a quartz surface covered with hydrogel. In some examples, the substrate may contain raised or recessed regions with substantially the same 3-D shape and / or height. In some examples, the substrate may be chosen to provide appropriate light absorption characteristics.For example, the substrate can be any of the following: polymerized Langmuir Blodgett thin film, functionalized glass (e.g., glass with controllable porosity), silica, titanium dioxide, alumina, indium tin oxide (ITO), Si, Ge, GaAs, GaP, SiO2, SiN4, modified silicon, top dielectric layer on the surface of a semiconductor integrated circuit (IC), or any of various gels or polymers (such as (poly)tetrafluoroethylene, (poly)vinylidene fluoride, polystyrene, polycarbonate, polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA), polycyclic olefins, or combinations thereof).

[0298] The substrate may include a polymer coating or gel, such as a polyacrylamide gel or a PDMS gel. Gels and coatings may additionally include components to modify their physicochemical properties, such as hydrophobicity. For example, a polyacrylamide gel or coating may contain modified acrylamide monomers, such as ethoxylated acrylamide monomers, phosphorylcholine acrylamide monomers, betaine acrylamide monomers, and combinations thereof, in its polymer structure.

[0299] The terms “determine,” “measure,” “evaluate,” “assess,” “determine,” and “analyze” are used interchangeably herein to refer to forms of measurement. These terms include determining (e.g., detecting) the presence of an element (component or element). These terms can include quantitative determination, qualitative determination, or a combination of both. Assessment can be relative or absolute. Depending on the context, in addition to determining the presence of something, “detecting presence” can also include determining the quantity of that thing present.

[0300] Example The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0301] Example 1. Design of a bilayer polymer perforated membrane Silicon substrates for cell electroporation / nanoelectroporation (CEP / CNP) have several limitations, including high cost, brittleness, and labor-intensive cleanroom semiconductor manufacturing protocols. While silicon-based electroporation / nanoelectroporation surfaces are effective for proof-of-concept studies and small-scale mRNA production in extracellular vesicles (EVs), efforts to scale up the production and efficiency of CEP / nanoelectroporation will greatly benefit from low-cost, mass-producible polymer alternatives. Bilayer polymeric CEP / CNP surfaces also offer superior production performance for EV mRNA drugs compared to track-etched films, especially for large mRNA molecules.

[0302] We have invented a polymer CEP / CNP surface design based on a photoresist-coated silicon (Si) film. Instead of the thin photoresist film used for patterning in Si CEP / CNP surface production, we apply a thick photoresist film (approximately 10 µm thick) onto the silicon film, and the film can be demolded after patterning to form a freestanding CEP / CNP surface. This method eliminates the need to fabricate “back-side” holes on the Si film and the expensive deep reactive ion etching (DRIE) for silicon etching. Compared to Si CEP / CNP surfaces, in addition to cost savings, the entire surface can be used for greater cell loading and EVmRNA production. While many photoresist resins are applicable, we demonstrate this approach here using the epoxy-based positive photoresist SU-8. SU-8 is an epoxy-based photoresist that can be used to photolithographically fabricate microstructures of single-pass perforated films at low cost and offers several advantages. It enables the creation of high aspect ratio micro / nanoscale patterns with excellent mechanical properties, thermal stability, and chemical resistance in a single photolithography step. In this study, we demonstrated a bilayer SU-8 film for polymer-based CNP surfaces using SU-8 TF6002 and SU-8 3010 photoresist resins.

[0303] Fabrication of double-layer perforated membranes: such as Figure 1 As shown, the manufacturing method involves several key steps: (1) spin-coating photoresist onto a silicon film to create the first layer of a bilayer perforated film; (2) soft-baking the first photoresist layer to evaporate the solvent and make the SU-8 photoresist more robust; (3) masking the photoresist with an array of pores about 1 µm in diameter to cause the negative photoresist to form nanopores in the first layer of the bilayer perforated film; (4) exposure-after-baking (PEB); (5) repeating steps (1) to (4) on the surface of the first layer with a second photoresist to form the second layer of the bilayer perforated film with an array of larger diameter pores (e.g., 2 µm to about 8 µm in diameter); and (6) simultaneously developing both photoresist layers to dissolve the unexposed photoresist to form smaller pores (i.e., nanochannels or pores about 1 µm in diameter) in the first layer, which are continuous with the larger diameter pores (i.e., microchannels) in the second layer to form a bilayer perforated film with “bottle”-shaped pores spanning the thickness of the two photoresist layers. Figure 2 and Figure 3 ).

[0304] Briefly, a 2 µm thick layer of SU-8 TF 6002 was first spin-coated onto a Si film coated with an OmniCoat release layer at 4000 rpm. After spin-coating with SU-8 TF 6002, the SU-8 TF 6002 was soft-baked at 110 °C for 3 minutes. A first mask with a 1 µm pore array was used for UV exposure, followed by PEB at 110 °C for 2 minutes. Then, an 8 µm thick layer of SU-8 3010 was spin-coated on top of the exposed but undeveloped SU-8 TF 6002 layer, followed by soft-baking at 95 °C for 7 minutes. A second mask with a larger pore array, patterned with the first mask, was used for UV exposure, followed by PEB (1 minute at 65 °C and 2 minutes at 95 °C). After that, both SU-8 layers were developed simultaneously. SU-8 developer dissolves in areas not exposed to UV light during each exposure step (blocked by the dark areas of the photomask). Finally, the OmniCoat release layer is removed to release the SU-8 bilayer film.

[0305] Design considerations and challenges: Two key design constraints were addressed: (1) the membrane needed to have a minimum pore size of 1 µm for the CNP surface, and (2) the final membrane thickness was approximately 10 µm to ensure sufficient mechanical strength for the treatment.

[0306] Achieving a pore size of 1 µm in a single-layer SU-8 photoresist film with a thickness greater than 2 µm (aspect ratio > 2:1) is technically challenging. As film thickness increases, UV light penetration across the entire film becomes more difficult, especially at smaller feature sizes. To overcome this, we implemented a two-layer approach. SU-8 TF 6002 photoresist (designed for high-resolution thin structures) was selected for the first (thin) layer to achieve nanopores with a pore diameter of approximately 1 µm. Additionally, SU-8 3010 photoresist was selected for the second (thick) layer to provide additional mechanical strength, resulting in a total film thickness of approximately 10 µm. The larger-diameter micropores on the thick layer were carefully aligned with the approximately 1 µm nanopores on the thin layer to achieve a CEP / CNP surface design with a “bottle-like” pore structure. Bottle-like pore structures are advantageous because they reduce cell damage from electroporation through small nanopores in contact with donor cells and minimize plasmid DNA aggregation and / or pore (i.e., channel) blockage due to contact between larger diameter micropores and plasmid DNA solution. This is particularly important for delivering very large transfection cargoes (e.g., DNA plasmids, RNA, and small molecules) into donor cells.

[0307] like Figure 4A and Figure 4BAs shown, we also fabricated a double-layer perforated film that uses SU-8 2002 photoresist as a thin layer to form channels with a smaller diameter of about 1 µm and SU-8 2005 photoresist as a thick layer to form microchannels with a larger diameter (e.g., 3-4 µm). The fabrication method of this double-layer perforated film is similar to that described for the SU-8 TF 6002-SU-8 3010 double-layer perforated films.

[0308] An added 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 used for the thin layer (forming nanochannels) is 110°C, while the SU-8 3010 photoresist used for the thick layer (forming microchannels) is processed at a lower PEB temperature of 95°C. This temperature difference allows the thin layer to undergo additional PEB during the processing of the thick layer, which further improves the structural integrity of the bilayer perforated film. This bilayer polymer design circumvents the high aspect ratio challenges we face in monolayer fabrication.

[0309] It is conceivable that multilayers could be used to create pores (i.e., channels) with more complex structures. For large silicon films, the UV exposure rate across the entire film surface may not be uniform. To address this, a thin barrier layer (such as silicon oxide (SiO2), gold, or silver) can be coated onto the layer surface after UV exposure and before adding the next photoresist layer. The presence of the barrier layer prevents overexposure into the underlying layer.

[0310] Example 2. Comparison of bilayer photoresist and silicon CEP / CNP perforated membrane in generating extracellular vesicles loaded with mRNA cargo. CEP / CNP assays were performed to evaluate the performance of the newly developed SU-8 bilayer perforated membrane in generating extracellular vesicles (EVs) encapsulating mRNA therapeutic cargo compared to our original silicon CEP / CNP membrane. To test this potential, approximately 24,000 human skin fibroblasts (from ABM) were seeded onto either a SU-8 bilayer membrane or a silicon membrane with a surface area of ​​0.9 cm × 0.9 cm. Skin fibroblasts were allowed to adhere to form contact with the openings of smaller diameter channels on the perforated membrane surface. After cell incubation, dystrophin (DMD) plasmid DNA was electroporated / nano-electroplated under identical conditions. The electric field applied during CEP / CNP was 222 V / cm at 100 V, with a 4.5 mm electrode-to-electrode distance and ten 10 ms pulses at 0.1 sec intervals. Cell culture was collected after 24 hours. After purification of the supernatant, RT-qPCR was used to quantify the mRNA copy number in the collected EVs. Figure 5The study showed that SU-8 and silicon surfaces produced similar dystrophin mRNA copy numbers / electroporation in fibroblasts. It should be noted that the electric field strength of 222 V / cm was optimized for the silicon perforated membrane, not the SU-8 bilayer perforated membrane. With optimized electroporation conditions, the EV mRNA yield of the SU-8 bilayer perforated membrane could potentially exceed that of the silicon perforated membrane.

[0311] Example 3. Imprinting of polymer CEP / CNP perforated membranes based on a sacrificial template Silicon-based 3D CEP / CNP films are highly brittle. They are also expensive due to the use of silicon (Si) films and the associated labor-intensive semiconductor manufacturing methods that require stringent cleanroom conditions. While Si-based CEP / CNP films are suitable for proof-of-concept studies and small-scale EV production, low-cost, mass-producible polymeric CEP / CNP film alternatives to Si CEP / CNP films are necessary for large-scale cell transfection and EV production for the development of cell- and EV-based gene therapies. Polymers can be more durable and cheaper than silicon films. Furthermore, the microfabrication / nanofabrication of polymers in non-cleanroom conditions is less expensive than cleanroom semiconductor manufacturing. Compared to commercially available polymeric CEP / CNP films with micropores and / or nanopores (e.g., track-etched films), silicon CEP / CNP films offer a clear and uniformly distributed pore size, pattern, and density. These characteristics are crucial for effective cell transfection as they provide high cell viability, especially for primary human cells. Highly efficient CEP / CNP can lead to robust and high-volume EV production with improved loading of genetic cargo or transfection reagents. Conventional polymer fabrication methods (such as injection molding and embossing) do not work well for polymer films with large arrays of micropores and / or nanopores having high aspect ratios (i.e., a pore length / pore diameter ratio much greater than 1), because the mold / master mold and the resulting polymer film can be easily broken due to the high stress generated during manufacturing, processing, and handling.

[0312] Manufacturing Method: This paper describes a novel method for fabricating polymer 3D CEP / CNP films. Although direct imprinting can be used to produce polymer films with channels (e.g., microchannels, nanochannels), our experience shows that the molds do not last very long due to the extremely fragile nano / micropillar structures. Figure 6 As shown, a two-step method is first applied to generate a large number of non-photoresist polymer CEP / CNP surfaces. Here, a patterned photoresist master mold on a silicon film with an array of nanopores / micropores is first designed and fabricated, such as... Figure 1 As shown in the diagram. Many negative molds are first fabricated as transition molds using soft lithography. For example... Figure 7AAs shown, this master mold can be used to prepare numerous PVA sacrificial template molds using water-soluble polymers (such as poly(vinyl alcohol) (PVA)), thereby casting an aqueous solution of PVA onto an SU-8 photoresist master mold. After drying, the sacrificial PVA template replicating the SU-8 master mold can be peeled off from the master mold. Then, a photocurable poly(dimethylsiloxane) (PDMS) resin liquid is imprinted onto the PVA sacrificial mold to form a PDMS film with a clear array of micropores and / or nanopores, such as... Figure 7B As shown in the diagram. Weights can be used to apply uniform force to improve the clarity of micropores and / or nanopores. A layer of PDMS or plastic spacers is added to the back side of the thin layer having a channel array (e.g., a microchannel array, a nanochannel array) to enhance the mechanical strength of the final polymer CEP / CNP. Imprinting can also be performed by spin-coating a liquid resin (such as PDMS or acrylic resin) onto a PVA sacrificial template. The 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 slightly less than the height of the smaller tip on the sacrificial template. After the resin cures, the polymer layer with the channel array can be demolded by dissolving the PVA in water. In some embodiments, the channel array may comprise a microchannel array or preferably a nanochannel array. PMMA nanonozzle arrays (200 nm in diameter) are limited by very thin films (less than 3 µm thick), which are too brittle to process large numbers of cells on a large surface area (S. Wang, C. Zeng, S. Lai, YJ Juang, Y. Yang and LJ Lee, “Polymeric Nanonozzle Array Fabricated by Sacrificial Template Imprinting”, Advanced Materials, 17, 1182-1186 (2005)). Furthermore, fiber bundle-based molds / master molds do not offer design flexibility in pore size, patterning, and density.

[0313] Design Considerations and Challenges: The sacrificial mold is made of a water-soluble polymer, such as polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or PVP / VA copolymers, while the curable polymer resin includes polydimethylsiloxane (PDMS) and acrylic acid. PVA is preferred for both the sacrificial mold and release film on the imprinted surface 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 recycled for reuse. PDMS is the preferred resin material for CNP surfaces due to its widely recognized biocompatibility in both in vitro and in vivo medical applications.

[0314] Spin coating is a simple method, but the challenge lies in controlling the precise PDMS film thickness across the entire surface of the sacrificial mold. If the film is too thick, it covers the protruding structures; however, if it is too thin, it misses smaller pores. In contrast, embossing offers better control over the PDMS film thickness. However, the challenge here is to ensure complete penetration of the protruding structures across the entire surface of the sacrificial mold through the partially cured PDMS resin film. To address this, a soft substrate, such as a rubber layer, is used during embossing to ensure complete contact with the protruding structures across the entire surface of the sacrificial mold. To protect the protruding structures on the surface of the sacrificial mold during embossing, the PVA coated on the rubber layer as a release film can be softened by exposure to hot water vapor or by heating above its glass transition temperature (Tg = 76°C). Alternatively, stronger water-soluble polymers, such as PVP and PVP / VA copolymers (Tg = 100–175°C, depending on molecular weight and copolymer ratio), can be used for the sacrificial mold. Figure 7A and Figure 7B Optical microscopy images of PDMS CEP / CNP surfaces prepared by STI with large pores and PVA-based sacrificial templates are presented.

[0315] Example 4. A porous electroporation cell chamber array device for high-throughput cell electroporation Cells and their derivatives (extracellular vesicles (EVs)) hold immense potential as next-generation drugs. Precise cell transfection for genetic modification / editing is crucial in cell therapy; however, existing cell transfection methods are less than satisfactory. EVs surpass synthetic nanoparticles and viral vectors in mRNA therapy due to their ability to overcome physiological barriers, reduce immunogenicity, and minimize reactiveness. However, cells naturally secrete a limited number of EVs containing relatively little functional mRNA. Our patented technology, Cell Nanoelectroporation (CNP), for gene delivery through nanoscale and submicron-scale pores, enables significant efficacy and high accuracy in cell transfection. CNP also generates a large number of EVs containing substantial amounts of endogenous mRNA. Such engineered EVs have been successfully demonstrated in various mouse models, including cancer therapy, anti-aging, and bone repair, and are currently in Phase I-II clinical trials in humans. For future clinical use, automated high-throughput cell transfection and EV generation are essential to make CNP technology, engineered cells, and their secreted EVs scalable, affordable, and sustainable.

[0316] This disclosure provides a porous cell culture well array attached to a large electroporation / nanoporation perforated membrane. An automated high-throughput CEP / CNP system is achieved by combining an electrode array design with automated cell loading and culture medium collection devices. As previously described, the electroporation / nanoporation surface is a perforated membrane having micropores and / or nanopores and is adhered to an array of polymer spacers that form the boundaries of the cell culture wells. Cells are cultured within individual pores of the porous cell culture spacers, and CEP / CNP is performed when an electrical pulse or multiple electrical pulses are applied to donor cells through openings in channels within the perforated membrane.

[0317] Figure 8 A schematic diagram and photographic example of a CEP / CNP film based on a pore array layer is shown. The film consists of a CEP / CNP layer made of microfabricated / nanofac-manufactured Si film with a clear array of cell transfection pores. Figure 10A ) or commercially available perforated films (such as track-etched films) Figure 10B The spacer array is made of a plate-like material attached to the pore layer / spacer via a bio-adhesive. The circuitry for cell electroporation consists of a cathode, a plate-like material in contact with a negatively charged reagent solution, and an anode, a coil-like or rod-like material, inserted into a cell loading well on the spacer. Different spacer array sizes and pore patterns can be designed. Preferred pore array layers have dimensions matching standard 6-well, 24-well, 96-well, or 384-well microtiter plates to allow the use of commercially available biolab equipment for automating cell and reagent loading prior to electroporation and EV collection in cell culture medium after electroporation.

[0318] Cells are cultured inside each well of the layer / spacer array to adhere to the surface of the CEP / CNP layer. Prior to electroporation, transfection reagents such as plasmid DNA or CRISPR / Cas9 reagents with the desired concentration and composition are prepared and loaded into the reagent reservoir beneath the CEP / CNP layer. A voltage with the desired pulse length, number, and interval is then selected from the electroporation power supply to stimulate the cells adhering in the wells of the chamber layer and deliver the transfection reagent via the pores on the CEP / CNP layer. A coil-like or rod-like anode is then moved from one well to another until cells in all wells are sequentially transfected. This well-to-well sequential electroporation is designed to maintain a low current during electroporation (which depends on the number of pores in each cell well). Otherwise, the high Joule heating caused by high current would lead to severe cell death. EVs secreted by the cells are collected from the cell culture medium after a selected incubation time following electroporation.

[0319] Example 5. A porous electroporation chamber array device for the generation of multiple high-throughput cell electroporation and extracellular vesicles. This disclosure provides a CEP / CNP device based on a dual-pore array chamber layer, comprising a CEP / CNP perforated membrane sandwiched between two polymer spacer arrays. Figure 11A and Figure 11B Here, a polymeric spacer array is attached to the surface of a large CEP / CNP perforated membrane for loading the same or different donor cells into individual cell culture wells defined by openings within the spacer array. The cell culture wells (i.e., the first spacer array) contact the surface of the CEP / CNP perforated membrane with openings having nanochannels. Furthermore, a second polymeric spacer array is attached to the surface of the large CEP / CNP membrane opposite to the first spacer array. The pores within the second spacer array define the dimensions of isolated electroporation reagent chambers, which are then used to load different cell transfection reagents (e.g., plasmid DNA, RNA, CRISPR / Cas9 components, small molecules, etc.) or the same cell transfection reagent at different concentrations and compositions into individual electroporation reagent wells. In summary, this system allows for multiplexed cell transfection and EV generation analysis using different cell types and transfection reagents on a single CNP membrane.

[0320] Unlike Figure 8 In the single-array CEP / CNP device, the plate electrode is the anode and is placed on the cell-side well array chamber layer. After loading the same or different cell types into individual wells and adhering as a monolayer to the surface of the CEP / CNP layer, the membrane is inverted so that different transfection reagents with different concentrations and / or compositions, or the same transfection reagent, can be loaded into individual wells on the reagent side of the chamber layer. Figure 11B and Figure 12 Then, a rod-shaped or coil-shaped cathode array is used. Figure 13 The reagent is inserted into the reagent wells to apply an electric current through the channels of the perforated membrane for automated cell transfection via sequential electroporation. After a specific incubation time, any remaining transfection reagent is removed from each well, followed by removal of the reagent-side chamber layer. Individual transfected cells in each well of the cell-side array layer can be analyzed on the membrane by cell staining or off the membrane by cell removal for further analysis, such as flow cytometry, PCR, and / or Western blotting. If desired, the cell-side chamber layer / spacer array can also be removed to allow for more detailed examination of cells adhering to the surface under a fluorescence microscope at higher magnification.

[0321] Example 6. Comparison of patterned channel array silicon CEP / CNP membranes and track-etched CEP / CNP membranes regarding mRNA production in extracellular vesicles. To test whether CEP / NEP membranes and / or channel arrangements differently affect the mRNA levels of electroporated genetic cargo in extracellular vesicles, neonatal human dermal fibroblasts (nHDF) cells were cultured on patterned channel array silicon CEP / CNP membranes or track-etched CEP / CNP membranes, and then subjected to CEP / CNP with the COL1A1 DNA plasmid. Twenty-four hours after CEP / CNP treatment, extracellular vesicles (EVs) secreted by neonatal human dermal fibroblasts (nHDF) cells were collected from the culture medium on both silicon and track-etched CEP / CNP membranes. The COL1A1 mRNA levels in the EVs collected from both silicon and track-etched CEP / CNP membranes were then quantified by qPCR. Figure 14 As shown, the mean copy number of COL1A1 mRNA in extracellular vesicles (EVs) secreted by neonatal human dermal fibroblasts (nHDF) cells undergoing electroporation on a silica membrane (Si-CNP; CNP) was approximately 54% higher than the mean copy number of COL1A1 mRNA in EVs secreted by neonatal human dermal fibroblasts (nHDF) cells undergoing electroporation on a track-etched membrane (TEP). CEP / CNP via a silica membrane (Si-CNP; Si) demonstrated to induce EV mRNA production more effectively than track-etched membranes. All data are presented as mean ± SD; CTR: control for untreated cells.

[0322] Example 7. Automated system for high-throughput cell electroporation This disclosure provides an automated system for high-throughput cell electroporation or high-throughput cell electroporation / nano-electroporation (HIT-CEP / CNP). This system includes... Figure 15A The diagram shows a CEP / CNP membrane setup based on a dual-well array, a newly developed sequential CEP / CNP electrical pulse generation system (which automates the output of electrical pulses to individual pores within the dual-well array-based CEP / CNP membrane), and optional accessory components. The CEP / CNP electrical pulse generation system can also be configured to deliver electrical pulses to cells in all or some of the electroporation chambers, but sequential electrical stimulation is preferred. This automated HIT-CEP / CNP system provides the ability to perform large-scale, multiplex cell transfections and generate extracellular vesicles (EVs) carrying transfection cargo of interest (e.g., DNA plasmids, RNA, or small molecules) with minimal manual intervention.

[0323] First, a prototype sequential CEP / CNP power system is described, which automates the sequential electrical pulses output to individual orifices of a dual-orifice array chamber. The CEP / CNP power system consists of an electrical pulse generator, an impulse pulse transducer, and an impulse chamber to deliver highly controllable electrical stimulation to donor cells to mediate CEP / CNP (…). Figure 15A Electrical pulse generator ( Figure 15B The device generates continuous and / or triggered square-wave electrical pulses to be applied to the high-throughput cell electroporation apparatus of HIT-CEP / CNP for donor cells. The electrical pulse train (or more than one train if needed) to be used for CEP / CNP can be adjusted (e.g., voltage, pulse width, and pulse interval) to modify the parameters of HIT-CEP / CNP. The electrical pulse generator is operatively coupled to and sends electrical pulses to the impulse pulse converter. Figure 15C The impulse pulse converter then converts the electrical output signal of the electrical pulse generator into electrical pulses, which are then sent to the impulse box to mediate the CEP / CNP. As an example, the impulse pulse converter sequentially sends 10 electrical output pulses to each CEP / CNP electrode via the impulse box's 25-pin connector. Impulse Box ( Figure 15D It features an automated button to initiate CEP / CNP transfection in individual wells via the electrode array. After cells in the first HIT-CEP / CNP chamber are transfected with CEP / CNP via the first electrode on the electrode array, the button is automatically triggered to transfect cells in the second well via the second electrode on the electrode array. The cycle is repeated until cells in all 25 wells are sequentially transfected with CEP / CNP.

[0324] In addition to the automated sequential power system and the CEP / CNP membrane based on a dual-well array chamber layer, optional accessories are used to further enhance the automation of the HIT-CEP / CNP system. For example, commercially available robotic pipetting systems can be used for automated washing, cell loading, reagent loading, and EV collection within each well of the HIT-CEP / CNP system to reduce contamination and human error.

[0325] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, alterations, and substitutions will now appear to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. It is intended that the appended claims define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0326] Implementation Plan Implementation Scheme 1. A perforated membrane comprising: A first layer, comprising a plurality of first passageways arranged through the first layer; and A second layer in contact with the first layer, the second layer comprising a plurality of second channels arranged through the second layer; in: The first average thickness of the first layer is different from the second average thickness of the second layer; The first channel of the plurality of first channels is in fluid communication with the second channel of the plurality of second channels; The first average diameter of the first channel is different from the second average diameter of the second channel.

[0327] Implementation Scheme 2. The perforated membrane as described in Implementation Scheme 1, wherein the first average thickness is greater than the second average thickness.

[0328] Implementation Scheme 3. The perforated membrane as described in Implementation Scheme 2, wherein the first average thickness is from about 1 µm to about 200 µm.

[0329] Implementation Scheme 4. The perforated membrane as described in Implementation Scheme 2 or 3, wherein the first average thickness is at least twice the second average thickness.

[0330] Implementation Scheme 5. The perforated membrane as described in any one of Implementation Schemes 2-4, wherein the first average thickness is at least three times the second average thickness.

[0331] Implementation Scheme 6. The perforated membrane as described in any one of Implementation Schemes 2-5, wherein the second average thickness is from about 100 nm to about 10 µm.

[0332] Implementation Scheme 7. The perforated membrane as described in any one of Implementation Schemes 1-6, wherein the first average diameter is greater than the second average diameter.

[0333] Implementation Scheme 8. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the first average diameter is from about 1 µm to about 20 µm.

[0334] Implementation Scheme 9. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the first average diameter is at least twice the second average diameter.

[0335] Implementation Scheme 10. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the first average diameter is at least three times the second average diameter.

[0336] Implementation Scheme 11. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the first average diameter is from about two to about three times the second average diameter.

[0337] Implementation Scheme 12. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the first average diameter is approximately three to approximately four times the second average diameter.

[0338] Implementation Scheme 13. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the second average diameter is from about 100 nm to about 5 µm.

[0339] Implementation Scheme 14. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the first average diameter is about half of the first average thickness.

[0340] Implementation Scheme 15. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the first average diameter is about one-third of the first average thickness.

[0341] Implementation Scheme 16. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the second average diameter is not more than half of the second average thickness.

[0342] Implementation Scheme 17. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the second average diameter is no more than one-third of the second average thickness.

[0343] Implementation Scheme 18. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the second average diameter is from about one-half to about one-third of the second average thickness.

[0344] Implementation Scheme 19. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the second average diameter is from about one-third to about one-quarter of the second average thickness.

[0345] Implementation Scheme 20. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the second average diameter is about half of the second average thickness.

[0346] Implementation Scheme 21. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the second average diameter is not more than half of the second average thickness.

[0347] Implementation Scheme 22. The perforated membrane as described in any one of Implementation Schemes 1-7, wherein the second average diameter is no more than about 25% of the second average thickness.

[0348] Implementation Scheme 23. The perforated membrane as described in any one of Implementation Schemes 1-22, wherein the first layer comprises a first polymer material.

[0349] Implementation Scheme 24. The perforated membrane as described in Implementation Scheme 23, wherein the first polymer material comprises a first photoreactive polymer.

[0350] Implementation Scheme 25. The perforated membrane as described in Implementation Scheme 23, wherein the first polymer material comprises a first crosslinked polymer.

[0351] Implementation Scheme 26. The perforated membrane as described in Implementation Scheme 23, wherein the first polymer material comprises a first synthetic polymer.

[0352] Implementation Scheme 27. The perforated membrane as described in Implementation Scheme 23, wherein the first polymer material comprises a first thermosetting polymer.

[0353] Implementation Scheme 28. The perforated membrane as described in Implementation Scheme 23, wherein the first polymer material comprises a first photocurable polymer or a first thermocurable polymer.

[0354] Implementation Scheme 29. The perforated membrane as described in Implementation Scheme 23, wherein the first polymer material comprises a first photoresist.

[0355] Implementation Scheme 30. The perforated film as described in Implementation Scheme 23, wherein the first polymer material comprises a first positive photoresist.

[0356] Implementation Scheme 31. The perforated film as described in Implementation Scheme 23, wherein the first polymer material comprises a first negative photoresist.

[0357] Implementation Scheme 32. The perforated film as described in Implementation Scheme 23, wherein the first polymer material comprises a first SU-8 photoresist.

[0358] Implementation Scheme 33. The perforated film as described in Implementation Scheme 23, wherein the first polymer material comprises a first SU-8 3000 series photoresist.

[0359] Implementation Scheme 34. The first perforated film as described in Implementation Scheme 23, wherein the first polymer material comprises SU-8 3010 photoresist polymer or SU-8 3005 photoresist polymer.

[0360] Implementation Scheme 35. The perforated film as described in Implementation Scheme 23, wherein the first polymer material comprises a first SU-8 2000 series photoresist.

[0361] Implementation Scheme 36. The perforated film as described in Implementation Scheme 23, wherein the first polymer material comprises SU-82005 photoresist.

[0362] Implementation Scheme 37. The perforated membrane as described in any one of Implementation Schemes 1-36, wherein the second layer comprises a second polymer material.

[0363] Implementation Scheme 38. The perforated membrane as described in Implementation Scheme 37, wherein the second polymer material comprises a second photoreactive polymer.

[0364] Implementation Scheme 39. The perforated membrane as described in Implementation Scheme 37, wherein the second polymer material comprises a second crosslinked polymer.

[0365] Implementation Scheme 40. The perforated membrane as described in Implementation Scheme 37, wherein the second polymer material comprises a second synthetic polymer.

[0366] Implementation Scheme 41. The perforated membrane as described in Implementation Scheme 37, wherein the second polymer material comprises a second thermosetting polymer.

[0367] Implementation Scheme 42. The perforated membrane as described in Implementation Scheme 37, wherein the second polymer material comprises a second photocurable polymer or a second thermocurable polymer.

[0368] Implementation Scheme 43. The perforated film as described in Implementation Scheme 37, wherein the second polymer material comprises a second photoresist.

[0369] Implementation Scheme 44. The perforated film as described in Implementation Scheme 37, wherein the second polymer material comprises a second positive photoresist.

[0370] Implementation Scheme 45. The perforated film as described in Implementation Scheme 37, wherein the second polymer material comprises a second negative photoresist.

[0371] Implementation Scheme 46. The perforated film as described in Implementation Scheme 37, wherein the second polymer material comprises a second SU-8 photoresist.

[0372] Implementation Scheme 47. The perforated film as described in Implementation Scheme 37, wherein the second polymer material comprises SU-8TF 6000 series photoresist.

[0373] Implementation Scheme 48. The perforated film as described in Implementation Scheme 37, wherein the second polymer material comprises SU-8TF 6002 photoresist.

[0374] Implementation Scheme 49. The perforated film as described in Implementation Scheme 37, wherein the second polymer material comprises a second SU-8 2000 series photoresist.

[0375] Implementation Scheme 50. The perforated film as described in Implementation Scheme 37, wherein the second polymer material comprises SU-82002 photoresist.

[0376] Implementation Scheme 51. The perforated membrane as described in any one of Implementation Schemes 1-50, wherein the first layer comprises at least one component that is different from the composition of the second layer material.

[0377] Implementation Scheme 52. The perforated membrane as described in any one of Implementation Schemes 1-50, wherein the first layer is made of a material different from the material of the second layer.

[0378] Implementation Scheme 53. The perforated film as described in Implementation Scheme 52, wherein the first layer comprises the first photoresist, and wherein the second layer comprises the second photoresist.

[0379] Implementation Scheme 54. The perforated film as described in Implementation Scheme 52, wherein the first layer comprises the first negative photoresist, and wherein the second layer comprises the second negative photoresist.

[0380] Implementation Scheme 55. The perforated film as described in Implementation Scheme 52, wherein the first layer comprises the first SU-8 photoresist, and wherein the second layer comprises the second SU-8 photoresist.

[0381] Implementation Scheme 56. The perforated film as described in Implementation Scheme 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.

[0382] Implementation Scheme 57. The perforated membrane as described in Implementation Scheme 52, wherein the first tensile strength of the first layer is greater than the second tensile strength of the second layer.

[0383] Implementation Scheme 58. The perforated film as described in Implementation Scheme 52, wherein the first layer and the second layer are made of a photoresist polymer.

[0384] Implementation Scheme 59. The perforated membrane as described in any one of Implementation Schemes 1-37, wherein the first layer is made of the same material as the second layer.

[0385] Implementation Scheme 60. The perforated membrane of Implementation Scheme 59, wherein the first layer and the second layer comprise the first photoreactive polymer.

[0386] Implementation Scheme 61. The perforated membrane as described in Implementation Scheme 59, wherein the first layer and the second layer comprise the first crosslinked polymer.

[0387] Implementation Scheme 62. The perforated membrane as described in Implementation Scheme 59, wherein the first layer and the second layer comprise the first synthetic polymer.

[0388] Implementation Scheme 63. The perforated membrane as described in Implementation Scheme 59, wherein the first layer and the second layer comprise the first photocurable polymer.

[0389] Implementation Scheme 64. The perforated membrane as described in any one of Implementation Schemes 1-63 is configured to electroporate donor cells with a transfection reagent.

[0390] Implementation Scheme 65. The perforated membrane as described in Implementation Scheme 64, wherein the transfection reagent comprises DNA, RNA, or a small molecule.

[0391] Implementation Scheme 66. A method for fabricating a perforated film from a photoresist material, comprising: (a) Spin-coating a first layer of the first photoresist material onto the substrate; (b) The first layer obtained in softening (a); (c) Using a first photomask, expose the softened first layer obtained in (b) to first UV radiation; (d) After (c), spin-coat a second layer of the second photoresist material onto the first layer; (e) The second layer obtained in (d) is softened during baking; (f) Using a second photomask, expose the softened second layer obtained in (e) to another UV radiation; (g) Performing post-exposure baking; and (h) After (g), develop both the first and second layers. This allows for the creation of a perforated membrane consisting of two layers.

[0392] Implementation Scheme 67. The method of Implementation Scheme 66 further includes: performing another post-exposure bake after (c) but before (d).

[0393] Implementation Scheme 68. The method as described in Implementation Scheme 66 or 67, wherein the substrate is a silicon film coated with a release layer.

[0394] Implementation Scheme 69. The method of Implementation Scheme 68, wherein the spin coating in (a) coats the first layer onto the release layer.

[0395] Implementation Scheme 70. The method of Implementation Scheme 69 further includes: removing the release layer after (h).

[0396] Implementation Scheme 71. The method as described in Implementation Scheme 66, wherein both the first photoresist material and the second photoresist material are negative photoresists.

[0397] Implementation Scheme 72. The method as described in Implementation Scheme 66, wherein the first photoresist material is different from the second photoresist material.

[0398] Implementation Scheme 73. The method of Implementation Scheme 72, wherein the first photoresist material has a higher resolution than the second photoresist material.

[0399] Implementation Scheme 74. The method of Implementation Scheme 66, wherein the first photomask comprises a first pattern, and wherein the second photomask comprises a second pattern.

[0400] Implementation Scheme 75. The method of Implementation Scheme 74, wherein the first pattern is configured to generate a plurality of first channels arranged in the first layer after (h), and wherein the second pattern is configured to generate a plurality of second channels arranged in the second layer after (h).

[0401] Implementation Scheme 76. The method of Implementation Scheme 66 further includes: applying a barrier layer on top of the first layer after (c) but before (d).

[0402] Implementation Scheme 77. The method of Implementation Scheme 76, wherein the barrier layer comprises silicon oxide, gold, or silver.

[0403] Implementation Scheme 78. The method of any one of Implementation Schemes 66-77, wherein the perforated membrane comprises The top layer, comprising a plurality of top passages traversing the top layer arrangement; and A bottom layer in contact with the top layer, the bottom layer comprising a plurality of bottom channels arranged through the bottom layer; in: The first average thickness of the top layer is different from the second average thickness of the bottom layer; The first channel of the plurality of top channels is in fluid communication with the second channel of the plurality of bottom channels; The first average diameter of the first channel is different from the second average diameter of the second channel.

[0404] Implementation Scheme 79. A method for producing a perforated membrane from a sacrificial template, comprising: (a) Provide a negative mold containing a recessed pattern; (b) Preparing a sacrificial male mold based on the female mold; and (c) A perforated membrane is prepared based on the sacrificial male mold. The perforated membrane described therein contains a different pattern.

[0405] Implementation Scheme 80. The method as described in Implementation Scheme 79, wherein the other pattern is substantially the same as the recessed pattern.

[0406] Implementation Scheme 81. The method as described in Implementation Scheme 79 or 80, wherein the preparation in (c) includes imprinting.

[0407] Implementation Scheme 82. The method of implementation scheme 81, wherein the imprinting includes providing a substrate and a weight.

[0408] Implementation Scheme 83. The method of Implementation Scheme 82, wherein the substrate is coated with a release layer.

[0409] Implementation Scheme 84. The method of Implementation Scheme 83, wherein the release layer comprises a releaseable polymer.

[0410] Implementation Scheme 85. The method as described in Implementation Scheme 79, wherein the preparation in (c) comprises spin coating.

[0411] Implementation Scheme 86. The method of any one of Implementation Schemes 79-85, wherein the preparation in (c) comprises applying a polymer resin to the sacrificial male mold.

[0412] Implementation Scheme 87. The method of Implementation Scheme 86, wherein the polymer resin is thermosetting.

[0413] Implementation Scheme 88. The method of Implementation Scheme 87 further includes: thermosetting the polymer resin.

[0414] Implementation Scheme 89. The method of Implementation Scheme 86, wherein the polymer resin is photocurable.

[0415] Implementation Scheme 90. The method of Implementation Scheme 87 further includes: photocuring the polymer resin.

[0416] Implementation Scheme 91. The method of any one of Implementation Schemes 79-90, wherein the preparation in (c) further comprises: removing the sacrificial male mold.

[0417] Implementation Scheme 92. The method of any one of Implementation Schemes 79-91, wherein the sacrificial male mold is made of a water-soluble polymer.

[0418] Implementation Scheme 93. The method of Implementation Scheme 92, wherein the water-soluble polymer is polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or polyvinylpyrrolidone / vinyl alcohol copolymer (PVP / VA copolymer).

[0419] Implementation Scheme 94. The method of any one of Implementation Schemes 86-93, wherein the polymer resin is a polydimethylsiloxane (PDMS) resin or an acrylic resin.

[0420] Implementation Scheme 95. The method of any one of Implementation Schemes 84 or 86-94, wherein the demoldable polymer is polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or polyvinylpyrrolidone / vinyl alcohol copolymer (PVP / VA copolymer).

[0421] Implementation Scheme 96. The method of any one of Implementation Schemes 82-84 or 86-95, wherein the substrate is a rubber layer.

[0422] Implementation Scheme 97. The method of any one of Implementation Schemes 79-96, wherein the perforated membrane comprises: Multiple channels are arranged through the perforated membrane; The average diameter of the channel; and Average thickness.

[0423] Implementation Scheme 98. A system for high-throughput cell electroporation, comprising: a) a perforated membrane, wherein the perforated membrane comprises: i. A first layer, the first layer comprising a plurality of first passages arranged through the first layer; and ii. A second layer in contact with the first layer, the second layer comprising a plurality of second channels arranged through the second layer; in: The first average thickness of the first layer is different from the second average thickness of the second layer; The first channel of the plurality of first channels is in fluid communication with the second channel of the plurality of second channels; The first average diameter of the first channel is different from the second average diameter of the second channel; and b) At least one donor cell in contact with the perforated membrane.

[0424] Implementation Scheme 99. The system as described in Implementation Scheme 98, wherein the first average thickness is greater than the second average thickness.

[0425] Implementation scheme 100. The system as described in implementation scheme 99, wherein the first average thickness is from about 1 µm to about 200 µm.

[0426] Implementation Scheme 101. The system as described in Implementation Scheme 99 or 100, wherein the first average thickness is at least twice the second average thickness.

[0427] Implementation Scheme 102. The system of any one of Implementation Schemes 99-101, wherein the first average thickness is at least three times the second average thickness.

[0428] Implementation Scheme 103. The system of any one of Implementation Schemes 99-102, wherein the second average thickness is from about 100 nm to about 10 µm.

[0429] Implementation Scheme 104. The system of any one of Implementation Schemes 98-103, wherein the first average diameter is greater than the second average diameter.

[0430] Implementation Scheme 105. The system of any one of Implementation Schemes 98-104, wherein the first average diameter is from about 1 µm to about 20 µm.

[0431] Implementation Scheme 106. The system of any one of Implementation Schemes 98-104, wherein the first average diameter is at least twice the second average diameter.

[0432] Implementation Scheme 107. The system of any one of Implementation Schemes 98-104, wherein the first average diameter is at least three times the second average diameter.

[0433] Implementation Scheme 108. The system of any one of Implementation Schemes 98-104, wherein the first average diameter is from about two to about three times the second average diameter.

[0434] Implementation Scheme 109. The system of any one of Implementation Schemes 98-104, wherein the first average diameter is about three to about four times the second average diameter.

[0435] Implementation Scheme 110. The system of any one of Implementation Schemes 98-104, wherein the second average diameter is from about 100 nm to about 5 µm.

[0436] Implementation Scheme 111. The system of any one of Implementation Schemes 98-104, wherein the first average diameter is about half of the first average thickness.

[0437] Implementation Scheme 112. The system of any one of Implementation Schemes 98-104, wherein the first average diameter is about one-third of the first average thickness.

[0438] Implementation Scheme 113. The system of any one of Implementation Schemes 98-104, wherein the second average diameter is no more than half of the second average thickness.

[0439] Implementation Scheme 114. The system of any one of Implementation Schemes 98-104, wherein the second average diameter is no more than one-third of the second average thickness.

[0440] Implementation Scheme 115. The system of any one of Implementation Schemes 98-104, wherein the second average diameter is from about one-half to about one-third of the second average thickness.

[0441] Implementation Scheme 116. The system of any one of Implementation Scheme-104, wherein the second average diameter is from about one-third to about one-quarter of the second average thickness.

[0442] Implementation Scheme 117. The system of any one of Implementation Schemes 98-104, wherein the second average diameter is about half of the second average thickness.

[0443] Implementation Scheme 118. The system of any one of Implementation Schemes 98-104, wherein the second average diameter is no more than half of the second average thickness.

[0444] Implementation Scheme 119. The system of any one of Implementation Schemes 98-118, wherein the first layer comprises a first polymer material.

[0445] Implementation Scheme 120. The system of Implementation Scheme 119, wherein the first polymer material comprises a first photoreactive polymer.

[0446] Implementation Scheme 121. The system as described in Implementation Scheme 119, wherein the first polymer material comprises a first crosslinked polymer.

[0447] Implementation Scheme 122. The system as described in Implementation Scheme 119, wherein the first polymer material comprises a first synthetic polymer.

[0448] Implementation Scheme 123. The system of Implementation Scheme 119, wherein the first polymer material comprises a first thermosetting polymer.

[0449] Implementation Scheme 124. The system of Implementation Scheme 119, wherein the first polymer material comprises a first photocurable polymer or a first thermocurable polymer.

[0450] Implementation Scheme 125. The system of Implementation Scheme 119, wherein the first polymer material comprises a first photoresist.

[0451] Implementation Scheme 126. The system of Implementation Scheme 119, wherein the first polymer material comprises a first positive photoresist.

[0452] Implementation Scheme 127. The system of Implementation Scheme 119, wherein the first polymer material comprises a first negative photoresist.

[0453] Implementation Scheme 128. The system of Implementation Scheme 119, wherein the first polymer material comprises a first SU-8 photoresist.

[0454] Implementation Scheme 129. The system of Implementation Scheme 119, wherein the first polymer material comprises a first SU-8 3000 series photoresist.

[0455] Implementation Scheme 130. The system of Implementation Scheme 119, wherein the first polymer material comprises a first SU-8 3010 photoresist or a first SU-8 3005 photoresist.

[0456] Implementation Scheme 131. The system of Implementation Scheme 119, wherein the first polymer material comprises a first SU-8 2000 series photoresist.

[0457] Implementation Scheme 132. The system of Implementation Scheme 119, wherein the first polymer material comprises a first SU-8 2005 photoresist.

[0458] Implementation Scheme 133. The system as described in Implementation Scheme 132, wherein the second layer comprises a second polymer material.

[0459] Implementation Scheme 134. The system as described in Implementation Scheme 133, wherein the second polymer material comprises a second photoreactive polymer.

[0460] Implementation Scheme 135. The system of Implementation Scheme 133, wherein the second polymer material comprises a second crosslinked polymer.

[0461] Implementation Scheme 136. The system of Implementation Scheme 133, wherein the second polymer material comprises a second synthetic polymer.

[0462] Implementation Scheme 137. The system of Implementation Scheme 133, wherein the second polymer material comprises a second thermosetting polymer.

[0463] Implementation Scheme 138. The system of Implementation Scheme 133, wherein the second polymer material comprises a second photocurable polymer or a second thermocurable polymer.

[0464] Implementation Scheme 139. The system of Implementation Scheme 133, wherein the second polymer material comprises a second photoresist.

[0465] Implementation Scheme 140. The system of Implementation Scheme 133, wherein the second polymer material comprises a second positive photoresist.

[0466] Implementation Scheme 141. The system as described in Implementation Scheme 133, wherein the second polymer material comprises a second negative photoresist.

[0467] Implementation Scheme 142. The system as described in Implementation Scheme 133, wherein the second polymer material comprises a second SU-8 photoresist.

[0468] Implementation Scheme 143. The system of Implementation Scheme 133, wherein the second polymer material comprises a second SU-8 TF 6000 series photoresist.

[0469] Implementation Scheme 144. The system of Implementation Scheme 133, wherein the second polymer material comprises SU-8TF 6002 photoresist.

[0470] Implementation Scheme 145. The system of Implementation Scheme 133, wherein the second polymer material comprises a second SU-8 2000 series photoresist.

[0471] Implementation Scheme 146. The system of Implementation Scheme 133, wherein the second polymer material comprises SU-82002 photoresist.

[0472] Implementation Scheme 147. The system of any one of Implementation Schemes 98-146, wherein the first layer comprises at least one component that is different from the composition of the second material.

[0473] Implementation Scheme 148. The system of any one of Implementation Schemes 98-147, wherein the first layer is made of a material different from the material of the second layer.

[0474] Implementation Scheme 149. The system of Implementation Scheme 148, wherein the first layer comprises the first photoresist, and wherein the second layer comprises the second photoresist.

[0475] Implementation Scheme 150. The system of Implementation Scheme 148, wherein the first layer comprises the first negative photoresist, and wherein the second layer comprises the second negative photoresist.

[0476] Implementation Scheme 151. The system of Implementation Scheme 148, wherein the first layer comprises the first SU-8 photoresist, and wherein the second layer comprises the second SU-8 photoresist.

[0477] Implementation Scheme 152. The system of Implementation Scheme 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.

[0478] Implementation Scheme 153. The system as described in Implementation Scheme 152, wherein the first tensile strength of the first layer is greater than the second tensile strength of the second layer.

[0479] Implementation Scheme 154. The system as described in Implementation Scheme 152, wherein the first layer and the second layer are made of photoresist polymer.

[0480] Implementation Scheme 155. The system of any one of Implementation Schemes 98-154, wherein the first layer is made of the same material as the second layer.

[0481] Implementation Scheme 156. The system as described in Implementation Scheme 155, wherein the first layer and the second layer comprise the first photoreactive polymer.

[0482] Implementation Scheme 157. The system as described in Implementation Scheme 155, wherein the first layer and the second layer comprise the first crosslinked polymer.

[0483] Implementation Scheme 158. The system as described in Implementation Scheme 155, wherein the first layer and the second layer comprise the first synthetic polymer.

[0484] Implementation Scheme 159. The system as described in Implementation Scheme 155, wherein the first layer and the second layer comprise the first photocurable polymer.

[0485] Implementation Scheme 160. The system as described in any one of Implementation Schemes 98-159 is configured to electroporate donor cells with a transfection reagent.

[0486] Implementation Scheme 161. The system of any one of embodiments 98-160, further comprising a first spacer array, wherein the first spacer array comprises: a) A thickness greater than the diameter of the donor cell; and b) A plurality of first holes, wherein the first hole of the plurality of first holes includes a first hole width and a second hole width perpendicular to the first width. The first hole of the first spacer array has a cross-sectional area defined by the material of the first spacer array.

[0487] Implementation Scheme 162. The system as described in Implementation Scheme 161, wherein the first spacer array is in contact with the perforated membrane.

[0488] Implementation Scheme 163. The system as described in Implementation Scheme 161 or 162, wherein the first aperture width ...

Claims

1. A perforated membrane comprising: A first layer, comprising a plurality of first passageways arranged through the first layer; and A second layer in contact with the first layer, the second layer comprising a plurality of second channels arranged through the second layer; in: The first average thickness of the first layer is different from the second average thickness of the second layer; The first channel of the plurality of first channels is in fluid communication with the second channel of the plurality of second channels; The first average diameter of the first channel is different from the second average diameter of the second channel.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

16. The perforated membrane according to any one of claims 1-7, wherein the second average diameter is not more than half of the second average thickness.

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

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

21. The perforated membrane according to any one of claims 1-7, wherein the second average diameter is not more than half of the second average thickness.

22. The perforated membrane according to any one of claims 1-7, wherein the second average diameter is not more than about 25% of the second average thickness.

23. The perforated membrane according to 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 photoreactive polymer.

25. The perforated membrane of claim 23, wherein the first polymer material comprises a first crosslinked 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 photocurable polymer or a first thermocurable polymer.

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

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

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

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

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

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

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

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

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

38. The perforated membrane of claim 37, wherein the second polymer material comprises a second photoreactive polymer.

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

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

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

42. The perforated membrane of claim 37, wherein the second polymer material comprises a second photocurable polymer or a second thermocurable polymer.

43. The perforated film of claim 37, wherein the second polymer material comprises a second photoresist.

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

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

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

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

48. The perforated film of claim 37, wherein the second polymer material comprises SU-8 TF 6002 photoresist.

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

50. The perforated film of claim 37, wherein the second polymer material comprises SU-8 2002 photoresist.

51. The perforated membrane according to any one of claims 1-50, wherein the first layer comprises at least one component that is different from the composition of the second layer material.

52. The perforated membrane according to any one of claims 1-50, wherein the first layer is made of a material different from that of the second layer.

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

54. The perforated film 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 film 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 film 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 the first tensile strength of the first layer is greater than the second tensile strength of the second layer.

58. The perforated film of claim 52, wherein the first layer and the second layer are made of a photoresist polymer.

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

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

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

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

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

66. A method for fabricating a perforated film using a photoresist material, comprising: (a) Spin-coating a first layer of the first photoresist material onto the substrate; (b) The first layer obtained in softening (a); (c) Using a first photomask, expose the softened first layer obtained in (b) to first UV radiation; (d) After (c), spin-coat a second layer of the second photoresist material onto the first layer; (e) The second layer obtained in (d) is softened during baking; (f) Using a second photomask, expose the softened second layer obtained in (e) to another UV radiation; (g) Perform baking after exposure; as well as (h) After (g), develop both the first and second layers. This allows for the creation of a perforated membrane consisting of two layers.

67. The method of claim 66, further comprising: After (c) but before (d), perform another exposure followed by baking.

68. The method of claim 66 or 67, wherein the substrate is a silicon film 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), the release layer is removed.

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 74, wherein the first pattern is configured to generate a plurality of first channels arranged in the first layer after (h), and wherein the second pattern is configured to generate a plurality of second channels arranged in the second layer after (h).

76. The method of claim 66, further comprising: After (c) but before (d), the barrier layer is applied 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 The top layer includes a plurality of top passages through which the top layer is arranged; as well as A bottom layer in contact with the top layer, the bottom layer comprising a plurality of bottom channels arranged through the bottom layer; in: The first average thickness of the top layer is different from the second average thickness of the bottom layer; The first channel of the plurality of top channels is in fluid communication with the second channel of the plurality of bottom channels; The first average diameter of the first channel is different from the second average diameter of the second channel.

79. A method for producing a perforated membrane from a sacrificial template, comprising: (a) Provide a negative mold containing a recessed pattern; (b) Preparing a sacrificial male mold based on the female mold; and (c) A perforated membrane is prepared based on the sacrificial male mold. The perforated membrane described therein contains a different pattern.

80. The method of claim 79, wherein the other pattern is substantially the same as the recessed pattern.

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

82. The method of claim 81, wherein the imprinting includes 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 releaseable polymer.

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

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

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

88. The method of claim 87, further comprising: The polymer resin is thermosetting.

89. The method of claim 86, wherein the polymer resin is photocurable.

90. The method of claim 87, further comprising: The polymer resin is photocured.

91. The method of any one of claims 79-90, wherein the preparation in (c) further comprises: Remove the sacrificial positive mold.

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

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

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

95. The method of any one of claims 84 or 86-94, wherein the demoldable polymer is polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or 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 rubber layer.

97. The method of any one of claims 79-96, wherein the perforated membrane comprises Multiple channels are arranged through the perforated membrane; The average diameter of the channel; and Average thickness.

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

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

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

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

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

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

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

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

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

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

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

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-quarter 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 photoreactive polymer.

121. The system of claim 119, wherein the first polymer material comprises a first crosslinked 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 photocurable polymer or a first thermocurable 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-8 2000 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 photoreactive polymer.

135. The system of claim 133, wherein the second polymer material comprises a second crosslinked 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 photocurable polymer or a second thermocurable 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 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 SU-8 2002 photoresist.

147. The system of any one of claims 98-146, wherein the first layer comprises at least one component that is different from the composition of the second material.

148. The system of any one of claims 98-147, wherein the first layer is made of a material different from that 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 the first tensile strength of the first layer is greater than the second tensile strength of the second layer.

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

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

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

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

160. The system of any one of claims 98-159, configured to electroporate donor cells 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 the diameter of the donor cell; and b) A plurality of first holes, wherein the first hole of the plurality of first holes includes a first hole width and a second hole width perpendicular to the first width. The first hole of the first spacer array has a cross-sectional area defined by the 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 aperture width and the second aperture width of the first aperture of the first spacer array are smaller than the surface area of ​​the first surface of the perforated membrane having a first plurality of channel openings.

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

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

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

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

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

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

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

173. The system of claim 172, wherein the first aperture of the first spacer array is separated by a first aperture distance of at least about 1 mm and other first apertures adjacent to the first aperture of the first spacer array.

174. The system of claim 172, wherein the first aperture of the first spacer array is separated by a first aperture distance from about 1 mm to about 5 cm and other first apertures adjacent to the first aperture of the first spacer array.

175. The system of claim 172, wherein the first holes of the first array spacers are separated by a first hole spacing from about 1 mm to about 1.5 cm and other first holes adjacent to the first spacer array.

176. The system of claim 172, wherein the first hole of the first array spacer is separated by a first hole spacing of about 5 mm and other first holes adjacent to the first hole of the first spacer array.

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 the width of the 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 two 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 crosslinked polymer, a photocurable polymer, a thermosetting polymer, a photoreactive 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, polydimethylsiloxane (PDMS), polydimethylsiloxane (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 the diameter of the donor cell; b) A plurality of second holes, wherein the second holes of the second spacer array include a first hole width and a second hole width perpendicular to the first width; and The second hole of the second spacer array has a cross-sectional area defined by the 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 aperture of the second spacer array does not exceed the surface area of ​​the surface of the perforated membrane having a second plurality of channel openings.

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

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

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

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

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

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

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

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

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

201. The system of any one of claims 186-196, wherein the second holes of the second spacer array are separated by a second hole spacing of 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 the width of the 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 two 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 from the first thickness of the first spacer array.

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

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

212. The system of any one of claims 186-208, wherein the distance between the second holes of the second spacer array is different from the distance between at least one first hole 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 from 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 conductive material that is 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 the surface of the first spacer array or the second spacer array.

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

220. The system of claim 215 or 216, wherein the cathode comprises a cathode coil array.

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

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

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

224. The system of claim 221, wherein the cathode coil array is configured to position individual cathode coils within at least five holes of the first spacer array or at least five 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 conductive material that is 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 the 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 a wire anode array configured to position individual wire anodes of the wire anode array within the individual holes of the first spacer array or the second spacer array.

231. The system of claim 230, wherein the wire anode array is configured to position individual wire anodes of the wire anode array 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 electrical pulse generation system.

233. The system of claim 232, wherein the electrical pulse generation system comprises: a) Electrical pulse generator; b) Impulse pulse converter; and c) Current output device.

234. The system of claim 232 or 233, wherein the electrical pulse generation system is configured to adjust the 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 electrical pulse generation system is configured to sequentially deliver electrical pulses to at least one of the individual line anodes of the anode line or the line anode array.

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

237. The system of any one of claims 232-236, further comprising an automated liquid handling or liquid dispensing device configured to: a) Arrange the at least one donor cell in at least one individual pore of the first spacer array; b) Adding or removing electroporation buffer within at least one individual pore of the second spacer array; or c) Adding or removing cell culture medium in at least one individual well of the first spacer array.

238. A system for high-throughput cell electroporation, comprising: a) A perforated membrane, wherein the perforated membrane includes channels disposed through the perforated membrane; b) First array spacers; c) the second array spacer; and d) At least one donor cell, 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 film comprises a silicon film, a track-etched film, a polymer film, a single-layer polymer film, or a double-layer polymer film.

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

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

242. The system of claims 238-241, wherein the width of the first aperture and the width of the second aperture of the first spacer array are smaller than the surface area of ​​the perforated membrane having a first plurality of channel openings.

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

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

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

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

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

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

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

252. The system of claim 251, wherein the first aperture of the first spacer array is separated by a first aperture distance of at least about 1 mm and other first apertures adjacent to the first aperture of the first spacer array.

253. The system of claim 251, wherein the first aperture of the first spacer array is separated by a first aperture distance from about 1 mm to about 1.5 cm or from about 1 mm to about 5 cm and other first apertures adjacent to the first aperture of the first spacer array.

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

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 the width of the 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 two 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 crosslinked polymer, a photocurable polymer, a thermosetting polymer, a photoreactive 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, polydimethylsiloxane (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 the diameter of the donor cell; and b) A plurality of second holes, wherein the second holes of the second spacer array include a first hole width and a second hole width perpendicular to the first width. The second hole of the second spacer array has a cross-sectional area defined by the 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 claims 238-265, wherein the second aperture of the second spacer array does not exceed the surface area of ​​the surface of the perforated membrane having a second plurality of channel openings.

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

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

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

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

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

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

275. The system of any one of claims 238-274, wherein the second aperture of the second spacer array is separated by the distance between the second apertures and other second apertures adjacent to the second aperture of the second spacer array.

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

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

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

279. The system of any one of claims 238-274, wherein the second aperture of the second spacer array is separated by a second aperture spacing of about 5 mm and other second apertures adjacent to the second aperture of the second spacer array.

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 the width of the 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 two 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 equivalent 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 from the first thickness of the first spacer array.

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

290. The system of any one of claims 238-288, wherein the distance between the second holes separating the second hole and other second holes adjacent to the second hole of the second spacer array is different from the distance between the first holes separating the first hole and other first holes adjacent 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 from 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 crosslinked polymer, a second photocurable polymer, a second thermocurable polymer, a second photoreactive polymer, a second silicon polymer, a second rubber, or a second photoresist polymer.

294. The system of any one of claims 238-293, further comprising a cathode.

295. The system of claim 294, wherein the cathode comprises a conductive material that is 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 the surface of the first spacer array or the second spacer array.

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

299. The system of claim 294 or 295, wherein the cathode comprises a cathode coil array.

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

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

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

303. The system of claim 300, wherein the cathode coil array is configured to position individual cathode coils within at least five holes of the first spacer array or at least five 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 conductive material that is 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 anode is configured to contact the 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 a wire anode array configured to position individual wire anodes of the wire anode array within the individual holes of the first spacer array or the second spacer array.

310. The system of claim 309, wherein the wire anode array is configured to position individual wire anodes of the wire anode array 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 electrical pulse generation system.

312. The system of claim 311, wherein the electrical pulse generation system comprises: a) Electrical pulse generator; b) Impulse pulse converter; and c) Current output device.

313. The system of claim 311 or 312, wherein the electrical pulse generation system is configured to adjust the 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 electrical pulse generation system is configured to sequentially deliver electrical pulses to at least one of the individual line anodes of the anode line or the line anode array.

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

316. The system of any one of claims 238-315, further comprising an automated liquid handling or liquid dispensing device configured to: a) Arrange the at least one donor cell in at least one individual pore of the first spacer array; b) Adding or removing electroporation buffer within at least one individual pore of the second spacer array; or c) Adding or removing cell culture medium in at least one individual well of the first spacer array.

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

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

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

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

321. The system of claim 320, wherein the first average thickness is from about 1 µm to about 200 µm.

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

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

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

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

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

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

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

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

332. The system of any one of claims 319-325, wherein the first average 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-quarter 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 photoreactive polymer.

343. The system of claim 341, wherein the first polymer material comprises a first crosslinked 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 photocurable polymer or a first thermocurable 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 SU-8 3010 photoresist.

353. The system of claim 341, wherein the first polymer material comprises a first SU-8 2000 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 photoreactive polymer.

357. The system of claim 355, wherein the second polymer material comprises a second crosslinked 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 photocurable polymer or a second thermocurable 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 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 SU-8 2002 photoresist.

369. The system of any one of claims 238-368, wherein the first layer comprises at least one component that is different from the composition of the second material.

370. The system of any one of claims 238-369, wherein the first layer is made of a material different from that 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 the first tensile strength of the first layer is greater than the 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 of a photoresist polymer.

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

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

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

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

383. A method for generating and collecting extracellular vesicles, comprising: a) Provides an electroporation device comprising an array of cell electroporation pores, wherein the electroporation device comprises: i. A perforated membrane located between a first spacer array and a second spacer array, wherein the perforated membrane comprises a plurality of channels that span a distance between a first surface of the perforated membrane and a second surface of the perforated membrane; ii. A first spacer array, which defines a cell culture chamber array; iii. A second spacer array defining an electroporation reagent chamber array, wherein a second arrangement of the electroporation reagent chamber array of the second spacer array is complementary to a first arrangement of the cell culture chamber array of the first spacer array; When the cell culture chamber array of the first spacer array and the electroporation reagent chamber array of the second spacer array are spatially aligned, and when the first spacer array and the second spacer array subsequently come into contact with the perforated membrane, at least two isolated cell electroporation chambers are created. The cell culture chambers of the cell culture chamber array include a portion of the perforated membrane; and The electroporation reagent chamber of the electroporation reagent chamber array includes a portion of the perforated membrane; The cell culture chambers of the cell culture chamber array are fluidly coupled to the electroporation reagent chambers of the electroporation reagent chamber array via a channel of a portion of the perforated membrane; b) Introduce donor cells into at least one cell culture chamber of the cell culture chamber array; c) Introducing a polynucleotide, DNA, RNA, vector, or plasmid into at least one electroporation buffer chamber of the electroporation buffer chamber array; d) Applying an electric field, current, or voltage through the electroporation device to electroporate the donor cell in at least one cell electroporation pore of the cell electroporation pore array; and e) Collect extracellular vesicles (EVs) produced by the donor cells.

384. The method of claim 383, wherein the extracellular vesicle (EV) is an exosome, an apoptotic body, or a microvesicle.

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

386. The method of claim 385, wherein the polynucleotide, DNA, or RNA encodes Col1A.

387. The method of any one of claims 383-386, wherein the perforated film is a silicon film, a track-etched film, a single-layer polymer film, or a double-layer polymer film.

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 sequentially applying the electric field to at least two cell electroporation pores prior to (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) to (e).

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

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

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

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