Electroporation devices and methods for cell transfection

Abstract

This invention provides improved electroporation systems and methods, particularly for high-throughput, large-scale manufacturing processes. [Solution] A system and method are provided for transfecting cells, such as mammalian cells and non-mammalian cells, using an electroporation apparatus having an electroporation chamber, which includes a first electrode, a second electrode, and a defined pathway within the electroporation chamber. The electroporation apparatus includes a first input that allows cells and cargo to pass into the electroporation chamber, and a first output that allows electroporated cells to pass out of the electroporation chamber.

Description

Detailed Description of the Invention

[0001] [Technical Field] Cross - reference to Related Applications This application is a PCT international patent application claiming the benefit and priority of U.S. Patent Application No. 17 / 513,007, filed on October 28, 2021. U.S. Patent Application No. 17 / 513,007 is a partial continuation application of U.S. Patent Application No. 16 / 923,606, filed on July 8, 2020. U.S. Patent Application No. 16 / 923,606 is a partial continuation application of U.S. Patent Application No. 16 / 506,190, filed on July 9, 2019, and claims the benefit of U.S. Provisional Patent Application No. 62 / 695,436, filed on July 9, 2018. The entire disclosure of each of the above - cited applications is hereby incorporated by reference herein.

[0002] The field of the invention relates to transfection systems and methods, particularly electroporation systems and methods.

[0003] [Background Art] The background description includes information that may be useful in understanding the methods and techniques presented herein. It is not admitted that any of the information provided herein is prior art or that any publication related to or explicitly or implicitly referenced in the subject matter presented herein is prior art.

[0004] Transfection may be used to introduce nucleic acids into cells to create genetically - modified cells. There are various physical, chemical, and viral methods for transfecting cells, including optoperforation, polymer - based methods using calcium phosphate, microinjection, electroporation, viral transduction, and lipid - mediated methods (e.g., using liposome - DNA complexes).

[0005] Electroporation involves applying controlled direct current (DC) electrical pulses to cells for relatively short durations. The electrical pulses are thought to induce a membrane potential that causes the reversible breakdown of the cell membrane's regular structure, leading to pore formation in the membrane. The target molecule can then enter the cell through these pores, typically within milliseconds to seconds, until the pores close. Pore formation can be controlled by adjusting various parameters, particularly the gap width (e.g., the distance between parallel electrode plates), the shape of the electrical pulse wave, the electric field strength, temperature, and the length of the electrical pulse.

[0006] Electroporation is generally used on a laboratory scale (e.g., using small cuvettes with a capacity of approximately 0.5 mL), but such laboratory-based techniques are unsuitable for large-scale clinical-grade production due to their low efficiency and high cost. Other transfection methods, such as lipid-based techniques using lipofectamine 2000™, are also frequently used, but they suffer from similar drawbacks due to their high cost. In addition, lipid-based methods do not function adequately for some cell types of clinical targets, such as NK cells including haNK. NK cells are transfected using mRNA and electroporation to express chimeric antigen receptors (see CAR-Leuk Res. (2009) 33:1255-9) or autocrine growth-stimulating cytokines (see Cytotherapy (2008) 10:265-74) by genetically manipulating primary NK cells, for example. While commercially available products such as Maxcyte exist for large-scale transfections (e.g., reaction sizes larger than 1 mL), these products are also expensive and / or lack high-throughput performance. Similarly, these commercial products are not optimal for transfecting NK cells and often result in problems associated with low yield and / or low cell viability. Typical electroporation rates can result in DNA transfection efficiencies of 2–5%, along with cell viability of approximately 10–20%.

[0007] In conventional techniques, electroporation is performed using two parallel plate electrodes, as shown in Figure 1A. In this configuration, cells typically flow between the two parallel plates. However, the electric field is non-uniform, with cells in the center of the chamber shown in (a) receiving a more uniform field, while cells near the electrodes shown in (b) receive a non-uniform field, particularly the electric field action potential generated at the electrode boundary. In other techniques, parallel plates of micromesh electrodes are used, as shown in Figure 1B (Selmeczi, D. et al., “Efficient large volume electroporation of dendritic cells through micrometer scale manipulation of flow in a disposable polymer chip,” Biomed Microdevices, 13:383-392 (2011)), where cells pass through upper and lower micromesh electrodes. As indicated by the similar trajectories of cells in the center of the chamber (a) and near the chamber boundary (b), this technique provides a more uniform electric field than passing between two parallel electrode plates, while the width of the chamber or the distance between the upper and lower micromesh electrodes is limited, resulting in lower transfection efficiency. In the configurations shown in Figures 1A and 1B, electric field strengths of approximately 1–1.3 kV / cm (e.g., 200 V / 2 mm for the parallel plates and 40 V / 400 μm for the parallel micromesh electrodes) are utilized.

[0008] In another embodiment, a single-cell electroporation chip is used for transfection. In this technique, the silicon chip may include multiple linear channels, each channel having a counter electrode positioned on the opposite side of the channel. Cells pass through the channels and are exposed to an electric field. In this type of technique, a DNA transfection efficiency of approximately 68% is achieved, along with a cell viability of approximately 79%.

[0009] [Overview of the prefecture] [Problems the invention aims to solve] Although various transfection systems and methods exist for mammalian cells, these techniques suffer from one or more disadvantages. Therefore, there is still a need to provide improved electroporation systems and methods, especially for high-throughput, large-scale manufacturing processes.

[0010] [Means for solving the problem] The technologies presented herein relate to various devices, systems, and methods for the electroporation of cells, such as mammalian and non-mammalian cells, for example, for large-scale continuous manufacturing processes. In particular, systems and methods for the electroporation of cells using an electroporation apparatus that includes a first input separated from a first output by an offset distance can provide high efficiency and viability.

[0011] In some embodiments, a method is provided for electroporating a cargo with cells. This method involves flowing cells together with the cargo into an electroporation chamber. The electroporation chamber includes a first electrode, a second electrode, a first input that allows cells and cargo to pass into the electroporation chamber, a first output that allows electroporated cells to pass out of the electroporation chamber, and a fluid channel region having a theredefined pathway between the first electrode and the second electrode for cells and cargo to flow. Furthermore, the cells are suspended in an electroporation medium and approximately 1 × 10⁶ 6 Cells per ml ~ approximately 500 x 10 6 The cell density may be 10

[0012] In another embodiment, an apparatus for electroporating a cargo into cells is provided. The apparatus includes one or more electroporation chambers. Each electroporation chamber includes a first electrode, a second electrode, a first input that allows cells and cargo to pass into the electroporation chamber, a first output that allows electroporated cells to pass out of the electroporation chamber, and a fluid channel region having a theredefined path between the first electrode and the second electrode for the cells and cargo to flow.

[0013] In another embodiment, a kit for cell transfection is provided. The kit includes an electroporation apparatus as described herein, a first container for containing cells to be transfected and cargo, a second container for containing electroporated cells, tubing for fluidly connecting the first and second containers to the apparatus for electroporating the cells, and optionally, reagents, such as cells to be transfected, electroporation medium, or a combination thereof.

[0014] The various subjects, features, aspects and advantages of the subject matter described herein will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawings in which figures represent components.

[0015] Brief explanation of the drawing The patent or application file shall include at least one color drawing. The publication of this patent or patent application containing the color drawing shall be made available by the Patent Office upon request and payment of the necessary fees. [Brief description of the drawing]

[0016] [Figures 1A-1B] A typical electroporation apparatus is shown, including parallel plate electrodes (as shown in Figure 1A) and micromesh electrodes with no offset between input and output (as shown in Figure 1B). [Figure 2A-2B] shows embodiments of an electroporation apparatus according to some embodiments provided herein. [Figure 2C] shows embodiments of the first and second electrodes according to some embodiments provided herein. [Figure 2D] A block diagram of an apparatus for generating and delivering electrical pulses to cells in an electroporation apparatus as described herein, according to some embodiments provided herein. [Figure 2E] A block diagram of a further apparatus for generating and delivering electrical pulses to cells in an electroporation apparatus as described herein, according to some embodiments provided herein. [Figures 3A-3D] These figures illustrate embodiments of an electroporation apparatus, including an electroporation device, according to some embodiments provided herein. Figure 3A shows a diagram of the structure of an input / output chamber offset electroporation device. Figure 3B shows a diagram of the electric field of the input / output chamber offset electroporation device. Figure 3C shows the electric field strength as a function of the cell migration distance through the input / output chamber offset electroporation device. Figure 3D shows an alternative arrangement of the electroporation device according to some embodiments provided herein. [Figure 3E-3G] shows an example of a pathway within an electroporation chamber according to some embodiments provided herein. [Figure 3H-3J] shows alternative arrangements of an electroporation apparatus according to some embodiments provided herein. [Figure 3K-3L] An example of a microfluidic chamber for sorting cells, according to some embodiments provided herein, is illustrated. [Figure 3M-3S] This drawing shows various aspects of the geometric shape and positioning of a post in a microfluidic chamber according to some embodiments provided herein. [Figure 3T] An example of a microfluidic chamber for modifying a buffer solution, according to some embodiments provided herein, is illustrated. [Figure 3U] An example of a microfluidic chamber having two separate chambers, such as one chamber for cell sorting and another chamber for buffer change, according to some embodiments provided herein. [Figure 3V-3W] shows alternative arrangements of an electroporation apparatus according to some embodiments provided herein. [Figure 3X] shows a modular arrangement of an electroporation system including an electroporation apparatus according to some embodiments provided herein. [Figure 4] A table of parameters related to a chamber offset electroporation apparatus according to some embodiments provided herein is shown. [Figure 5] shows examples of fluid flow waveforms (pump) and electrical waveforms (stimulation) that may be applied to cells passing through a chamber offset electroporation apparatus according to some embodiments provided herein. [Figure 6] Shows various examples of micromesh formed from different materials suitable for use in chamber offset electroporation apparatus, according to some embodiments provided herein. [Figures 7A-7E] show the results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein. [Figures 8A-8D] Further results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein are shown. [Figures 9A-9D] show the results of electroporation experiments with EC7 cells and corresponding parameters using the methods and devices described herein. [Figures 10A-10E] Further results of electroporation experiments with EC7 cells and corresponding parameters using the methods and devices described herein are shown. [Figure 11A-11C] Microscopic images of electroporated EC7 cells are shown. [Figures 12A-12B] Further results of transfected EC7 cells using the methods and devices described herein are shown. [Figures 13A-13D] Show various transfection efficiencies in different cell lines using the methods and devices described herein. [Figures 14A-14E] show the results of transfection experiments for introducing mRNA into T cells using PbAE as described herein. [Figures 15A-15B] The results of transfection experiments for introducing mRNA into T cells in the presence and absence of electroporation, as described herein, are shown. [Figures 16A-16D] show the results of transfection experiments with adipose-derived mesenchymal stem cells (AD-MSCs) and corresponding parameters using the methods and devices described herein. [Figure 17] This is a block diagram of an electroporation apparatus according to another embodiment of the present disclosure. [Figure 18] This is an exploded view of an electroporation channel that may be used in the electroporation apparatus of Figure 17, according to another embodiment of the present disclosure. [Figure 19] This is an isometric view of the electroporation chamber shown in Figure 18, with the layers assembled together. [Figure 20] This is a front view of the fluid channel layer of the electroporation chamber shown in Figure 18. [Figure 21] This is a front view of a fluid channel layer having a different fluid volume from the fluid channel layer in Figure 20, according to another embodiment of the present disclosure. [Figure 22] This is a front view of a fluid channel layer having a linear fluid channel according to another embodiment of the present disclosure. [Figure 23] This is a front view of the fluid channel layer of Figure 20, showing an example of fluid channel dimensions.

[0017] [Modes for carrying out the invention] Systems and methods for the electroporation of cells, such as mammalian cells (e.g., NK cells, EC-7 cells, T cells, etc.) and non-mammalian cells, are provided along with corresponding electroporation protocols that provide high efficiency and viability. In some embodiments, the electroporation protocol involves applying multiple electrical pulses, a stepwise fluid flow, an electroporation buffer with low conductivity and low volume molar osmotic concentration, a relatively moderate volume, and / or a relatively moderate time constant to the cells.

[0018] In various embodiments, apparatuses are provided for electroporating cargo into mammalian cells. The apparatus may include one or more electroporation chambers, each of which includes a first electrode, a second electrode, and a fluid channel region having a defined path between the first and second electrodes. Each of the first and second electrodes may be a solid electrode or a mesh electrode, such as a micromesh electrode. The apparatus also includes a first input that allows cells and cargo to pass into the electroporation chamber, and a first output that allows electroporated cells to pass out of the electroporation chamber. As used herein, “electroporated cells” include transfected cells, dead cells, and untransfected cells. In some embodiments, the first input and the first output may be separated by an offset distance. The first electrode may be surrounded by a first material, and the second electrode may be surrounded by a second material, which may be the same or different.

[0019] For example, Figure 2A illustrates an exemplary apparatus for electroporating a cargo to cells, including an electroporation apparatus 700a. The electroporation apparatus 700a includes an electroporation chamber 720 which includes two electrodes, such as a first electrode 740(1) and a second electrode 740(2), which are shown here to be in a straight line with each other. The electroporation apparatus 700a further includes a first input 710 which allows cells and cargo to pass into the electroporation chamber 720 and a first output 730 which allows electroporated cells to pass out of the electroporation chamber 720. The first input 710 and the first output 730 may be separated by an offset distance (d) to facilitate the flow of cells laterally through the electroporation chamber 720. A fluid channel region 725 is located within the electroporation chamber 720 and includes a defined path 735 between the first electrode 740(1) and the second electrode 740(2) for the flow of cells and cargo. Although not shown, pathway 735 may have a pathway inlet corresponding to a first input for allowing cells and cargo to pass into pathway 735, and a pathway outlet corresponding to a first output 730 for allowing electroporated cells to pass out of pathway 735.

[0020] In any embodiment, the first input may be located in or defined on the first electrode, the second electrode, or the fluid channel region. In addition, the first output may be located in or defined on the first electrode, the second electrode, or the fluid channel region. Both the first input and the first output may be located on either the first electrode or the second electrode. For example, as shown in Figure 2A, both the first input 710 and the first output 730 are located on the second electrode 740(2). Alternatively, the first input may be located on the first electrode and the first output on the second electrode, or the first input may be located on the second electrode and the first output on the first electrode. In this specification, in the case of a fluid channel region, the first input, the first output, or both may be located, for example, on the sidewall of the fluid channel region.

[0021] As further illustrated in Figure 2B, within the electroporation apparatus 700b, the upper surface 706 of the first electrode 740(1) may be surrounded by or connected to the first material 715(1), and the lower surface 711 of the second electrode 740(2) may be surrounded by or connected to the second material 715(2). For example, since the first and second materials contain cells within the internal chamber of the electroporation apparatus, particularly within the electroporation chamber between the parallel first and second electrodes, they may be non-porous materials such as polydimethylsiloxane (PDMS) acrylic, polyethylene, polypropylene, or acrylic glass. Additionally or alternatively, the electroporation apparatus 700b may further include a first external input 750 for further enabling the passage of cells and cargo into the electroporation chamber 720, and a first external output 760 for further enabling the passage of electroporated cells from the electroporation chamber 720. A first external input may be present in or defined in a first material, and a first external output may be present in or defined in a second material. Alternatively, a first external input may be present in or defined in a second material, and a first external output may be present in or defined in a first material. In further embodiments, both the first external input and the first external output may be present in or defined in either a first or second material. For example, as illustrated in Figure 2B, both the first external input 750 and the first external output 760 are present in or defined in the second material 715(2), and the arrows indicate the flow direction of cells and cargo through the electroporation apparatus and the electroporated cells exiting the electroporation apparatus. Other openings or holes shown in Figures 2A and 2B, which are not referenced, represent openings for connecting various components of the electroporation apparatus, for example, via screws and / or plugs, as is known to those skilled in the art.

[0022] Each of the first and second electrodes may be a solid electrode, such as a solid plate electrode, or a porous mesh electrode, such as a micromesh electrode. In one embodiment, both the first and second electrodes are solid electrodes, such as solid plate electrodes. In any embodiment, when the first electrode, the second electrode, or both are solid electrodes, the first electrode, the second electrode, or both include a plurality or a series of solid metal plates. For example, as illustrated in Figure 2C, the first electrode 741(1) and the second electrode(741)(2) include a plurality of solid metal plates 745. Each solid metal plate 745 may be formed to apply an independent electric field to create a specific electric field pattern. Suitable materials on which the first and second electrodes may be formed include, but are not limited to, stainless steel, polyimide, silicon, precious metals, metals of group 4, conductive materials, and combinations thereof. Examples of suitable precious metals include, but are not limited to, ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), osmium (Os), iridium (Ir), platinum (Pt), and gold (Au). Examples of metals in Group 4 include titanium (Ti), zirconium (Zr), hafnium (Hf), and rutherfordium (Rf). Examples of suitable conductive materials include, but are not limited to, indium tin oxide (ITO), carbon nanotubes (CNTs), and conductive polymers, such as poly(3,4-ethylenedioxythiophene)polystyrene sulfonic acid (PEDOT:PSS).

[0023] In any embodiment, any one of the electroporation apparatuses disclosed herein may include components for supplying electrical pulses to cells during an electroporation process with the electroporation parameters described herein. For example, Figure 2D illustrates apparatus 852 for generating and supplying electrical pulses to cells as described herein. Specifically, as shown in Figure 2D, apparatus 852 includes a power supply 854 and a control circuit 858 connected to the power supply 854. The power supply 854 supplies electrical pulses to the electroporation apparatus 860 (e.g., any one of the electroporation apparatus examples disclosed herein).

[0024] In the example shown in Figure 2D, the power supply 854 includes a power source 856, a power converter 862 connected to the power source 856, and a pulse circuit 864 connected between the power converter 862 and the electroporation device 860. The power converter 862 may include a DC-DC power converter and / or an AC-DC power converter, depending on the type of power provided by, for example, the power source 856. For example, the power converter 862 may receive DC power from the power source 856 (e.g., one or more batteries) and output a regulated DC voltage to the pulse circuit 864. In such an example, the power converter 862 may include a DC-DC power converter with any preferred conversion topology (e.g., buck, boost, etc.). In another example, the power converter 862 may receive AC power from the power source 856 (e.g., AC main). In such an example, the power converter 862 may include an AC-DC power converter with any preferred conversion topology (e.g., AC-DC rectifier, PFC boost, etc.).

[0025] The pulse circuit 864 in Figure 2D receives a DC voltage from the power converter 862 and converts the DC voltage into DC electrical pulses for the electroporation device 860. For example, the pulse circuit 864 may include one or more switching elements (not shown) for reading the DC voltage from the power converter 862 and generating electrical pulses. In other examples, the pulse circuit 864 may include other suitable circuits for generating electrical pulses for the electroporation device 860.

[0026] As shown in Figure 2D, the control circuit 858 includes a controller 866 and a pulse generator 868 coupled to the controller 866. The controller 866 receives a feedback signal 870 representing an output parameter (e.g., output voltage) of the power converter 862 and generates one or more control signals 872 for the power converter 862 to control one or more switching elements (not shown) within the power converter 862 based on the feedback signal 870. For example, the controller 866 may control the switching elements of the power converter to adjust the output voltage of the power converter to a specified value (e.g., a desired amplitude of an electrical pulse). In some examples, the controller 866 may decrease or increase the output voltage based on the feedback signal 870 and / or other signals disclosed herein.

[0027] In addition, as shown in Figure 2D, the controller 866 may generate a signal 874 for the pulse generator 868, and the pulse generator 868 may generate one or more control signals 876 for the pulse circuit 864 for generating electrical pulses based on the signal received from the controller 866. For example, the signal 874 may be a start and / or stop signal that commands the pulse generator 868 to start generating electrical pulses. In some examples, the pulse generator 868 may adjust the frequency, pulse width, duty cycle, etc., of the electrical pulses based on the signal received from the controller 866.

[0028] In some examples, the control circuit 858 may receive one or more additional signals to control the power converter 862 and / or the pulse generator 868. For example, as also shown in Figure 2D, the pulse generator 868 in the control circuit 858 may optionally receive one or more signals 878a. Signals 878a are feedback signals from the pulse circuit 864, the electroporation device 860, etc., and may be used to control the pulse circuit 864 for generating electrical pulses. In addition, as also shown in Figure 2D, the controller 866 in the control circuit 858 may optionally receive one or more signals 880 representing feedback signals from the pulse circuit 864, the electroporation device 860, etc., and / or one or more signals 882 representing user-defined commands from the user interface. In such examples, signals 880 and / or signals 882 may be used to control the pulse circuit 864 and / or the power converter 862, monitor pulse timing, etc. In some examples, the apparatus 852 may include one or more detectors (not shown) for monitoring the chip voltage and / or chip current associated with the electroporation apparatus 860. In such examples, the pulse generator 868 and / or controller 866 may receive the detected chip voltage and / or detected chip current via signals 878a, 880 and then control the pulse circuit 864 and / or power converter 862 based on the detected chip voltage and / or current.

[0029] The control circuit 858 may also generate one or more additional signals for control purposes. For example, the controller 866 may optionally generate one or more signals 884 for other components of the electroporation apparatus and / or components outside the electroporation apparatus. In such an example, the signals 884 may represent user feedback provided to the user interface, control signals for controlling the liquid pump, peristaltic pump, and pinch valve motors (e.g., stepping motors) within the apparatus, etc. In addition, the pulse circuit 864 may optionally generate one or more signals 878b for other components of the electroporation apparatus and / or components outside the electroporation apparatus. For example, the pulse circuit may generate a signal 878b for the detector to instruct the detector to begin monitoring voltage and current.

[0030] Figure 2E illustrates another example apparatus 886 for generating and supplying electrical pulses to cells as described herein. As shown, apparatus 886 includes a power supply 888 connected to an electroporation apparatus 860 and a control circuit 890 for controlling the power supply 888. The power supply 888 and control circuit 890 in Figure 2E are similar to the power supply 854 and control circuit 858 in Figure 2D, but include additional components. For example, the power supply 888, along with the power supply 856, power converter 862 and pulse circuit 864 in Figure 2D, includes a filter 892 connected between the power supply 856 and the power converter 862. The filter 892 may include one or more components (not shown), such as a capacitor or inductor, for smoothing the electrical signal from the power supply 856.

[0031] In addition, as also shown in Figure 2E, the control circuit 890, along with the controller 866 and pulse generator 868 in Figure 2D, includes one or more gate drivers 894 coupled to the pulse generator 868. In the example in Figure 2E, the gate driver 894 receives a signal from the pulse generator 868 and generates one or more pulse-width modulation (PWM) control signals 896 for controlling one or more switching elements (e.g., MOSFETs) in the pulse circuit 864. In addition, the control circuit 890 may receive and / or generate one or more of the various signals referenced in Figure 2D. For example, the controller 866 may receive a feedback signal 870 and generate a control signal 872 for controlling one or more switching elements (e.g., MOSFETs) in the power converter 862, as described above.

[0032] The control circuits disclosed herein may include hardware components and / or programmed software components for performing various functions, including those disclosed herein. For example, one of the control circuits 858, 890 in Figures 2D and 2E may include one or more sensors for monitoring parameters of power supplies 854, 888 (as described herein), parameters related to the electroporation apparatus 860, etc. The parameters may include electrical parameters related to power supplies 854, 888 and / or the electroporation apparatus 860, fluid parameters related to the electroporation apparatus 860, etc. In such an example, the control circuits 858, 890 may control other components of power supplies 854, 888 and / or apparatuses 852, 886 (e.g., liquid pumps) based on the parameters. In this specification, it is considered that any one of the power supplies 854, 888 and any one of the control circuits 858, 890 described herein may provide electrical pulses and electroporation parameters such as waveform, electric field strength, voltage, duration between each electrical pulse, pulse width, duration of each electrical pulse, etc. For example, any one of the control circuits 858, 890 may provide one or more of the following: (i) each electrical pulse has the form of an exponential discharge waveform or a square waveform; (ii) a plurality of electrical pulses are applied with an electric field strength of about 0.3 kV / cm to about 3 kV / cm; (iii) the electric field strength is applied with a voltage of about 15 V to about 100 V; (iv) the duration between each electrical pulse is about 0.1 seconds to about 10 seconds; and (v) each electrical pulse has a pulse width of about 10 μs to about 10,000 μs.

[0033] In some embodiments, the first electrode may be an upper micromesh electrode, and the second electrode may be a lower micromesh electrode. In such embodiments, the electroporation chamber may include an upper micromesh electrode, a lower micromesh electrode, and a defined path between the upper and lower micromesh electrodes for the flow of cells and cargo. Each of the upper and lower micromesh electrodes has porosity to allow cells to pass through the micromesh electrode either into or out of the electroporation chamber. For example, each of the upper and lower micromesh electrodes may have porosity such as an opening area of ​​about 30% to about 50%. The width of the pore openings of the upper and lower micromesh electrodes may be about 70 μm to about 140 μm. The upper micromesh electrode may be surrounded by a first material, and the lower micromesh electrode may be surrounded by a second material. The first material includes a first input that allows cells to pass into the electroporation chamber, and the second material includes a first output that allows electroporated cells to pass out of the electroporation chamber. Additionally or alternatively, instead of cells and / or cargo moving through the pores of the upper micromesh electrode and entering the electroporation chamber, the upper micromesh electrode may optionally include a second input to allow the passage of cells and / or cargo into the electroporation chamber. The second input may be substantially aligned with the first input in the first material. Additionally or alternatively, instead of electroporated cells moving through the pores of the lower micromesh electrode and leaving the electroporation chamber, the lower micromesh electrode may include a second output to allow the passage of electroporation from the electroporation chamber to the outside. The second output may be substantially aligned with the first output in the second material.

[0034] For example, Figure 3A illustrates an exemplary apparatus for electroporating a cargo into cells, which includes an IOCO electroporation apparatus 200 comprising micromesh electrodes. The IOCO electroporation apparatus 200 comprises two micromesh electrodes shown here aligned with each other, such as an upper micromesh electrode 240(1) and a lower micromesh electrode 240(2). The upper surface of the upper micromesh electrode 240(1) may be surrounded by a first material, and the lower surface of the lower micromesh electrode 240(2) may be surrounded by a second material. For example, the first and second materials may be non-porous materials such as polydimethylsiloxane (PDMS) acrylic, polyethylene, or polypropylene, since the cells are contained within the internal chambers of the electroporation apparatus, particularly within the chamber 220 between the two parallel micromesh electrodes. The first input 210 is formed on the upper micromesh electrode 240(1) (in the region without PDMS), and the first output 230 is formed on the lower micromesh electrode 240(2) (in the region without PDMS). Thus, in this example, the upper micromesh electrode 240(1) and the lower micromesh electrode 240(2) extend to the length of the electroporation chamber 220, surrounded by the first material (upper PDMS layer) and the second material (lower PDMS layer), in order to direct the cell flow along the lateral distance of the chamber. The openings in the first material (upper PDMS layer) (first input 210) and the openings in the second material (lower PDMS layer) (first output 230) are separated by an offset distance (d) to facilitate the lateral passage of the cell flow through the electroporation chamber 220.

[0035] In any embodiment, the electroporation chamber described above may have any desired shape, including, but is not limited to, circular, oval, square, rectangular, or polygonal.

[0036] In any embodiment, the electroporation chamber width (w) may be in the range of approximately 0.01 mm to 15 mm, 0.01 mm to 10 mm, 0.01 mm to 7.5 mm, 0.01 mm to 5 mm, approximately 0.05 mm to 5 mm, approximately 0.1 mm to 15 mm, 0.1 mm to 10 mm, 0.1 mm to 7.5 mm, approximately 0.1 mm to 5 mm, approximately 1 mm to 15 mm, approximately 1 mm to 10 mm, approximately 1 mm to 7.5 mm, approximately 1 mm to 5 mm, approximately 2 mm to 10 mm, approximately 2 mm to 5 mm, approximately 0.01 mm to 4 mm, approximately 0.1 mm to 4 mm, approximately 1 mm to 4 mm, approximately 0.01 mm to 2 mm, approximately 0.05 mm to 1 mm, approximately 0.1 mm to 0.5 mm, or approximately 0.2 mm to 0.4 mm. In some embodiments, the chamber width (w) is approximately 0.3 mm or approximately 4 mm.

[0037] In some embodiments, the offset distance (d) between the first input and the first output may be in the range of approximately 0.1 cm to approximately 10 cm, approximately 0.1 cm to approximately 5 cm, approximately 0.1 cm to approximately 4 cm, or approximately 1 cm to approximately 3 cm. In some embodiments, the offset distance (d) is approximately 2 cm. In some embodiments, there is no overlap between the input and the output.

[0038] The first input and / or second input are fluidically connected to the electroporation chamber, and the electroporation chamber is fluidly connected to the first output and / or second output. The inputs, outputs, and electroporation chamber may also include one or more valves, regulators, pumps, or any other microfluidic components to control the flow of cells through the electroporation chamber.

[0039] In some embodiments, both the first input and the first output may be located in a first material on a higher micromesh electrode, provided that a suitable offset distance is maintained between the first input and the first output. Additionally or alternatively, both the second input and the second output may be located on the higher micromesh electrode. In other embodiments, both the first input and the first output may be located in a second material on a lower micromesh electrode, provided that a suitable offset distance is maintained between the first input and the first output. Additionally or alternatively, both the second input and the second output may be located on the lower micromesh electrode.

[0040] In some embodiments, cells may enter a first input and flow in a first direction. Once they have passed the first or second electrode, the cell flow may change direction and flow laterally along a path parallel to the length of the electroporation chamber, perpendicular or substantially perpendicular to the first direction. Once they reach the first output, the cell flow may change direction again and be discharged from the electroporation chamber in a second direction parallel to the first direction.

[0041] In some embodiments, a voltage suitable for generating an electric field in the range of about 0.3 kV / cm to about 3 kV / cm is applied between parallel first and second electrodes. Typical electric fields are in the range of about 1 to 1.3 kV / cm (e.g., 40 V / 300 μm) for haNKS and about 0.3 to 1 kV / cm for EC-7 cells. In some embodiments, the voltage may be about 10 V, about 20 V, about 30 V, about 40 V, about 50 V, about 75 V, about 100 V, or about 200 V, or any preferred range therein, or higher.

[0042] In some embodiments, the diameters of the first input, second input, first output, and second output may be approximately the same. In other embodiments, the diameter of the first input may be larger or smaller than the diameter of the first output, and / or the diameter of the second input may be larger or smaller than the diameter of the second output. For example, the diameter of the first input and / or the diameter of the second input may be in the range of about 0.1 mm to about 10 mm, about 1 mm to about 7 mm, about 3 mm to about 5 mm, or about 4 mm. The diameter of the first output and / or the diameter of the second output may be in the range of about 0.1 mm to about 10 mm, about 1 mm to about 7 mm, about 3 mm to about 5 mm, or about 4 mm.

[0043] In any embodiment, the length (l) of the electroporation chamber may be in the range of approximately 2 mm to 100 mm, approximately 2 mm to 80 mm, approximately 2 mm to 60 mm, approximately 2 mm to 40 mm, approximately 2 mm to 20 mm, approximately 5 mm to 100 mm, approximately 5 mm to 80 mm, approximately 5 mm to 60 mm, approximately 5 mm to 40 mm, approximately 5 mm to 20 mm, approximately 5 mm to 15 mm, approximately 8 mm to 12 mm, or approximately 10 mm.

[0044] In any embodiment, the height (h) of the electroporation chamber may be in the range of approximately 100 μm to approximately 1000 μm, approximately 100 μm to approximately 750 μm, approximately 100 μm to approximately 500 μm, approximately 100 μm to approximately 300 μm, approximately 100 μm to approximately 200 μm, approximately 200 μm to approximately 1000 μm, approximately 200 μm to approximately 750 μm, approximately 200 μm to approximately 500 μm, or approximately 200 μm to approximately 300 μm. In some embodiments, the chamber height (h) is approximately 284 μm.

[0045] In any embodiment, the ratio of length (l) to width (w) (l / w) may be about 1 to about 50, about 1 to about 40, about 1 to about 20, or about 1 to about 10.

[0046] In any embodiment, once cells enter the electroporation chamber, they are exposed to a uniform electric field until they leave the electroporation chamber. For example, Figure 3B is a diagram of the electric field in the IOCO electroporation apparatus 200. Position (1) refers to a cell at the input (first input 210), position (2) refers to a cell inside the electroporation chamber, and position (3) refers to a cell at the output (first output 230).

[0047] Figure 3C is a plot of electric field strength against cell migration distance through the IOCO electroporation apparatus 200. At position (1), the input (first input 210), the cell is not exposed to the electric field until it passes through the micromesh electrode and enters the electroporation chamber. Within the electroporation chamber, indicated by position (2), the cell moves along the length of the chamber while receiving a uniform (maximum intensity) electric field. Once it enters the output (first output 230), indicated by position (3), the cell is no longer exposed to the electric field.

[0048] Figure 3D shows an alternative configuration of the electroporation apparatus presented herein, including electroporation apparatus 310-330. In this embodiment, cells may have curved or circular (horizontal) channels between the upper and lower meshes, rather than having linear (horizontal) channels between the upper and lower meshes. Each of the linear and curved channels may include at least one segment extending parallel to a plane defined by at least one surface of the upper and lower meshes.

[0049] In this example, various layers 310-330 of the electroporation apparatus are shown in Figure 3D. Solid outer plates 310(1) and 310(2) (e.g., plastic, metal, or any other suitable material) surround the apparatus. An adhesive layer or other suitable material (e.g., double-sided tape, pressure-sensitive adhesive material, etc.) 320(1) (first material) is provided between the upper solid outer plate 310(1) and the upper mesh 330(1), and an adhesive layer or other suitable material (e.g., double-sided tape, pressure-sensitive adhesive layer, etc.) 330(2) (second material) is provided between the lower outer plate 310(2) and the lower mesh 330(2). Another adhesive layer (e.g., pressure-sensitive adhesive material or double-sided tape, etc.) (340) containing curved channels or curved pathways 335 through which cells and cargo may flow is located between the upper mesh 330(1) and the lower mesh 330(2). The fluid may be driven by a syringe 350 through the electroporation apparatus 310-330. The electroporation apparatus 310-330 may also face an electroporator pulser 360. In any embodiment, the electroporation apparatus 310-330 may be a continuous electroporation apparatus through which cells flow in a continuous manner.

[0050] The pathways through which cells and cargo flow through the electroporation chamber are defined within the electroporation chamber, for example, within the fluid channel region described above between the first electrode (e.g., a solid electrode or an upper micromesh electrode) and the second electrode (e.g., a solid electrode or an lower micromesh electrode). It is understood that any preferred pathway is considered by embodiments provided herein, provided that it includes at least one horizontal flow segment parallel to at least one of the first electrode (e.g., a solid electrode or an upper micromesh electrode) and the second electrode (e.g., a solid electrode or an lower micromesh electrode). The horizontal flow segment may be longer or shorter than 1 / 10, 1 / 4, 1 / 2, 3 / 4, 7 / 8, or 9 / 10 of the length of the pathway extending between the upper and lower micromesh electrodes. In some cases, the pathway may be linear, curved, branched, detoured, meandering, or any combination thereof. Examples of curved pathways include, but are not limited to, circular pathways, helical pathways, conical pathways, or any combination thereof. For example, as illustrated in Figure 3B, when arrows illustrate cell flow, the path is linear. As illustrated in Figures 2A, 2B, 3D, and 3E, when arrows illustrate cell flow, the path is curved. As illustrated in Figure 3F, when arrows illustrate cell flow, the path is linear and may have one or more changes or switchbacks in direction. As illustrated in Figure 3G, the path is circular and may have one or more branches. As shown in Figures 3E to 3G, the path may include an inlet 380 (out-of-plane) which may correspond to a first input or be substantially aligned with the first input, and one or more outlets 390 (in-plane) which may correspond to a first output or be substantially aligned with the first output. The length of the path may be adjusted, for example, shortened or lengthened, depending on the specific characteristics of the electroporation process.

[0051] In any embodiment, the electroporation apparatus described herein may include means for generating a flow of cells from the sample tube to the electroporation chamber of the electroporation apparatus, through the apparatus, and to the collection tube. For example, as illustrated in Figure 3H, the flow generating means 520 is fluidly connected to the sample tube 510 containing the cells and / or cargo to be transfected and the electroporation apparatus 530, for example, via a tube. The flow generating means 520 can generate a flow of cells and / or cargo through the tube from the sample tube 510, through the electroporation apparatus 530, and into the collection tube 550. Although not shown, an air filter device may be fluidly connected to the contents of the sample tube 510, the collection tube 550, or both. The flow generating means may be any suitable pump, such as a peristaltic pump or a syringe pump. In this specification, it is conceivable that two or more flow generating means 520 may be present in the electroporation apparatus. For example, one or more flow generating means 520 may be fluidly connected between the sample tube 510 and the electroporation apparatus 530, or one or more flow generating means 520 may be fluidly connected between the collection tube 550 and the electroporation apparatus 530. Additionally or alternatively, the electroporation apparatus 530 and / or flow generating means 520 may also face a controller 540, for example, which includes an electroporator pulser.

[0052] In any embodiment, the electroporation apparatus described herein may also include a priming buffer tube fluidly connected to the electroporation apparatus for delivering priming buffer as needed. Suitable priming buffers include, but are not limited to, distilled water, deionized water, isotonic buffers, and combinations thereof. For example, as illustrated in Figure 3I, the priming buffer tube 560 is fluidly connected to the electroporation apparatus 530. Valve 570 may be opened to deliver priming buffer to the electroporation apparatus 530 via the flow generating means 520, or valve 570 may be closed to stop delivery. Valve 575 may be opened to deliver cells and / or cargo to the electroporation apparatus 530 via the flow generating means 520, or valve 575 may be closed to stop delivery. In some embodiments, both valves 570 and 757 may be opened to deliver cells and priming buffer. In some embodiments, priming buffer may be delivered first, followed by cells and / or cargo.

[0053] In any embodiment, the electroporation apparatus described herein may also include a first microfluidic device for sorting cells before they enter the electroporation chamber of the electroporation apparatus. For example, as illustrated in Figure 3J, the first microfluidic device 580 may be fluidly connected to the electroporation apparatus 530, a sample tube 510 containing cells to be transfected, and optionally a buffer sorting tube 577. A flow generating means 520 may generate a flow of cells from the sample tube 510 and / or buffer from the sorting tube 577, passing through the electroporation apparatus 530 and into a collection tube 550. The first microfluidic device 580 is capable of pre-sorting cells from the sample tube 510 to the electroporation apparatus 530, for example, based on cell size. Additionally or alternatively, the first microfluidic device 580 is capable of causing a buffer change of cells from the sample tube 510. Flow 590 contains cells that have been sorted and removed and are not delivered to the electroporation apparatus 530, and may be a waste flow or may be delivered to another electroporation apparatus using different parameters. Additionally or alternatively, a second microfluidic device 585 may be fluidly connected to the electroporation apparatus 530 and the collection tube 550. The second microfluidic device 585 has the ability to sort the electroporated cells, for example, based on the size of the electroporated cells, before collection in the collection tube 550. Flow 595 contains electroporated cells that have been sorted and removed and are not delivered to the collection tube 550.

[0054] Although two microfluidic devices are shown in Figure 3J, in this specification, only one microfluidic device may be present in the electroporation apparatus, for example, to sort cells before they enter the electroporation chamber or to sort electroporated cells after they leave the electroporation chamber.

[0055] Figure 3K is a diagram of an example of a microfluidic device (e.g., microfluidic device 580, microfluidic device 585). In this example, a solution containing cells 100 (e.g., cells to be transfected, electroporated cells, transfected cells) is entered into a chamber 105 by an input mechanism 110. Cells are shown as circles. The cells pass through the chamber 105, which contains a matrix of posts 115, shown here as rectangular structures distributed along multiple lines with a gradient. As the cells pass between diagonally oriented rows of posts within the chamber, they are deflected laterally toward a second output mechanism 122. A side view of part of the chamber is shown in Figure 3L, where the chamber has a floor 160 and optionally a ceiling 150, which may be made of the same or different material as the posts 115. Cells (black circles) are shown passing through the matrix of posts. The rows of posts may be further arranged in a curved manner, which is understood to result in the cells being guided toward the side of the chamber.

[0056] In this example, two output mechanisms are shown for the chamber. The solution exiting the chamber via the first output mechanism 120 (which may also be referred to as the third output mechanism when two or more microfluidic devices are present) is depleted of cells, while the solution exiting the chamber via the second output mechanism 122 (which may also be referred to as the fourth output mechanism when two or more microfluidic devices are present) is concentrated of cells. In any embodiment, the second output mechanism may be fluidly connected to the electroporation chamber, for example, to the first input. Depletion refers to a state in which the cell concentration in the solution exiting the chamber via the output mechanism 120 is 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower than the cell concentration in the solution entering the chamber via the input mechanism 110. Enrichment or concentration refers to a state in which the concentration of cells in the solution exiting the chamber via the output mechanism 122 is increased by 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more compared to the concentration of cells in the solution entering the chamber via the input mechanism 110. For cells induced to the output mechanism 122, the chamber's deflection point 130 must be positioned so that any existing cells are directed to the output mechanism 122 (and not to the output mechanism 120). Generally, the output mechanism 122 is formed to have a larger cross-sectional area than the output mechanism 120. Generally, the output mechanism has a cross-sectional area large enough for the fluid flow at the flow rates described herein so as not to introduce high pressures that would damage cells or devices.

[0057] In some embodiments, the width of the output mechanism 120 is greater than the width of the output mechanism 122. For example, the width of the output mechanism 120 is 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5 times that of the output mechanism 122. In other embodiments, the sum of the cross-sectional areas of the output mechanisms may be greater than or equal to the sum of the cross-sectional areas of the input mechanisms.

[0058] In some embodiments, the microfluidic device is formed to allow a solution containing cells to flow through the device by enabling connection to a standard pump or a microfluidic device such as a syringe. Parameters that may affect the flow of solution through the device include the dimensions of the chamber 105, the in-plan and out-of-plan dimensions of the posts 115, the spacing of posts in a row (dx), the rotation of posts (Φ), the distance between post rows (dy), the diameter of the input mechanism, and the diameter of the output mechanism (see Figures 3M to 3Q below). In some embodiments, the dimensions of these parameters are selected to accommodate the applied pressure from the manual operation of a syringe or standard pump (e.g., peristaltic pump, diaphragm pump, syringe pump, lobe pump, etc.) that drives the flow of solution through the device. Various pressures are acceptable, provided that the pressure does not damage the microfluidic device or cells.

[0059] Generally, cells may flow into a microfluidic device either continuously (single-in-one within a specific pathway) or in multiples (multiple cells flowing through multiple pathways).

[0060] Figure 3M is a top-down view drawing of a cross-section of chamber 105, where each cross-section contains two rows of seven posts. Cell flow is shown along pathways (p). This drawing shows the positioning of posts 115 within the cross-section of the chamber of the microfluidic device. Generally, the posts are aligned along lines with a gradient such that the spatial spacing (dx) is defined by an angle (θ), and may be fixed or variable, provided that the variation does not result in cells evading the pathways (p) between the posts.

[0061] In some embodiments, the width of the chamber is 1 mm, 2.5 mm, 5 mm, 7.5 mm, 10 mm, 12.5 mm, 15 mm, 17.5 mm, 20 mm, 30 mm, 40 mm, 50 mm, or more, or any size in between. The width of each path (p) controls the characteristics of the streamlines. For example, several paths such as 1, 2, 4, 8, 12, 16, 32, or any range in between, may be used depending on the width of the chamber. In other embodiments, the fluid velocity through the chamber may be 1 mL / hour, 2 mL / hour, 3 mL / hour, 4 mL / hour, 5 mL / hour, 6 mL / hour, 7 mL / hour, 8 mL / hour, 9 mL / hour, 10 mL / hour, 15 mL / hour, 20 mL / hour, 25 mL / hour, 30 mL / hour, 40 mL / hour, 50 mL / hour, etc. The chamber is preferably rectangular, but may also be circular, semicircular, V-shaped, or any other suitable shape.

[0062] The angle (θ) is the angle between a horizontal axis with a 0 gradient and a line with a gradient on which the posts are distributed (in this example, the angle provides a measure of the negative gradient (tan(θ) = Δy / Δx of the row of posts)). In this example, since each row of posts is arranged diagonally, it is understood that the posts may have a positive or negative gradient. Here, it is understood that the diagonal may encompass any orientation that is not parallel or perpendicular to the chamber, for example, angles between 1° and 89°, between 91° and 179°, between 181° and 269°, or between 271° and 359° in clockwise or counterclockwise directions. Other exemplary angle ranges may be 1°~10°, 11°~20°, 21°~30°, 31°~40°, 41°~50°, 51°~60°, 61°~70°, 71°~80°, 81°~90°, 91°~100°, 101°~110°, 111°~120°, 121°~130°, 131°~140°, 141°~150°, 151°~160°, 161°~170°, or 171°~180°.

[0063] Generally, multiple rows of posts are present within the chamber, each row having the same or substantially the same gradient as defined by an angle (θ). When cells 100 enter the chamber 105 of the microfluidic device, the cells circulate laterally toward the side of the chamber 105 through multiple paths (p) between the posts 115 until they reach the side of the chamber. When enriching cells, the cells generally do not traverse the rows of posts, and typically move along specific paths (p). The cells then exit the microfluidic chamber via the output mechanism 122. In other embodiments, the posts may have curved components along the length of the chamber 105.

[0064] As used herein, the term “post” refers to an internal structure of a chamber having relevant planar dimensions, external dimensions, rotation angle, and shape. Internal dimensions may refer to the length (l) and width (w) of the post, while external dimensions may refer to the height (h) of the post. Angle of inclination or rotation angle (Φ) refers to the rotation of the post relative to the chamber.

[0065] The shape refers to the three-dimensional characterization of the post, e.g., cylindrical, conical, pyramidal, cubic, or cubic. Generally, the post is positioned such that its axis (external plane dimension) is perpendicular to the surface to which it is attached. In some embodiments, all posts within a chamber are the same dimensions. In other embodiments, the dimensions of the post vary as a function of space depending on the position, measured, for example, along the height (h) of the post. Generally, the post may be of any shape and is not limited to any specific geometric shapes presented herein. The post shape may be arbitrary, as shown in Figure 3R, represented by the length (l) and width (w) of the axis, in addition to a number of radii (r4~r12) associated with bends, positioned, for example, at repeating fixed or repeating variable intervals along the length of the axis. Several different shapes may be described by the number of radii. In addition, referring to Figures 3R and 3S, one or more sides of the post may be of a straight shape. Thus, the post may have any preferred shape consisting of curves and / or straight lines.

[0066] In some embodiments, the posts are arranged at intervals (dx) along a gradient line, where the intervals are regularly spaced or fixed. For example, the posts may be placed at intervals of 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 85 μm, 90 μm, 95 μm, 100 μm (including any value between these ranges), depending on the size of the cells to be enriched.

[0067] In other embodiments, the posts are distributed along a gradient line such that the spacing is not fixed but varies between any two consecutive posts. Any spacing (dx) is acceptable, provided it is small enough to prevent cells from evading the pathway (p). For example, in the case of variable spacing, the first post may be positioned at a specific location, the second post 5 μm from the first post, and the third post 4 μm from the second post. The spacing can be selected based on the size of the cells.

[0068] In some embodiments, the spacing may be selected to be large enough to allow enrichment of specific types of cells while allowing smaller cells to exit through the output mechanism 120. For example, a blood sample may contain several different cell types of relatively small size, e.g., erythrocytes, neutrophils (e.g., 12–14 μm in diameter), eosinophils (e.g., 12–17 μm in diameter), basophils (e.g., 14–16 μm in diameter), lymphocytes (e.g., 10–14 μm in diameter), and monocytes (e.g., 20 μm in diameter). Other types of cells in the human body are much larger, e.g., in the range of 40–100 μm or more in diameter. In such cases, the posts may be separated to allow passage of erythrocytes and leukocytes while enriching larger cells (e.g., normal cells, tumor cells, etc.). The arrangement (spacing) of the posts is determined based on the type of cells being isolated. The ratio of different sheath flow to sample flow may affect the size-based sorting performance.

[0069] Each post has a corresponding length (l), width (w), and height (h), also referred to as the in-plane dimension (see also Figure 3N). In some embodiments, the width of the post may be 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 85 μm, 90 μm, 95 μm, 100 μm, etc., or any value in between. The length of the post may be 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 85 μm, 90 μm, 95 μm, 100 μm, etc., or any value in between. In general, the post may be any shape (in terms of top-down orientation and in relation to dimensions w and l), for example, circular, oval, square, rectangular, etc., but are not limited to these.

[0070] In the examples presented herein, the posts have an oval or quadrangular shape. In some embodiments, the length of the post is 1.1 to 10 times greater than the width of the post; 1.1 to 5 times greater than the width of the post; 2 to 4 times greater than the width of the post; 3 to 4 times greater than the width of the post; or 2 to 3 times greater than the width of the post. In some embodiments, the length and width may have a ratio greater than or less than any of the following ratios: 20:1, 18:1, 16:1, 14:1, 12:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.1:1, or any smaller or larger ratio.

[0071] In some embodiments, the cross-section of the post may be defined by the intersection of the post and a plane parallel to the original wall, which extends with height (h). The cross-section may be symmetrical in one or both of the length (l) and width (w) directions.

[0072] In yet another embodiment, the length and width of each post in the matrix are the same. In yet another embodiment, if the posts are circular, the radius of each post in the matrix is ​​the same.

[0073] Further features include the angle of rotation of the post (Φ), the height or external dimension of the post, the spacing between rows (dy), and the offset of the rows (xo), which are described in more detail in Figures 3N to 3Q and throughout this application. As used herein, the term “angle of rotation” refers to the degree of rotation of the post relative to its position in the chamber. In some embodiments, the post has a length greater than its width. In these embodiments, an angle of rotation of 0 means that the length of the post is aligned with an axis perpendicular to the side of the chamber. To describe different angles of rotation, the post may be rotated in a clockwise or counterclockwise direction. As an example, the post in Figures 3M to 3Q is rotated by approximately 35° in a counterclockwise motion to achieve the geometric shape of the post shown in these figures.

[0074] Referring to Figure 3N, each post has a height (h) or out-of-plan dimension related to the post being tall enough to prevent cells from escaping from pathway (p) by flowing through an upward pathway (e.g., over the top of the post to reach a different pathway). For example, in some embodiments, the post will extend to the height of the microfluidic chamber, such as being in contact with the chamber floor 160 and the chamber ceiling 150. In other embodiments, the height or out-of-plan dimension of the post may extend to a portion of the total height of the microfluidic device. For example, the post may have a height of 1 μm, 3 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 85 μm, 90 μm, 95 μm, 100 μm, etc., or any value in between.

[0075] Referring to Figure 3O, each post has an associated rotation angle (Φ) or inclination, as shown in the top-down view. In this example, the rotation of the post is determined by rotating the post counterclockwise, for example, approximately 35° counterclockwise in this example, until the desired rotation is reached, based on an orientation in which the length of the post is aligned with a first axis perpendicular to the side of the chamber and the width of the post is aligned with a second axis parallel to the side of the chamber. Any rotation angle that facilitates the flow of cells into pathway (p) may be used, and all such rotation angles are considered herein. In some embodiments, the rotation angle is between 1° and 179°, between 1° and 89°, between 5° and 85°, between 10° and 80°, between 15° and 75°, between 20° and 70°, between 25° and 65°, between 30° and 60°, between 35° and 55°, between 40° and 50°, between 30° and 40°, between 32° and 38°, between 34° and 36°, or 35°. In other embodiments, the rotation angle may be between 91° and 179°, between 95° and 175°, between 100° and 170°, between 105° and 165°, between 110° and 160°, between 115° and 155°, between 120° and 150°, between 125° and 145°, between 130° and 140°, or 135°.

[0076] As previously shown, the posts are distributed along a line with a negative gradient. By orienting the cells along this line, the cells flowing through pathway (p) are guided laterally toward the side of the chamber, while the solution in which the cells were initially suspended flows in a certain manner (e.g., nearly horizontally or horizontally) and can exit the microfluidic chamber via the output mechanism 120.

[0077] Referring to Figure 3P, in yet another embodiment, the post rows are spaced apart by intervals (dy). In some embodiments, the posts are arranged at intervals (dy) along a vertical line, where the intervals are regularly spaced or fixed. For example, the posts may be placed at intervals of 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 85 μm, 90 μm, 95 μm, 100 μm, etc. (including any values ​​in between), depending on the size of the cells to be enriched.

[0078] In other embodiments, the posts are distributed along a vertical line such that the spacing (dy) is not fixed but varies between any two consecutive rows of posts. For example, the first row of posts may be positioned at a specific location, the second row may be 5 μm from the first row, and the third row may be 4 μm from the second row, all aligned with respect to the vertical line. The spacing (dy) can be selected based on the size of the cells to be enriched. Any spacing (dy) is acceptable, provided that it does not require high pressure that could damage the microfluidic device and is wide enough to allow the flow of solution into the chamber. In some embodiments, consecutive rows of posts may have a zero offset (xo).

[0079] Referring to Figure 3Q, consecutive post columns may have an offset (xo) with respect to the interval (dx). For example, a column (r) may be determined to a specific position (e.g., using Cartesian coordinates x, y). The (r+1)th column may be displaced to the right by a certain amount (xo). The (r+2)th column may be displaced to the right by a certain amount (2xo). The (r+3)th column may be displaced to the right by a certain amount (3xo), and so on, until the column is displaced up to the interval (dx). The offset may be set in a fixed manner so that each consecutive column is displaced by a fixed amount in the same direction. Alternatively, the offset may be set in a variable manner so that each consecutive column is displaced by a variable amount (up to a maximum of (dx)) in the same direction. In other embodiments, the offsets may be applied in alternating directions such that the (r+1)th column has a fixed or variable offset in one direction relative to column (r), and the (r+2)th column has a fixed or variable offset in the opposite direction (for example, one offset is to the right and the next offset is to the left).

[0080] The post may be made of any suitable material, such as poly(dimethylsiloxane) (PDMS), glass, plastic, elastomer, or silicon. PDMS can generally be fabricated to have submicron resolution (e.g., features less than 0.1 μm). In some embodiments, the chamber 105 and / or post 115 may be made of PDMS. In other embodiments, the chamber may be made of glass and / or silicon and / or plastic, and the post may be made using PDMS (for example, the bottom of the chamber may be glass or silicon, and the top of the chamber may be glass or plastic). Lithography known in the art may be used to fabricate the chamber and post as described herein. Further materials having properties similar to PDMS may be used to construct the microfluidic devices shown herein.

[0081] In addition, specific embodiments may include several additional features, including valves (e.g., between an input mechanism and a chamber, between a chamber and an output mechanism, between an output mechanism and another input mechanism or another chamber), pumps, and mixers. In some embodiments, valves may be located during curation and in the PDMS.

[0082] Various techniques can be used to fabricate microfluidic devices. Microfluidic devices may be formed from one or more of the following materials: poly(methyl methacrylate) (PMMA), polycarbonate, polystyrene, polyethylene, polyolefin, silicone (e.g., poly(dimethylsiloxane) (PDMS)), silicon, and combinations thereof. Other materials are well known in the art.

[0083] Methods for fabricating chambers and microfluidic devices having posts using the materials referenced herein are known in the art and include, but are not limited to, embossing, laser micromachining, milling, molding (e.g., thermoplastic injection molding or compression molding), photolithography (e.g., stereolithography or X-ray photolithography), silicon micromachining, and wet or dry chemical etching.

[0084] Silicon manufacturing techniques using photolithography followed by wet (KOH) or dry etching (reactive ion etching with fluorine or other reactive gases) are available for glass materials. For example, glass masters can be formed by conventional photolithography and serve as master templates in molding techniques for fabricating plastic or PDMS-based devices.

[0085] The microfluidic device may be fabricated within one or more layers that are connected together, for example by adhesive, clamps, heat, or solvents. Alternatively, the microfluidic device may be fabricated as a single piece using, for example, stereolithography or other three-dimensional manufacturing techniques.

[0086] In some embodiments, due to the deformability of the cells, the gap distance may be selected to be about 5 μm or less relative to the size (e.g., diameter) of the cells to be enriched. In other embodiments, the gap distance may be selected to be about 5 μm or more relative to the size (e.g., diameter) of the cells to be sorted. In the case of rigid cells or microparticles having the same or similar diameter as the deformable cells, the gap distance may differ from the gap distance in deformable cells of the same diameter (e.g., larger in the case of rigid cells).

[0087] Figure 3T is a diagram of a microfluidic cell enrichment apparatus in another example. In this example, a first solution containing cells 100 enters the chamber 105 via a first input mechanism 110. The cells are shown as circles. The cells pass through the chamber 105, which contains a matrix of posts 115, shown here as rectangular structures distributed along a gradient line. As the cells pass between diagonally oriented rows of posts within the chamber, they are deflected laterally toward the sides of the chamber. Similar to Figure 3L, the chamber has a floor 160 (not shown) and an optional ceiling 150, which may be made of the same or different material as the posts 115.

[0088] In this example, a second input mechanism 112 is present in the system. The second solution (e.g., a buffer different from the first solution that enters the chamber through the first input mechanism 110), such as the buffer from buffer tube 577, enters the chamber through the second input mechanism 112. In some embodiments, the first and second solutions are substantially unmixed, and therefore the upper region 155 of the chamber contains the first solution and only a small amount of the second solution, while the lower region 165 of the chamber contains the second solution and only a small amount of the first solution. Thus, when the cells are deflected laterally and exit the chamber through the output mechanism 122, the solution exiting the chamber through the output mechanism 122 is present in the buffer of the second solution.

[0089] In some embodiments, buffer changes may be performed sequentially in a cascade design, as shown in Figure 3U. In this example, a first solution containing cells 100 enters a first chamber 105(1) via a first input mechanism 110(1). Cells are shown as circles. The cells pass through chamber 105(1), which contains a matrix of posts 115(1), shown here as rectangular structures distributed along a gradient line. As the cells pass between diagonally oriented rows of posts within the chamber, the cells are deflected laterally. Similar to Figure 3L, the chamber has a floor (160) (not shown) and an optional ceiling (150), which may be the same material as or different from the posts 115. The (enriched) cells exit the chamber via an output 122(1) that flows into input mechanism 110(2), if a second input mechanism 112 is the input to the system.

[0090] The second solution (e.g., buffer) enters the chamber through a second input mechanism (112), unlike the first solution which enters the chamber through a first input mechanism 110(1). In some embodiments, the first and second solutions are substantially unmixed, and therefore the upper region of the chamber (155) contains the first solution and only a small amount of the second solution, while the lower region of the chamber (165) contains the second solution and only a small amount of the first solution. The cells (concentrated and present in the buffer from the input mechanism 112) exit the chamber 105(2) through an output mechanism 122(2).

[0091] In other embodiments, buffer changes may be performed in parallel. For example, the flow path may be divided so that the sample flows into multiple chambers, each chamber including an input mechanism for a buffer. In this configuration, the same buffer may be supplied to each of the parallel chambers so that the cells are concentrated in the same buffer. Alternatively, different buffers may be supplied to each of the parallel chambers so that the cells are concentrated in different buffers, for example in different downstream assays.

[0092] Generally, the input mechanism can be driven by a syringe pump or by manual depressing of the syringe. In some embodiments, one or more syringe pumps may be used to automatically drive each of the multiple inputs. In other embodiments, one or more manual depressing of syringes may be used to manually drive each of the multiple inputs.

[0093] In any embodiment, electroporation apparatus capable of providing different electroporation effects is described herein. For example, an electroporation apparatus 600 having three electrodes 640(1), 640(2), and 640(3) is provided, as illustrated in Figure 3V. The electroporation apparatus 600 includes two pathways for the movement of cells and cargo: a first pathway between electrode 640(1) and electrode 640(2) and a second pathway between electrode 640(2) and electrode 640(3), where arrows indicate the flow of cells. Cells can enter the electroporation chamber at inlet 620 and exit through outlet 630. Although not illustrated, more than two outlets may be present, for example, each outlet may collect different types of cells. Different electrode gap lengths (L1, L2, L3, L4) are present within the electroporation apparatus 600. Different electrode gap lengths and different pathways result in different capacities; therefore, different electric field strengths and electroporation effects can be achieved in cells moving through each pathway. For example, L2 can be made larger than L4, and in such an example, the capacity in the first pathway (C1) can be made larger than the capacity in the second pathway (C2) (C1 > C2). In any embodiment, cells (of the same or different types) may be pre-sorted and / or selected, for example using the microfluidic device described above, before entering the first or second pathway.

[0094] This specification also considers that the electroporation apparatus described herein may exist as cartridges that are fluidically connected (e.g., connected, stacked) to provide pathways of desired length and shape. For example, as illustrated in Figure 3W, each of the cartridges 410 includes an electroporation chamber containing a first electrode 440(1) and a second electrode 440(2), where the cartridges may be sequentially stacked, and arrows indicate the flow of cells through the cartridges. For example, the outlet of the top cartridge 410 is fluidly connected to the inlet of an intermediate cartridge 410, and the outlet of the intermediate cartridge 410 is fluidly connected to the inlet of the bottom cartridge 410.

[0095] In further embodiments, a modular system including the electroporation apparatus described herein is provided. For example, as illustrated in Figure 3X, the modular system 800 includes three modules formed to be fluidically connected to each other, such as a first module 805, a second module 815, and a third module 825. The first module 805 includes a first container 807 (e.g., a sample tube) for containing cells and / or cargo to be transfected, and a tube 810 for fluidly connecting the first container 807 to the second module 815. The first module 805 may also optionally include an air filter device 809 and a tube 810 for fluidly connecting the air filter device 809 to the first container 807. The second module 815 includes an electroporation apparatus 820 and tubes 810 for fluidly connecting the electroporation apparatus to the first module 805, e.g., a first container 807, and the second module 825, as well as tubes 810 for fluidly connecting flow generating means (e.g., a peristaltic pump) as described herein to the electroporation apparatus 820 and the first module 805, e.g., the first container 807. The third module 825 includes a second container 830 (e.g., a collection tube) for collecting electroporated cells, and tubes 810 for fluidly connecting the second container 830 to the second module 815, e.g., the electroporation apparatus 820. The third module 825 may also optionally include an air filter apparatus 809 and tubes 810 for fluidly connecting the air filter apparatus 809 to the second container 830. Optionally, a connector 850, such as a Luer lock, may connect the modules together, for example, the first module 805 to the second module 815, and the second module 815 to the third module 825. In this specification, it is considered that each of the first module 805, the second module 815, and the third module 825 may be packaged and sterilized separately.

[0096] The electroporation apparatus described herein offers several advantages over mesh-based electroporation apparatuses, such as the configuration shown in Figure 1B. First, when measured from the first input to the first output of the electroporation chamber, the distance traveled through the electroporation chamber increases without a corresponding increase in the electric field. Therefore, cells are exposed to a uniform electric field for a longer period than in a strictly linear channel, resulting in improved electroporation efficiency without a corresponding decrease in viability.

[0097] In Figure 1B, the electroporation chamber width (w) between the upper and lower mesh electrodes increases, which can result in a longer travel distance. However, a wider electroporation chamber width results in higher impedance, requiring a higher voltage to generate a suitable electric field. In addition, higher voltages are more likely to have a detrimental effect on cell viability. Furthermore, in parallel plate electrode configurations, such as in Figure 1A, the electric field distribution becomes non-uniform.

[0098] Therefore, according to the embodiments, the electroporation apparatus described herein can have shorter electrode pairs by the offset input / output design, thereby increasing the length of the electroporation chamber and preventing an increase in the electrode pair distance.

[0099] Figure 4 presents a table illustrating the differences between the electroporation apparatus described herein and electroporation apparatuses in the prior art. Such differences include, but are not limited to, (1) the presence of an input-output offset distance, (2) the use of low-conductance, low-volume molar osmotic concentration electroporation media instead of commercially available electroporation media such as BTX and RPMI, (3) an exponential discharge waveform applied in a series of electrical pulses, and (4) a stepwise fluid flow scheme.

[0100] In the examples shown in Figures 3A to 3C, stainless steel micromesh electrodes are used, although the electrodes are not limited to any particular material and may be formed from any suitable material. For example, as shown in Figure 6, suitable materials include silicon and polyimide. Silicon and polyimide may be used to form suitable micromesh electrodes, but such materials are typically processed in a cleanroom facility using microfabrication protocols known in the art. In contrast, stainless steel micromesh electrodes are easy to set up and do not require dedicated facilities, such as a cleanroom.

[0101] It is understood that any suitable material may be used, provided that the micromesh has pores that allow cells to pass through (for example, the micromesh pores are larger than the cells) and the micromesh pores conform to the electrode patterning around the pore openings.

[0102] Electroporation method and parameters A method for electroporating a cargo with cells is provided herein, for example, with an electroporation apparatus as described above. This method may include flowing cells together with the cargo into an electroporation chamber. The electroporation chamber includes a first electrode (e.g., a solid electrode), a second electrode (e.g., a solid electrode), and a fluid channel region (all as described herein) having a defined path between the first and second electrodes. The apparatus also includes a first input that allows the passage of cells and cargo into the electroporation chamber as described herein, and a first output that allows the passage of electroporated cells from the electroporation chamber as described herein. In some embodiments, the first input and the first output may be separated by an offset distance. The cells may be suspended in an electroporation medium. The first electrode may be surrounded by a first material as described herein, and the second electrode may be surrounded by a second material as described herein, they may be the same or different. The first and second materials may also include a first external input and a first external output as described herein.

[0103] In some embodiments, the electroporation chamber includes an upper micromesh electrode as described herein, a lower micromesh electrode as described herein, and a defined path between the upper and lower micromesh electrodes for the flow of cells and cargo as described herein. Each of the upper and lower micromesh electrodes has porosity as described herein. In addition, the upper micromesh electrode may be surrounded by a first material as described herein. The first material may include a first input as described herein, which allows cells to pass into the electroporation chamber. The lower micromesh electrode may be surrounded by a second material. The second material may include a first output as described herein, which allows electroporated cells to pass from the electroporation chamber. The first input and the first output are separated by an offset distance as described herein. Cells can be suspended in an electroporation medium. Optionally, the upper micromesh electrode may further include a second input as described herein, and / or the lower micromesh electrode may further include a second output as described herein.

[0104] In any embodiment, the flow of cells and cargo may be carried out in a stepwise manner. For example, a flow rate of more than about half the total volume of the electroporation chamber may be pumped into the electroporation chamber at specified time intervals. In some embodiments, a flow rate approximately equal to the total volume of the electroporation chamber may be pumped into the electroporation chamber. Figure 5 shows an example of a series of electrical pulses (stimuli) and an example of a stepwise fluid flow scheme (pump). In the stepwise fluid flow scheme, the chamber volume is approximately 22 μL. Every second, 11 μL is pumped into the electroporation chamber, replacing half of the flow rate. This may be repeated for the duration required to process the desired amount of cells.

[0105] Additionally or alternatively, any suitable time interval may be used with any amount suitable for a stepwise fluid flow scheme. For example, the time interval may be in the range of about 0.1 seconds to about 10 seconds, about 0.5 seconds to about 5 seconds, about 1 second to about 2 seconds, or any time in between, or may include longer intervals. In some embodiments, the amount of fluid pumped into the electroporation chamber as a function of time may be three-quarters of the volume of the input chamber, half of the volume of the electroporation chamber, one-quarter of the volume of the electroporation chamber, one-eighth of the volume of the electroporation chamber, or less. Any suitable flow rates of cells and cargo can be used with the parameters described herein. For example, the flow rate of cells and cargo in the electroporation medium may be approximately 0.1 ml / min or more, approximately 0.5 ml / min or more, approximately 1 ml / min or more, approximately 2.5 ml / min or more, approximately 5 ml / min or more, approximately 7.5 ml / min or more, approximately 10 ml / min or more, approximately 12.5 ml / min or more, or approximately 15 ml / min or more, or approximately 0.1 ml / min to approximately 15 ml / min, approximately 0.5 ml / min to approximately 15 ml / min, approximately 1 ml / min to approximately 15 ml / min, approximately 1 ml / min to approximately 12.5 ml / min, approximately 1 ml / min to approximately 10 ml / min, approximately 1 ml / min to approximately 7.5 ml / min, approximately 1 ml / min to approximately 5 ml / min, or approximately 1 ml / min to approximately 2.5 ml / min.

[0106] In any embodiment, cells are exposed to a uniform or substantially uniform electric field within an electroporation chamber. Multiple electrical pulses may be applied to the cells within the electroporation chamber, where each electrical pulse may be the same or different. In some embodiments, direct current (DC) electrical pulses are applied. Additionally or alternatively, alternating current (AC) electrical pulses are applied. In any embodiment, each pulse may have the form of an exponential discharge waveform or a square waveform. In any embodiment, multiple electrical pulses may include both exponential discharge waveforms and square waveforms.

[0107] In the case of a series of electrical pulses, a voltage may be applied to the electrodes every 100ms to 5000ms (e.g., every 100ms, 250ms, 500ms, 1000ms, 2000ms, 3000ms, etc.) to generate an electric field of a given intensity. This may be repeated for the duration required to process the desired amount of cells. Thus, while the embodiment may be referred to as continuous flow, it is understood that continuous flow involves a stepwise fluid flow scheme in which the fluid is continuously pumped into the electroporation chamber according to predetermined time intervals.

[0108] Additionally or alternatively, any preferred duration between electrical pulses may be used in conjunction with any preferred duration of electrical pulses applied to cells in the electroporation chamber. For example, the duration between each pulse may be in the range of about 0.1 seconds to about 15 seconds, about 0.1 seconds to about 10 seconds, about 0.1 seconds to about 5 seconds, about 0.2 seconds to about 1 second, about 0.4 seconds to about 0.6 seconds, or any time in between, and may include longer intervals. In some embodiments, the duration of the electrical pulse may be in the range of approximately 10 ms to approximately 10 s, approximately 10 ms to approximately 5 s, approximately 10 ms to approximately 1 s, approximately 10 ms to approximately 500 ms, approximately 10 ms to approximately 250 ms, approximately 50 ms to approximately 150 ms, approximately 75 ms to approximately 125 ms, approximately 100 ms to approximately 10 s, approximately 100 ms to approximately 5 s, approximately 100 ms to approximately 1 s, approximately 100 ms to approximately 500 ms, approximately 100 ms to approximately 250 ms, or any range in between.

[0109] Regarding the preferred number of pulses and the interval between pulses, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 pulses may provide more desirable results than a single pulse. Therefore, the pulses may be in the range of 2-10 pulses, 2-6 pulses, 6-8 pulses, or 2-3 pulses. Most typically, pulses are separated from subsequent pulses by a relatively short interval, typically between 0.5 and 15 seconds, although longer intervals may be used in some cases.

[0110] In any embodiment, approximately 0.1kV / cm to approximately 5kV / cm, approximately 0.1kV / cm to approximately 3kV / cm, approximately 0.1kV / cm to approximately 2kV / cm, approximately 0.1kV / cm to approximately 1kV / cm, approximately 0.1kV / cm to approximately 0.5kV / cm, approximately 0.3kV / cm to approximately 5kV / cm, approximately 0.3kV / cm to approximately 3kV / cm, approximately 0.3kV / cm to approximately 2kV / cm, approximately 0.3kV / cm to approximately 1kV / cm, approximately 0.3kV / cm to approximately 0 Multiple electrical pulses may be applied at voltages suitable for generating electric field strengths of 0.5kV / cm, approximately 0.5kV / cm to approximately 3kV / cm, approximately 0.5kV / cm to approximately 2kV / cm, approximately 0.5kV / cm to approximately 1kV / cm, approximately 0.8kV / cm to approximately 3kV / cm, approximately 0.8kV / cm to approximately 2kV / cm, approximately 0.8kV / cm to approximately 1kV / cm, approximately 1kV / cm to approximately 3kV / cm, or approximately 1kV / cm to approximately 2kV / cm. Suitable voltages may be approximately 10V, 15V, 20V, 30V, 40V, 50V, 75V, 100V, or 200V, or approximately 10V to 200V, approximately 10V to 100V, approximately 10V to 75V, approximately 10V to 50V, approximately 10V to 40V, approximately 10V to 30V, approximately 15V to 200V, approximately 15V to 100V, approximately 15V to 75V, approximately 15V to 50V, approximately 15V to 40V, approximately 15V to 30V, approximately 20V to 50V, approximately 20V to 40V, approximately 20V to 30V, approximately 30V to 50V, approximately 30V to 40V, or any suitable range among them.

[0111] Additionally or alternatively, each electrical pulse may be approximately 10 μs or longer, approximately 50 μs or longer, approximately 75 μs or longer, approximately 100 μs or longer, approximately 250 μs or longer, approximately 500 μs or longer, approximately 750 μs or longer, approximately 1,000 μs or longer, approximately 5,000 μs or longer, or approximately 10,000 μs; or approximately 10 μs to approximately 10,000 μs, approximately 10 μs to approximately 5,000 μs, approximately 10 μs to approximately 1,000 μs, approximately 10 μs to approximately 750 μs, approximately 10 μs to approximately 50 The pulse width may be 0 μs, approximately 10 μs to approximately 250 μs, approximately 10 μs to approximately 100 μs, approximately 10 μs to approximately 50 μs, approximately 100 μs to approximately 1000 μs, approximately 100 μs to approximately 750 μs, approximately 100 μs to approximately 500 μs, approximately 100 μs to approximately 250 μs, approximately 250 μs to approximately 1000 μs, approximately 250 μs to approximately 750 μs, approximately 250 μs to approximately 500 μs, approximately 500 μs to approximately 1000 μs, or approximately 500 μs to approximately 750 μs.

[0112] Any suitable cells, e.g., mammalian cells, non-mammalian cells, or both, may be transfected with the cargo. Examples of suitable mammalian cells, but not limited to, include NK cells (e.g., haNK cells, primary NK cells, activated (aNK) cells), EC-7 cells (derivatives of HEK293 cells modified to produce adenovirus), T cells (e.g., primary CD4 / CD8 T cells), CHO-S cells, dendritic cells, embryonic cells, stem cells (e.g., adipose-derived mesenchymal stem cells (AD-MSCs)), epithelial cells, lymphocytes, macrophages, gamete cells, and fibroblasts. Examples of suitable non-mammalian cells, but not limited to, include bacterial cells and yeast cells. Any suitable cargo, e.g., nucleic acids, may be used. The nucleic acids may be RNA (e.g., synthetic RNA, mRNA, in vitro transcription RNA, GFP-mRNA, etc.) and / or DNA (e.g., synthetic DNA, GFP-DNA, plasmid DNA, etc.). In particular, NK cells, EC-7 cells (derivatives of HEK293 cells modified to produce adenovirus), and T cells can be transfected with RNA (e.g., synthetic RNA, mRNA, in vitro transcription RNA, GFP-mRNA, etc.) and / or DNA (e.g., synthetic DNA, GFP-DNA, plasmid DNA, etc.) using the electroporation apparatus described herein. Furthermore, it is considered that the electroporation apparatus described herein can be used to perform in vitro fertilization. For example, eggs and sperm can be passed through an electroporation apparatus like the one described herein to achieve egg fertilization.

[0113] In embodiments to be further considered, the culture medium or electroporation buffer used when cells are transfected is optionally a medium with low conductivity and low volume molar osmotic pressure concentration containing one or more nutrients. In any embodiment, the electroporation medium has a conductivity of approximately 0.05 mS / m or higher, approximately 1 mS / m or higher, approximately 5 mS / m or higher, approximately 10 mS / m or higher, approximately 15 mS / m or higher, approximately mS / m or higher, approximately 25 mS / m or higher, approximately 30 mS / m or higher, approximately 50 mS / m or higher, approximately 100 mS / m or higher, approximately 150 mS / m or higher, or approximately 200 mS / m It may have conductances of m or more; or approximately 0.05 mS / m to approximately 200 mS / m; approximately 1 mS / m to approximately 30 mS / m, approximately 1 mS / m to approximately 20 mS / m, approximately 1 mS / m to approximately 10 mS / m, approximately 1 mS / m to approximately 5 mS / m, approximately 3 mS / m to approximately 30 mS / m, approximately 5 mS / m to approximately 20 mS / m, or approximately 5 mS / m to approximately 15 mS / m. Additionally or alternatively, the electroporation medium may be approximately 50 mOsm / l or higher, approximately 100 mOsm / l or higher, approximately 150 mOsm / l or higher, approximately 200 mOsm / l or higher, approximately 250 mOsm / l or higher, approximately 300 mOsm / l or higher, approximately 350 mOsm / l or higher, or approximately 400 mOsm / l; or approximately 50 mOsm / l to approximately 400 mOsm / l, or approximately 50 mOsm / l to approximately 300 mOsm / l It may have a volume molar osmotic pressure concentration of approximately 50 mOsm / l to approximately 200 mOsm / l, approximately 100 mOsm / l to approximately 400 mOsm / l, approximately 100 mOsm / l to approximately 300 mOsm / l, approximately 200 mOsm / l to approximately 400 mOsm / l, approximately 200 mOsm / l to approximately 300 mOsm / l, approximately 250 mOsm / l to approximately 400 mOsm / l, or approximately 250 mOsm / l to approximately 300 mOsm / l.

[0114] In any embodiment, the electroporation medium may contain one or more salts, sugars, and buffers. Suitable salts include, but are not limited to, metal halide salts, phosphates, metal sulfates, and combinations thereof. Suitable metal halide salts include, but are not limited to, potassium chloride (KCl), sodium chloride (NaCl), lithium chloride (LiCl), calcium chloride (CaCl2), chromium chloride (CrCl3), potassium bromide (KBr), sodium bromide (NaBr), magnesium chloride (MgCl2), magnesium bromide (MgBr2), magnesium fluoride (MgF2), magnesium iodide (MgI2), lithium bromide (LiBr), potassium iodide (KI), sodium iodide (NaI), and lithium iodide (LiI). Suitable phosphates include, but are not limited to, monosodium phosphate (NaH2PO4), disodium phosphate (Na2HPO4), trisodium phosphate (Na3PO4), monomagnesium phosphate (Mg(H2PO4)2), dimagnesium phosphate (MgHPO4), trimagnesium phosphate (Mg3(PO4)2), monopotassium phosphate (KH2PO4), dipotassium phosphate (K2HPO4), tripotassium phosphate (K3PO4), monocalcium phosphate (Ca(H2PO4)2), dicalcium phosphate (CaHPO4), and phosphorus. Examples include tricalcium sulfate (Ca3(PO4)2) and chromium phosphate (CrPO4). Suitable metal sulfates include, but are not limited to, sodium sulfate (Na2SO4), magnesium sulfate (MgSO4), potassium sulfate (K2SO4), calcium sulfate (CaSO4), and chromium sulfate (Cr2(SO4)3). In some embodiments, one or more salts may be potassium chloride, magnesium chloride, disodium phosphate, monopotassium phosphate, magnesium sulfate, and combinations thereof.

[0115] Examples of suitable sugars include monosaccharides, disaccharides, or combinations thereof. Suitable monosaccharides include, but are not limited to, glucose, fructose, and galactose. Suitable disaccharides include, but are not limited to, disaccharides formed from two of glucose, fructose, and galactose. For example, suitable disaccharides may be sucrose, lactose, maltose, trehalose, or combinations thereof. Examples of suitable buffers include, but are not limited to, hepes, tris(hydroxymethyl), and PBS (phosphate-buffered saline).

[0116] In any embodiment, each of one or more salts may be present in the electroporation medium at concentrations of about 0.1 mM to about 200 mM, about 0.1 mM to about 150 mM, about 0.1 mM to about 100 mM, about 0.1 mM to about 80 mM, about 0.1 mM to about 60 mM, about 0.1 mM to about 40 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 1 mM to about 20 mM, or about 1 mM to about 10 mM. For example, salts such as KH2PO4, Na2HPO4, and MgSO4 may be present in the electroporation medium at concentrations of about 0.1 mM to about 20 mM. Sugars may be present in the electroporation medium at concentrations of approximately 20 mM to 300 mM, 20 mM to 200 mM, or 40 mM to 200 mM. Buffers may be present in the electroporation medium at concentrations of approximately 1 mM to 100 mM, 10 mM to 50 mM, or 15 mM to 30 mM.

[0117] Other suitable media include, but are not limited to, Cw100 (0.11 S / m, 0.12 osm / l) or Cw240, as shown in Table 1. Commercially available electroporation media may be used, but have been shown to be suboptimal. Such commercial media contain RPMI (1.37 S / m, 0.28 osm / l), BTX (8 mS / m, 0.27 osm / l), DMEM (Dulbecc's modified Eagle medium), diluted PBS, or PBS, in or out of the presence of hepes. The media are generally conductive media and may be further sterile.

[0118]

Table 1

[0119] In any embodiment, the cells in electroporation are about 1×10 6 cells / ml or more, about 10×10 6 cells / ml or more, about 50×10 6 cells / ml or more, about 100×10 6 cells / ml or more, about 150×10 6 cells / ml or more, about 200×10 6 cells / ml or more, about 250×10 6 cells / ml or more, about 300×10 6 cells / ml or more, about 400×10 6 cells / ml or more, or about 500×10 6 cells / ml; or about 1×10 6 cells / ml to about 300×10 6 cells / ml, about 1×10 6 cells / ml to about 250×10 6 cells / ml, about 1×10 6 cells / ml to about 200×10 6 cells / ml, about 1×10 6 cells / ml to about 150×10 6 cells / ml, about 1×10 6 cells / ml to about 100×10 6 cells / ml, about 1×10 6 cells / ml to about 50×10 6 cells / ml, about 1×10 6 cells / ml to about 300×10 6 cells / ml, about 1×10 6 cells / ml to about 250×10 6 cells / ml, about 10×10 6 cells / ml to about 200×10 6 cells / ml, about 10×10 6 cells / ml to about 150×10 6 cells / ml, about 10×10 6 cells / ml to about 100×106 cells / ml, approximately 10 × 10 6 Cells per ml ~ approximately 50 x 10 6 cells / ml, approximately 100 x 10 6 Cells per ml ~ approximately 300 x 10 6 cells / ml, approximately 100 x 10 6 Cells per ml ~ approximately 250 x 10 6 cells / ml, approximately 100 x 10 6 Cells per ml ~ approximately 200 x 10 6 cells / ml, or approximately 100 x 10⁶ 6 Cells per ml ~ approximately 150 x 10 6 They may be present in the electroporation medium at a cell density of 1 cell / ml.

[0120] Regarding suitable capacitance, it is considered that capacitance may be in the range of approximately 1 μF to approximately 150 μF. In some embodiments, capacitance may be in the range of approximately 1 to 100 μF, approximately 5 to approximately 75 μF, approximately 5 to approximately 50 μF, approximately 10 to approximately 40 μF, or approximately 10 to approximately 30 μF, or approximately 10 to approximately 25 μF. In other embodiments, capacitance is approximately 10 μF.

[0121] In some embodiments, multiple electrical pulses may be used with corresponding short time constants. In some embodiments, time constants of less than 30 milliseconds, less than 20 milliseconds, less than 10 milliseconds, or less than 5 milliseconds may be used. In other embodiments, the time constants may be in the range of about 0.5 to 30 milliseconds, about 1 to 20 milliseconds, and about 5 to 15 milliseconds; or about 10 milliseconds.

[0122] In some embodiments, the electric field strength for electroporation is between approximately 0.3 and 3 kV / cm. Lower electric field strengths (e.g., approximately 0.5 to 1 kV / cm) have been found to be suitable for EC-7 cells, while higher electric field strengths (e.g., approximately 1 to 3 kV / cm) have been found to be suitable for Hank cells. Generally, electric field strengths for electroporation in the range of approximately 0.1 to approximately 5 kV / cm are considered herein. The voltage may be selected to produce a suitable electric field strength.

[0123] Generally, the impedance (Rp) can range from approximately 200Ω to infinity, or otherwise from approximately 200Ω to a maximum of approximately 1kΩ.

[0124] The concentration of the cargo added to the electroporation reaction, for example, the material to be transported into the cell, is approximately 50 μg / ml or more, approximately 100 μg / ml or more, approximately 200 μg / ml or more, approximately 300 μg / ml or more, approximately 400 μg / ml or more, or approximately 500 μg / ml or more; approximately 50 μg / ml to approximately 500 μg / ml, approximately 50 μg / ml to approximately 400 μg / ml, approximately 50 μg / ml to approximately 300 μg / ml, approximately 50 μg / ml to approximately 200 μg / ml, approximately 50 μg / ml to approximately 100 μg / ml, approximately 100 μg / ml to approximately 500 μg / ml, approximately 100 μg / ml to approximately 400 μg / ml, approximately 100 μg / ml to approximately 300 μg / ml, or approximately 100 μg / ml to approximately 200 μg / ml. In some embodiments, GFP-mRNA or GFP-DNA was added to the electroporation medium at concentrations of approximately 50 μg / ml, 60 μg / ml, 100 μg / ml, 200 μg / ml, 100-300 μg / ml, and 50-100 μg / ml, while in other embodiments, dextran 500k was added to the electroporation reaction at a concentration of approximately 50 μg / ml.

[0125] In some embodiments, 4 × 10 in a single chamber device 7 Cells per ml (13.2 mL (5.28 × 10)) 8 A maximum of 7.33 uL / s can be achieved in a single cell within 30 minutes. 9 To achieve individual cells, the cell density may be increased fourfold, and / or parallel processing may be carried out using multiple electroporation chambers.

[0126] In some embodiments and even in the examples provided below, the electronics and parameters of electroporation may be designed specifically for the electroporation of NK cells (e.g., haNK cells, primary NK cells, activated (aNK) cells), EC7 cells, dendritic cells, CHO-S cells, T cells (e.g., primary CD4 / CD8 T cells), and stem cells (e.g., AD-MSC cells).

[0127] In some embodiments, the cells are one or more of the following: NK cells (e.g., haNK cells, primary NK cells, activated (aNK) cells), EC7 cells, dendritic cells, CHO-S cells, T cells (e.g., primary CD4 / CD8 T cells), and stem cells (AD-MSC cells), and one or more of the electroporation parameters are as follows: (i) the electric field strength is applied at a voltage of approximately 20V to approximately 50V; (ii) the pulse width is approximately 10μs to approximately 750μs; and (iii) the cell density of the cells in the electroporation medium is approximately 5 × 10⁻⁶ 6 Cells per ml ~ approximately 300 x 10 6 It is one cell / ml.

[0128] In some embodiments, the cells are EC-7 cells, the cargo is DNA, and one or more of the electroporation parameters are as follows: (i) the electric field strength is applied at a voltage of approximately 20V to 50V, 20V to 40V, or 20V to 30V; (ii) the pulse width is approximately 10μs to 100μs, 10μs to 75μs, or 10μs to 50μs; (iii) the number of pulses is 2 to 6 or 2 to 3; and (iv) the cell density of the cells in the electroporation medium is approximately 10 × 10 6 Cells per ml ~ approximately 250 x 10 6 cells / ml, approximately 10 × 10 6 Cells per ml ~ approximately 100 x 10 6 cells / ml, or approximately 50 × 10 6 Cells per ml ~ approximately 100 x 10 6This is the number of cells / ml. Additionally or alternatively, EC-7 cells with electroporated DNA cargo may have a viability of at least about 60%, at least about 70%, or at least about 80%, along with a transfection efficiency of at least about 70%, at least about 80%, or at least about 95%.

[0129] In some embodiments, the cells are CHO-S cells, the cargo is DNA, and one or more of the electroporation parameters are as follows: (i) the electric field strength is applied at a voltage of approximately 20V to 50V, or approximately 30V to 50V, or approximately 40V to 50V; (ii) the pulse width is approximately 100μs to 750μs, approximately 250μs to 750μs, or approximately 500μs to 750μs; (iii) the number of pulses is 2 to 6 or 2 to 3; and (iv) the cell density of the cells in the electroporation medium is approximately 50 × 10⁻¹⁶ 6 Cells per ml ~ approximately 250 x 10 6 cells / ml, approximately 50 x 10 6 Cells per ml ~ approximately 125 × 10 6 cells / ml, or approximately 50 × 10 6 Cells per ml ~ approximately 100 x 10 6 The cell density is 1 / ml. Additionally or alternatively, CHO-S cells with electroporated DNA cargo may have a viability of at least 60%, at least 70%, at least 80%, or at least 90%, with a transfection efficiency of at least about 70%, at least about 80%, or at least about 85%.

[0130] In some embodiments, the cells are T cells (e.g., primary CD4 / CD8 T cells), the cargo is DNA or RNA, and one or more of the electroporation parameters are as follows: (i) the electric field strength is applied at a voltage of approximately 20V to 50V, or approximately 30V to 50V, or approximately 40V to 50V; (ii) the pulse width is approximately 100μs to 750μs, approximately 100μs to 500μs, or approximately 200μs to 400μs; (iii) the number of pulses is 2 to 10 or 2 to 6; and (iv) the cell density of the cells in the electroporation medium is approximately 10 × 10 6 Cells per ml ~ approximately 300 x 10 6 cells / ml, approximately 20 x 10 6 Cells per ml ~ approximately 250 x 10 6 cells / ml, or approximately 10 × 10 6 Cells per ml ~ approximately 200 x 10 6 The cell density is 10000 cells / ml. Additionally or alternatively, T cells (e.g., primary CD4 / CD8 T cells) electroporated with DNA or RNA cargo may have a viability of at least 60%, at least 70%, at least 80%, or at least 90%, with a transfection efficiency of at least about 20%, at least about 80%, at least about 90%, or at least about 99%.

[0131] In some embodiments, the cells are NK cells (e.g., haNK cells, primary NK cells, activated (aNK) cells), the cargo is DNA or RNA, and one or more of the electroporation parameters are as follows: (i) the electric field strength is applied at a voltage of approximately 20V to 50V, or approximately 30V to 50V, or approximately 40V to 50V; (ii) the pulse width is approximately 100μs to 750μs, approximately 200μs to 750μs, or approximately 200μs to 600μs; (iii) the number of pulses is 2 to 6 or 2 to 3; and (iv) the cell density of the cells in the electroporation medium is approximately 10 × 10⁻¹⁶ 6 Cells per ml ~ approximately 300 x 10 6 cells / ml, approximately 10 × 10 6 Cells per ml ~ approximately 250 x 10 6 cells / ml, or approximately 10 × 106 Cells per ml ~ approximately 100 x 10 6 The cell density is 10000 cells / ml. Additionally or alternatively, NK cells (e.g., haNK cells, primary NK cells, activated (aNK) cells) to which DNA or RNA cargo has been electroporated may have a viability of at least 60%, at least 70%, at least 80%, or at least 90%, with a transfection efficiency of at least about 10%, at least about 50%, at least about 80%, at least about 90%, or at least about 95%.

[0132] In some embodiments, the cells are dendritic cells, the cargo is DNA or RNA, and one or more of the electroporation parameters are as follows: (i) the electric field strength is applied at a voltage of approximately 20V to 50V, or approximately 30V to 50V, or approximately 30V to 40V; (ii) the pulse width is approximately 100μs to 750μs, approximately 100μs to 500μs, or approximately 200μs to 400μs; (iii) the number of pulses is 2 to 10 or 6 to 8; and (iv) the cell density of the cells in the electroporation medium is approximately 10 × 10 6 Cells per ml ~ approximately 100 x 10 6 cells / ml, approximately 10 × 10 6 Cells per ml ~ approximately 50 x 10 6 cells / ml, or approximately 10 × 10 6 Cells per ml ~ approximately 25 x 10 6 The cell density is 1000 cells / ml. Additionally or alternatively, dendritic cells electroporated with DNA or RNA cargo may have a viability of at least 70%, at least 80%, or at least 90%, along with a transfection efficiency of at least about 70%, at least about 80%, or at least about 90%.

[0133] In some embodiments, the cells are stem cells (e.g., AD-MSC cells), the cargo is DNA, and one or more of the electroporation parameters are as follows: (i) the electric field strength is applied at a voltage of approximately 20V to 50V, or approximately 30V to 50V, or approximately 30V to 40V; (ii) the pulse width is approximately 50μs to 250μs, approximately 50μs to 100μs, or approximately 100μs to 200μs; (iii) the number of pulses is 2 to 10 or 6 to 8; and (iv) the cell density of the cells in the electroporation medium is approximately 10 × 10 6 Cells per ml ~ approximately 100 x 10 6 cells / ml, approximately 10 × 10 6 Cells per ml ~ approximately 50 x 10 6 cells / ml, or approximately 10 × 10 6 Cells per ml ~ approximately 25 x 10 6 This is the number of cells / ml. Additionally or alternatively, stem cells with electroporated DNA cargo (e.g., AD-MSC cells) may have a viability of at least about 70%, at least about 80%, or at least about 90%, along with a transfection efficiency of at least about 70%, at least about 80%, or at least about 90%.

[0134] The methods described herein can be applied to transient or stable transfections. The term “transient transfection” refers to the introduction or transfection of nucleic acids that are present in cells for a limited period of time and are not integrated into the genome. The term “stable transfection” refers to transfection that results in the permanent expression of a target gene through the integration of the transfected nucleic acid into the nuclear genome or through the maintenance of the transfected plasmid as an extrachromosomal replicating episome within the cell.

[0135] In any embodiment, the method described herein may further include a first cell sorting step and / or a second cell sorting step using, for example, a microfluidic device as described herein. The first cell sorting step can sort the cells before introduction into the electroporation chamber by applying pressure to cause the flow of a first solution containing cells through a microfluidic chamber as described herein, which includes a plurality of post rows as described herein, thereby deflecting the cells to the sides of the chamber by the post rows, depleting the cells from the solution exiting a first output mechanism as described herein, and concentrating the cells in the solution exiting a second output mechanism as described herein. The cells exiting the second output mechanism can be introduced into the electroporation chamber together with the cargo. The second cell sorting step involves applying pressure to cause the flow of the fourth solution containing the electroporated cells through a microfluidic chamber as described herein, which includes multiple post rows as described herein, thereby deflecting the electroporated cells to the sides of the chamber by the post rows, depleting the electroporated cells from the solution exiting the third output mechanism as described herein, and concentrating the electroporated cells in the solution exiting the fourth output mechanism as described herein, thereby sorting the electroporated cells after they leave the electroporation chamber.

[0136] The techniques and methods provided herein may be incorporated into various devices or platforms as part of a workflow. For example, an electroporation apparatus as described herein may be incorporated into an automated device, for example, in the form of a cartridge, where electroporation may be performed in conjunction with one or more other automated processes on cells. For example, automated electroporation may be performed in conjunction with automated cell culture and harvesting as described in U.S. Patent Application Publication No. 2017 / 0037357 (which is incorporated by reference in its entirety).

[0137] In other embodiments, in vivo transfection methods are provided, in which the above method is carried out, and transfected cells mixed with, for example, isotonic buffer may be administered to a patient to treat a disease or disorder. Examples of diseases, but not limited to, include mitochondrial disorders, cardiac dysfunction, heart failure, autism, diabetes, and deafness. It is also conceivable that the electroporation apparatus described herein may be adapted for application to target tissues to contribute to the delivery of drugs or formulations, for example, insulin or drugs to treat diabetes. For example, electrodes for electroporation may be applied to the skin surface, or a pair of needle electrodes may be applied subcutaneously. It is also conceivable that the electroporation apparatus and method may be used for adenovirus production, protein synthesis, cellular immunotherapy, or regenerative medicine.

[0138] This specification also provides kits. For example, a kit may include an electroporation apparatus as described herein, a first container for containing cells to be transfected and cargo, a second container for containing electroporated cells, tubing for fluidly connecting the first and second containers to the electroporation apparatus, and optionally suitable reagents. In some embodiments, a kit may include a first package including a first module (e.g., first module 805) containing, for example, a first portion of the first container and tubing. The kit may further include a separated second package including a second module (e.g., second module 815) containing, for example, a second portion of the electroporation apparatus and tubing. The kit may also include a separated third package including a third module (e.g., third module 825) containing, for example, a third portion of the second container and tubing. Optionally, reagents may reside in a separated fourth package. The first, second, third, and fourth packages may be sterile. Suitable reagents include, but are not limited to, transfection target cells as described herein, electroporation media as described herein, and combinations thereof. Additionally or alternatively, the kit may further include electroporation tips, connecting tubes, apparatus, electroporation tools, electroporation buffers, additives, reagents, and combinations thereof. [Examples]

[0139] Example 1. Transfection of haNK cells In some embodiments, the conditions for electroporation of haNK cells are shown in Table 2 below.

[0140] [Table 2]

[0141] Figures 7A to 7E show the experimental results of the electroporation reaction. Figure 7A shows various efficiencies and viability under various experimental conditions. In particular, using the IOCO electroporation apparatus, under conditions of 1.5 kv / cm, Rp 1k, and 10 uF, electroporation of GFP-mRNA into hank cells yielded an efficiency of over 80% with a viability of over 70%. The name "reg" refers to the original micromesh, while "offset" refers to the offset micromesh. Figure 7B shows a microscopic image of GFP-mRNA being electroporated into hank cells. Figures 7C and 7D show the results of cell sorting, where Figure 7C shows live cells and Figure 7D shows electroporated cells. Figure 7E shows a histogram corresponding to the cell sorting results, showing control cells and cells to which GFP was electroporated.

[0142] Figures 8A–8D show a set of different experimental results under various electroporation conditions, demonstrating that electroporation of GFP-mRNA to haNK cells achieved efficiency exceeding 50% and viability exceeding 70%. Figures 8B and 8C show the results of cell sorting, where Figure 8B shows live cells and Figure 8C shows electroporated cells. Figure 8D shows a histogram corresponding to the cell sorting results, showing control cells and cells to which GFP was electroporated. GFP expression was counted in live cells (propidium iodide negative, PI-). CTLs represent the control experiment.

[0143] Example 2. Transfection of EC7 cells Typical conditions for electroporation of EC7 cells are shown in Table 3 below.

[0144] [Table 3]

[0145] Figures 9A to 9D show the experimental results of the electroporation reaction in EC-7 cells. Figure 9A shows various efficiencies and viability under various experimental conditions. In particular, using an IOCO electroporation apparatus under conditions of 1 kv / cm, Rp200, and 10 uF, electroporation of GFP-DNA into EC-7 cells yielded an efficiency of over 90% and a viability of over 50%. Figures 9B to 9C show the results of cell sorting, where Figure 9B shows live cells and Figure 9C shows electroporated cells. Figure 9D is a histogram corresponding to the cell sorting results, showing control cells and cells electroporated with GFP.

[0146] Figures 10A–10E show a set of additional experimental results in EC-7 cells under various electroporation experimental conditions. Figures 10B, 10C, and 10D show the results of cell sorting, where Figure 10B shows live cells and Figures 10C and 10D show electroporated live cells. Figure 10E shows control cells and GFP-electroporated cells, along with histograms corresponding to the cell sorting results. GFP expression was counted in live cells (propidium iodide negative, PI-). CTLs represent the control experiment.

[0147] Figures 11A to 11C show images of GFP-DNA that was skillfully electroporated into EC-7 cells using the electroporation apparatus and offset chamber shown in Figures 3A to 3C.

[0148] Figure 11A shows the electroporation of GFP-DNA and GFP-AD5 (DNA encoding AD5 virus production) into EC-7 cells, obtained 20 hours after electroporation. Figure 11B shows the electroporation of GFP-DNA and DNA shuttle vector into EC-7 cells, obtained 44 hours after electroporation. Figure 11C shows a comparison of electroporation results using a micromesh versus a cuvette, obtained 6 days after electroporation.

[0149] According to embodiments presented herein, Figure 11C also shows comet formation during the early stages of AD5 virus production in EC7 cells. In this series of images, the cells form clusters, or "comet" shaped plaques, with each cell becoming more circular.

[0150] As shown in this series of images, EC-7 cells can successfully electroporate fluorescently tagged viral DNA (for AD5 virus production) and initiate viral production, demonstrating that the technologies and systems provided herein are suitable for adenovirus production (for example, for use in viral vaccines and for the production of viral proteins).

[0151] Figures 12A and 12B show further results of transfecting EC7 cells using the method and device described herein. Figure 12A shows a table of electroporation parameters when the cargo volume varied between electroporation runs. Figure 12B shows an image 6 days after electroporation. An efficiency of approximately 70–80% was observed by 14 days after electroporation. Multiple "comet" clusters were observed after transfection. Approximately 17–18 million cells / min may be transfected into EC7 cells at a cell concentration of approximately 40 m / ml in electroporation buffer cw100 with a conductance of approximately 240 mS / m.

[0152] Example 3. Electroporation of various cell types In other embodiments, the methods and systems provided herein may be used to transfect various different cell types. For example, the methods and systems provided herein may be used to transfect up to 10 8 One or more cells can be transfected. Methods and systems provided herein may be used to transfect EC7 cells, haNK cells, CHO cells, and T cells. For example, a flow rate of 0.6 ml / min and 4 × 10⁶ cells. 7To transfect EC7 cells using a cell concentration of 7 cells / ml, the methods and techniques presented herein may be used. In another example, to transfect haNK cells using a flow rate of 0.36 ml / min and a cell concentration of 3×10 7 cells / ml, the methods and techniques presented herein may be used. In yet another example, to transfect CHO cells using a flow rate of 0.45 ml / min and a cell concentration of 2.5×10 7 cells / ml, the methods and techniques provided herein may be used. In the case of T cells, to transfect T cells at a flow rate of 0.44 ml / min and a cell concentration of approximately 2×10

[0153] cells / ml, the methods and techniques presented herein may be used. These conditions may be further varied to achieve optimal conditions.

[0154] Example 4. Transfection of Primary T Cells In other embodiments, methods and systems presented herein are used to electroporate mRNA attached to poly(β-aminoester) polymers into primary T cells. Certain poly(β-aminoester) polymers have been shown to act as effective RNA transfection agents.

[0155] To deliver mRNA to T cells, nanoparticles containing RNA and poly(β-aminoester) polymers are used (see, e.g., Moffett, et al., Nature Communications (2017) 8:389). For example, in some embodiments, nanoparticles may be created using a polyglutamic acid (PGA) based shell. To target the nanoparticles to an appropriate target, a target molecule (e.g., an antibody binding domain) may be attached to the PGA molecule. For example, to target the nanoparticles to T cells, an anti-CD3 / anti-CD28 binding domain may be attached to the PGA molecule. For targeting, PGA or any other suitable negatively charged molecule may be used. Nanoparticle uptake may occur by specific targeting or association of a cation membrane.

[0156] The interior of the nanoparticles may contain mRNA and a carrier molecule such as poly(β-aminoester). Once the nanoparticles are taken up into the cell, the mRNA is released into the cell by degradation of the nanoparticles, osmotic expansion, or several other suitable processes, and the mRNA is transcribed into its respective proteins. In some cases, synthetic mRNA may be used to reduce mRNA degradation.

[0157] The methods provided herein may be used, for example, to transfect mRNA into cells using mRNA and a poly(β-aminoester) (PbAE) carrier, or using nanoparticles encapsulating mRNA and a poly(β-aminoester) carrier. Figures 14A to 14E show T cell transfection using PbAE complexed with mRNA (not free mRNA and without electroporation). Figure 13A shows a poly(β-aminoester) structure suitable for use by the techniques provided herein. Figure 14B shows the results of cell sorting by flow cytometry of T cells transfected with mRNA using PbAE. Figure 14C is a graph showing the results normalized to live cells based on various mRNA-to-carrier molecule ratios. Figures 14D and 14E show the cell viability results for GFP-mRNA transfection in stimulated T cells (Figure 14D) and unstimulated T cells (Figure 14E) under various conditions. In these experiments, a PbAE:RNA ratio of 60:1 resulted in a high level of viable cells while also achieving a high GFP delivery rate.

[0158] Figures 15A and 15B show GFP mRNA transfection of T cells using control cells (without electroporation) and electroporated cells. In particular, Figure 15A shows control cells (without electroporation), where mainly background fluorescence is observed. Figure 15B shows electroporated cells, where GFP-mRNA is detected in the majority of cells, as shown by the histogram.

[0159] Example 5. Transfection of adipose-derived mesenchymal stem cells (AD-MSCs) Adipose-derived mesenchymal stem cells (AD-MSCs) are approximately 1 x 10⁻⁶ 5Cells were detached and collected at a density of one million cells / ml. The cells were washed with PBS and electroporation buffer and suspended in electroporation buffer at a density of 3.3 million cells / ml. Under each condition, 30 μg of DNA was electroporated at a final cell density of 0.1 ml. The electroporation conditions were as follows: pulse width = 100 u, pulse duration = 340 ms, and electric field strength varied from 0.9 to 1.6 kV / cm as shown in Table 4 below.

[0160] [Table 4]

[0161] After electroporation, cells are 30 x 10 3 cells / cm 2 Seeds were seeded at the specified density. GFP expression efficiency was verified by flow cytometry 24 hours after electroporation. The results are shown in Figures 16A-16D. In each of Figures 16A-15C, the x-axis values ​​1-8 correspond to the electric field strength as shown in Table 4, and "CTL" corresponds to the control without electroporation. Figure 16A shows cell viability 24 hours after electroporation. As the electric field strength decreases, the number of viable cells increases. Figure 16B shows GFP (green fluorescent protein) expression read by flow cytometry in the measurement of cell transfection efficiency 24 hours after electroporation. As the electric field strength increased, a higher percentage of viable cells expressed GFP. Figure 16C shows the median GFP fluorescence, which indicates how bright GFP was in the flow cytometry measurement. Higher electric field strength resulted in the measurement of brighter GFP. The results suggest that electric field strengths between 1.1 and 1.3 kv / cm may show the best performance in terms of viability and efficiency. Note that this survival rate includes both adherent cells and supernatant, reflecting the true overall survival rate. Figure 16D shows photographs of the MSC transfection results. The photograph on the left shows the morphology of the MSCs, and the image on the right is a fluorescence image in the same light field, showing that the majority of the cells express green fluorescent protein (green).

[0162] Example 6. Transfection of EC-7 cells, CHO-S cells, primary CD4 / CD8 T cells, haNK cells, primary NK cells, activated NK cells, dendritic cells, and AD-MSC cells. EC-7 cells, CHO-S cells, primary CD4 / CD8 T cells, haNK cells, primary NK cells, activated NK cells, dendritic cells, and AD-MSC cells were electroporated using an apparatus similar to the one shown in Figure 2A, with the electroporation parameters shown in Table 5 below.

[0163] [Table 5]

[0164] The compositions of electroporation media ("Electro media") 1 and 2 used in Table 5 are shown in Tables 6 and 7 below.

[0165] [Table 6]

[0166] [Table 7]

[0167] Table 8 below shows the results of electroporating the cells in Table 5 according to the given parameters.

[0168] [Table 8]

[0169] [Table 9]

[0170] Figure 17 shows an example electroporation system 1700 according to another embodiment of the present disclosure. As shown in Figure 17, the system 1700 includes a mount for a pump 1701 and a tip assembly 1720 (which may be called an electroporation chamber) positioned between two I / O cartridges 1703. For example, the tip assembly 1720 may be connected to the output cartridge of one of the two I / O cartridges 1703 via a tube and one of a sterile connectors 1704. In various embodiments, a normally closed connector may be used to connect the tube. As shown in Figure 17, each I / O cartridge 1703 may be connected to a vent 1702 to allow excess air to escape from the I / O cartridge 1703.

[0171] Pump 1701 may be connected to an input cartridge of an I / O cartridge 1703 via a tube and one of a sterile connector 1704. One or more pumps 1701 may be used to generate a fluid flow through the chip assembly 1720. The chip assembly 1720 can be selectively coupled to and detached from the system 1700, thereby allowing different chip assemblies to be used in the system 1700, as will be further described later. For example, the system 1700 may include a chip holder structure for detachably mounting different chip assemblies, with optional chip sensors positioned adjacent to the chip holder structure.

[0172] System 1700 may include a scanner and user interface that can be programmed to identify or verify a chip assembly 1720 placed in System 1700 and select an action to be performed using the chip assembly 1720. In various embodiments, the chip assembly 1720 may be gamma-irradiation compatible. System 1700 may allow the use of various consumables, buffers, etc., in conjunction with the chip assembly 1720. In one embodiment, System 1700 is a closed system design with an input container and a normally closed Luer connector to the input container for easy and sterile connection and separation of cell samples. In various embodiments, when connected outside the hood, sterile connections may be provided using sterile connectors such as CPC AseptiQuick.

[0173] Figure 18 shows an exemplary apparatus for electroporating cells along with cargo, including an electroporation chamber 1820 (which may be called a tip or tip assembly). The electroporation chamber 1820 includes a first electrode 1840 and a second electrode 1842, with three fluid channel layers 1825, 1826, and 1827 located between electrodes 1840 and 1842. Each fluid channel layer 1825, 1826, and 1827 may contain a fluid channel 1835 (which may be called a patch). For example, a fluid containing cells (and optionally cargo) may flow laterally through the fluid channel 1835 through the electroporation chamber 1820, while the shape of the fluid channel 1835 defines a specific path through which the fluid travels.

[0174] As shown in Figure 18, the fluid channel layers 1825, 1826, and 1827 may align with each other so that the fluid channels 1835 of each layer 1825, 1826, and 1827 are also aligned. The electrodes 1840 and 1842 may align with each other and with the layers 1825, 1826, and 1827 so as to form a sandwich configuration in which the periphery of each electrode 1840 and 1842 and the periphery of each channel layer 1825, 1826, and 1827 are substantially the same. In various embodiments, the length and / or width of one or more channels 1825, 1826, and 1827 may be greater than the length and / or width of electrodes 1840 and 1842 so as to prevent the electrodes 1840 and 1842 from unintentionally coming into contact with each other and causing a short circuit.

[0175] The electroporation chamber 1820 may include a first support layer 1815 and a second support layer 1816. As shown in Figure 18, electrodes 1840 and 1842 and channel layers 1825, 1826, and 1827 may be located between support layers 1815 and 1816. The electroporation chamber 1820 also includes a first plate 1817 and a second plate 1818 (which may be called housing plates or cover plates). Plates 1817 and 1818 may be arranged as the outermost layers of the electroporation chamber 1820, as shown in Figure 18.

[0176] Support layers 1815 and 1816 and plates 1817 and 1818 can provide structural support to electrodes 1840 and 1842 and channel layers 1825, 1826, and 1827. Support layers 1815 and 1816 can be aligned with electrodes 1840 and 1842 and channel layers 1825, 1826, and 1827 such that the periphery of each layer has substantially the same footprint in the electroporation chamber 1820. In various embodiments, support layers 1815 and 1816 may have a longer length and / or width than electrodes 1840 and 1842, thereby preventing electrodes 1840 and 1842 from coming into contact with another conductive indicator and causing a short circuit.

[0177] Plates 1817 and 1818 may be aligned with support layers 1815 and 1816, electrodes 1840 and 1842, and channel layers 1825, 1826, and 1827 such that the perimeter of each layer has approximately the same footprint in the electroporation chamber 1820. In various embodiments, plates 1817 and 1818 may have a longer length and / or width than the other layers (for example, as shown in Figure 18) so as to prevent electrodes 1840 and 1842 or the other layers from coming into contact with the external chamber. Other embodiments of the electroporation chamber may have more or fewer layers than those shown in Figure 18. For example, more or fewer fluid channel layers, more or fewer support layers (or no support layers), etc., may be included.

[0178] As shown in Figure 18, the electroporation chamber includes an input 1810 and an output 1830. The input 1810 may allow cells and cargo to enter the electroporation chamber 1820, while the output 1830 may allow electroporated cells to exit the electroporation chamber 1820. The input 1810 and the output 1830 are separated by an offset distance, which may facilitate the lateral flow of cells through the electroporation chamber 1820, such as through the fluid channels 1835 of the channel layers 1825, 1826, and 1827.

[0179] For example, input 1810 and output 1830 may define openings in plate 1818 (which can be considered the upper plate when the electroporation chamber is received by the electroporation apparatus). As shown in Figure 18, the support layer 1816 and electrode 1842 also include openings aligned with input 1810 and output 1830. Thus, cells and cargo may flow into the electroporation chamber 1820 through input 1810, proceed to the corresponding openings in the support layer 1816 and electrode 1842, and reach the fluid channel 1835. The fluid containing cells and cargo then passes through the fluid channel 1835 as it undergoes electroporation and exits through output 1830 via the corresponding openings in electrode 1842 and support layer 1816.

[0180] Other unlabeled openings shown in Figure 18 may represent openings for connecting various components of the electroporation chamber via, for example, screws, plugs, etc. In one embodiment, plates 1817 and 1818 may include four holes for banana plugs and two screw holes for connecting Luer sockets (for example, supplying fluid to and from the electroporation chamber 1820 via tubing).

[0181] The channel layers 1825, 1826, and 1827 may contain any suitable material to enable fluid flow in the fluid channel 1835. For example, the channel layers 1825, 1826, and 1827 may contain polyethylene terephthalate (PET) material, pressure-sensitive adhesive (PSA) material, silicone gasket material, etc. In various embodiments, the central channel layer 1825 may contain PET material, while the outer channel layers 1826 and 1827 contain PSA material, and the channel layers 1825, 1826, and 1827 are bonded together.

[0182] For example, the outer channel layers 1826 and 1827 may be double-sided PSA material having a thickness of approximately 100 μm, and channel layer 1825 may be a PET film having a thickness of approximately 100 μm, thereby the entire channel structure having a thickness of approximately 300 μm and providing a seal between electrodes 1840 and 1842. In various embodiments, the height of the fluid channel 1835 (i.e., the thickness of channel layers 1825, 1826, and 1827) may be uniform over the entire length of the fluid channel 1835 (e.g., from inlet to outlet) to determine the electric field applied to the cells during electroporation. In other embodiments, any suitable fluid channel height such as approximately 100 μm, 200 μm, 400 μm, 500 μm, or 1 mm may be used.

[0183] Electrodes 1840 and 1842 may include solid electrodes such as solid plate electrodes or porous mesh electrodes such as micromesh electrodes. In one embodiment, both electrodes 1840 and 1842 are solid electrodes such as solid plate electrodes. In various embodiments, electrodes 1840 and 1842 may include arrays of solid metal plates, similar to the plate shown in Figure 2C. Each solid metal plate may be configured to apply an independent electric field to create a specific electric field pattern.

[0184] Suitable materials for forming electrodes 1840 and 1842 include, but are not limited to, stainless steel, silicon, precious metals, Group 4 metals, conductive materials, and combinations thereof. Suitable precious metals include, but are not limited to, ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), osmium (Os), iridium (Ir), platinum (Pt), and gold (Au). Examples of Group 4 metals include titanium (Ti), zirconium (Zr), hafnium (Hf), and rutherfogium (Rf). Suitable conductive materials include, but are not limited to, indium tin oxide (ITO), carbon nanotubes (CNT), and conductive polymers such as poly(3,4-ethylenedioxythiophene) polystyrene sulfonic acid (PEDOT:PSS). In one embodiment, electrodes 1840 and 1842 are each made from 316 stainless steel shim stock having a thickness of approximately 0.004 inches (approximately 0.1 mm).

[0185] The support layers 1815 and 1816 and plates 1817 and 1818 may include any suitable material that provides structural support to electrodes 1840 and 1842 containing a fluid containing cells in fluid channel layers 1825, 1826, and 1827. Examples include, but are not limited to, non-porous materials such as polydimethylsiloxane (PDMS) acrylic, polyethylene, polypropylene, metal plates, pressure-sensitive adhesives (PSA), PET materials, acrylic glass, and injection-molded plastics. In one embodiment, each of the support layers 1815 and 1816 includes a double-sided PSA material to bond plates 1817 and 1818 to electrodes 1840 and 1842, and each plate 1817 and 1818 is a 1 / 4 inch (approximately 6.35 mm) thick acrylic sheet.

[0186] Figure 19 shows the electroporation chamber 1820 (or chip assembly) in an assembled format with layers compressed together. As shown in Figure 19, plates 1817 and 1818 form the outer surface of the assembly, with the other layers sandwiched between plates 1817 and 1818. Plate 1818 includes an input 1810 for supplying a fluid containing cells to the electroporation chamber 1820 (e.g., via a tube or another suitable fluid delivery system) and an outlet 1830 for supplying electroporated cells from the electroporation chamber 1820 (e.g., via a tube or another suitable fluid delivery system).

[0187] The chip assembly shown in Figure 19 can be used in an electroporation apparatus such as the system 1700 in Figure 17. For example, multiple chip assemblies can be selectively inserted into the system to electroporate different types of cells. After each electroporation process, or when a new cell type is desired for electroporation, the chip assembly in Figure 19 can be removed from the electroporation system and replaced with another chip assembly.

[0188] As will be further described later, different chip assemblies may have fluid channels 1835 of different volumes, fluid channels 1835 of different shapes, etc. Various chip assemblies may have the same size or footprint, thereby allowing chip assemblies with different volumes or fluid channels to be interchangeable in the electroporation system. In one embodiment, chip assemblies having fluid channels of different volumes, such as a large-volume chip assembly (e.g., fluid channel 1835 volume of approximately 500 microliters), an intermediate-volume chip assembly (e.g., fluid channel 1835 volume of approximately 250 microliters), and a small-volume chip assembly (e.g., fluid channel 1835 volume of approximately 50 microliters), can be selectively inserted into the electroporation system. In other embodiments, designs of more or fewer chip assemblies can be used in conjunction with the electroporation system, and each chip assembly may have fluid channels of larger or smaller volumes than those provided above.

[0189] Figure 20 shows an example of a channel layer 2025 that may be part of an electroporation chamber, such as the electroporation chamber 1820 in Figure 18. The channel layer 2025 includes a fluid channel 2035 having multiple curved sections and multiple straight path sections (which may be called S-shaped paths). Figure 20 shows a fluid channel 2035 having eight curved sections and nine straight sections (having seven full lengths and two half lengths at each end of the fluid channel 2035). The fluid channel 2035 can be considered as four full cycles of a single S-shaped curve pattern.

[0190] Once the channel layer 2025 is received by the electroporation system, the fluid containing cells is supplied to the two inlet ends of the fluid channel 2035, travels through the S-curve of the fluid channel 2035 to undergo electroporation, and can exit through the other outlet end of the fluid channel. The volume of the fluid channel 2035 can determine the volumetric velocity of the fluid in the channel and can be synchronized with the electrical pulses of the electroporation process. In some embodiments, the fluid channel 2035 may have a volume of approximately 500 microliters (for example, it may be possible to process more than 200 million cells within a given time period). In other embodiments, the fluid channel 2035 may include larger or smaller fluid volumes (e.g., about 25 microliters, 50 microliters, 100 microliters, 200 microliters, 250 microliters, 300 microliters, 400 microliters, 750 microliters, 1 milliliter, etc.), more or fewer curves, thicker or narrower channel widths, longer or shorter channel lengths, and other preferred path shapes.

[0191] Figure 21 shows an example of a channel layer 2125 that may be part of an electroporation chamber, such as the electroporation chamber 1820 in Figure 18. The channel layer 2125 includes a fluid channel 2135 having multiple curved sections and multiple straight path sections (which may be called S-shaped paths). Similar to the fluid channel 2035 in Figure 20, Figure 21 shows a fluid channel 2135 having eight curved sections and nine straight sections (having seven full lengths and two half lengths at each end of the fluid channel 2135).

[0192] Compared to the fluid channel 2035 in Figure 20, the fluid channel 2135 in Figure 21 has a shorter length than the curved section. Therefore, the fluid channel 2135 may have a smaller fluid volume than the fluid channel 2135 in Figure 20. For example, the fluid channel 2135 may have a fluid volume of approximately 250 microliters (for example, it may be possible to process about 500 million cells within a specified time period). In other embodiments, the fluid channel 2135 may include a larger or smaller fluid volume, more or fewer curved sections, a thicker or narrower channel width, a longer or shorter channel length, and other preferred path shapes.

[0193] Figure 22 shows an example of a channel layer 2225 that may be part of an electroporation chamber, such as the electroporation chamber 1820 in Figure 18. The channel layer 2225 includes a fluid channel 2235 having one straight section (in contrast to the S-shaped curves of the fluid channels 2035 and 2135 in Figures 20 and 21, which have multiple curved sections). Thus, the fluid channel 2235 in Figure 22 may have a smaller fluid volume than the fluid channels 2035 and 2135 in Figures 20 and 21.

[0194] Once the channel layer 2225 is received by the electroporation system, the fluid containing cells is supplied to one end of the fluid channel 2235, travels through the straight line of the fluid channel 2235 to undergo electroporation, and can exit through the other end of the fluid channel. In some embodiments, the fluid channel 2235 may have a volume of approximately 50 microliters (for example, it may be possible to process about 500 million cells within a specified time period). In other embodiments, the fluid channel 2235 may include a larger or smaller fluid volume, more or fewer curves, a thicker or narrower channel width, a longer or shorter channel length, and other preferred path shapes.

[0195] Figure 23 shows a channel layer 2325 of another example including a fluid channel 2335. Dimensional examples are provided in Figure 23 for illustrative purposes only. For example, the fluid channel 2335 has a width of 3 millimeters. While the channel width may vary across different chip assemblies having different volumes, within one fluid channel 2335 of one channel layer 2325, the channel width may remain constant from the channel inlet to the outlet. This may facilitate the maintenance of a constant cell flow velocity within the fluid channel 2335. Based on the dimensions of the straight and curved sections of the fluid channel 2335 shown in Figure 23, the fluid channel 2335 may have a total fluid volume of approximately 500 microliters. In other embodiments, the channel layer 2325 and the fluid channel 2335 may have any other suitable dimensions. For example, in various embodiments, the width of the fluid channel 2335 may be approximately 1 millimeter, 2 millimeters, 4 millimeters, 5 millimeters, 10 millimeters, etc.

[0196] In some embodiments, numbers representing properties such as the amount, concentration, and reaction conditions of components used to describe and claim a particular embodiment should be understood as being modified with the term “approximately” in some examples. Therefore, in some embodiments, the numerical parameters shown herein and in the appended claims are approximations that may vary depending on the desired properties sought to be obtained by the particular embodiment. In some embodiments, the numerical parameters must be interpreted in light of the number of significant figures reported and by applying common rounding techniques. Despite the approximations of numerical ranges and parameters representing a broad range in some embodiments, the numbers shown in specific examples are reported to a pragmatically accurate extent. The numbers presented in some embodiments may necessarily contain certain errors resulting from the standard deviation found in their respective test measurements.

[0197] As used in this specification and throughout the claims described herein, the meanings of “a,” “an,” and “the” include multiple references unless the context explicitly states otherwise. Furthermore, as used in this specification, the meaning of “in” includes “in” and “on” unless the context explicitly states otherwise. Unless the context explicitly states otherwise, all scopes shown herein should be interpreted as encompassing their endpoints, and unrestricted scopes should be interpreted as encompassing commercially practical values. Similarly, all value lists should be considered as encompassing intermediate values ​​unless the context explicitly states otherwise.

[0198] It should also be obvious to those skilled in the art that many further modifications beyond those already described herein are possible, provided they do not deviate from the inventive concept herein. Furthermore, when interpreting both this specification and the claims, all terms should be interpreted in the broadest possible form that fits the context. In particular, the terms “comprises” and “comprising” should be interpreted in a non-exclusive manner to refer to elements, components, or steps, indicating that the referenced element, component, or step may exist with, be used with, or be combined with other elements, components, or steps that are not explicitly referenced. Where the claims herein refer to at least one selected from the group consisting of A, B, C... and N, the text should be interpreted as requiring only one element from that group, and not A+N, B+N, etc. [Brief explanation of the drawing]

[0199] [Figure 1A] A typical electroporation apparatus is shown, including parallel plate electrodes (as shown in Figure 1A) and micromesh electrodes with no offset between input and output (as shown in Figure 1B). [Figure 1B]A typical electroporation apparatus is shown, including parallel plate electrodes (as shown in Figure 1A) and micromesh electrodes with no offset between input and output (as shown in Figure 1B). [Figure 2A] This specification shows embodiments of an electroporation apparatus according to several embodiments provided herein. [Figure 2B] This specification shows embodiments of an electroporation apparatus according to several embodiments provided herein. [Figure 2C] This specification shows embodiments of the first and second electrodes according to several embodiments provided herein. [Figure 2D] This is a block diagram of an apparatus for generating and delivering electrical pulses to cells in an electroporation apparatus as described herein, according to some embodiments provided herein. [Figure 2E] This is a block diagram of a further apparatus for generating and delivering electrical pulses to cells in an electroporation apparatus as described herein, according to some embodiments provided herein. [Figure 3A] This specification describes embodiments of an electroporation apparatus, including an electroporation device, according to several embodiments provided herein. Figure 3A shows a diagram of the structure of an input / output chamber offset electroporation apparatus. [Figure 3B] This specification describes embodiments of an electroporation apparatus, including an electroporation device, according to several embodiments provided herein. Figure 3B shows a diagram of the electric field of an input / output chamber offset electroporation apparatus. [Figure 3C] This specification describes embodiments of an electroporation apparatus, including an electroporation device, according to several embodiments provided herein. Figure 3C shows the electric field strength as a function of cell migration distance through an input / output chamber offset electroporation apparatus. [Figure 3D]This specification shows embodiments of an electroporation apparatus, including an electroporation device, according to some embodiments provided herein. Figure 3D shows an alternative arrangement of an electroporation device according to some embodiments provided herein. [Figure 3E] This specification shows configurations of pathways within an electroporation chamber according to several embodiments provided herein. [Figure 3F] This specification shows configurations of pathways within an electroporation chamber according to several embodiments provided herein. [Figure 3G] This specification shows configurations of pathways within an electroporation chamber according to several embodiments provided herein. [Figure 3H] The following are alternative arrangements of electroporation apparatus according to some embodiments provided herein. [Figure 3I] The following are alternative arrangements of electroporation apparatus according to some embodiments provided herein. [Figure 3J] The following are alternative arrangements of electroporation apparatus according to some embodiments provided herein. [Figure 3K] An example of a microfluidic chamber for sorting cells, according to some embodiments provided herein, is illustrated. [Figure 3L] An example of a microfluidic chamber for sorting cells, according to some embodiments provided herein, is illustrated. [Figure 3M] These are drawings illustrating various aspects of the geometric shape and positioning of posts within a microfluidic chamber according to some embodiments provided herein. [Figure 3N] These are drawings illustrating various aspects of the geometric shape and positioning of posts within a microfluidic chamber according to some embodiments provided herein. [Figure 3O] These are drawings illustrating various aspects of the geometric shape and positioning of posts within a microfluidic chamber according to some embodiments provided herein. [Figure 3P] These are drawings illustrating various aspects of the geometric shape and positioning of posts within a microfluidic chamber according to some embodiments provided herein. [Figure 3Q] These are drawings illustrating various aspects of the geometric shape and positioning of posts within a microfluidic chamber according to some embodiments provided herein. [Figure 3R] These are drawings illustrating various aspects of the geometric shape and positioning of posts within a microfluidic chamber according to some embodiments provided herein. [Figure 3S] These are drawings illustrating various aspects of the geometric shape and positioning of posts within a microfluidic chamber according to some embodiments provided herein. [Figure 3T] An example of a microfluidic chamber for modifying a buffer solution, according to some embodiments provided herein, is illustrated. [Figure 3U] An example of a microfluidic chamber having two separate chambers, such as one chamber for cell sorting and another chamber for buffer change, according to some embodiments provided herein, is illustrated. [Figure 3V] The following are alternative arrangements of electroporation apparatus according to some embodiments provided herein. [Figure 3W] The following are alternative arrangements of electroporation apparatus according to some embodiments provided herein. [Figure 3X] This specification shows a modular arrangement of an electroporation system including an electroporation apparatus according to some embodiments provided herein. [Figure 4] A table of parameters related to a chamber offset electroporation apparatus according to some embodiments provided herein is shown. [Figure 5] Examples of fluid flow waveforms (pumps) and electrical waveforms (stimuli) that may be applied to cells passing through a chamber offset electroporation apparatus according to some embodiments provided herein are shown. [Figure 6] This specification presents various examples of micromeshes formed from different materials suitable for use in chamber offset electroporation apparatus, according to several embodiments provided herein. [Figure 7A] The results of electroporation experiments using the methods and devices described herein with haNK cells and corresponding parameters are shown. [Figure 7B] The results of electroporation experiments using the methods and devices described herein with haNK cells and corresponding parameters are shown. [Figure 7C] The results of electroporation experiments using the methods and devices described herein with haNK cells and corresponding parameters are shown. [Figure 7D] The results of electroporation experiments using the methods and devices described herein with haNK cells and corresponding parameters are shown. [Figure 7E] The results of electroporation experiments using the methods and devices described herein with haNK cells and corresponding parameters are shown. [Figure 8A] Further results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein are presented. [Figure 8B] Further results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein are presented. [Figure 8C] Further results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein are presented. [Figure 8D] Further results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein are presented. [Figure 9A]The results of electroporation experiments using the methods and devices described herein with EC7 cells and corresponding parameters are shown. [Figure 9B] The results of electroporation experiments using the methods and devices described herein with EC7 cells and corresponding parameters are shown. [Figure 9C] The results of electroporation experiments using the methods and devices described herein with EC7 cells and corresponding parameters are shown. [Figure 9D] The results of electroporation experiments using the methods and devices described herein with EC7 cells and corresponding parameters are shown. [Figure 10A] Further results of electroporation experiments using the methods and devices described herein with EC7 cells and corresponding parameters are presented. [Figure 10B] Further results of electroporation experiments using the methods and devices described herein with EC7 cells and corresponding parameters are presented. [Figure 10C] Further results of electroporation experiments using the methods and devices described herein with EC7 cells and corresponding parameters are presented. [Figure 10D] Further results of electroporation experiments using the methods and devices described herein with EC7 cells and corresponding parameters are presented. [Figure 10E] Further results of electroporation experiments using the methods and devices described herein with EC7 cells and corresponding parameters are presented. [Figure 11A] Microscopic images of electroporated EC7 cells are shown. [Figure 11B] Microscopic images of electroporated EC7 cells are shown. [Figure 11C] Microscopic images of electroporated EC7 cells are shown. [Figure 12A]Shows further results of transfected EC7 cells using the methods and devices described herein. [Figure 12B] Shows further results of transfected EC7 cells using the methods and devices described herein. [Figure 13A] Shows various transfection efficiencies in different cell lines using the methods and devices described herein. [Figure 13B] Shows various transfection efficiencies in different cell lines using the methods and devices described herein. [Figure 13C] Shows various transfection efficiencies in different cell lines using the methods and devices described herein. [Figure 13D] Shows various transfection efficiencies in different cell lines using the methods and devices described herein. [Figure 14A] Shows the results of transfection experiments for introducing mRNA into T cells using PbAE as described herein. [Figure 14B] Shows the results of transfection experiments for introducing mRNA into T cells using PbAE as described herein. [Figure 14C] Shows the results of transfection experiments for introducing mRNA into T cells using PbAE as described herein. [Figure 14D] Shows the results of transfection experiments for introducing mRNA into T cells using PbAE as described herein. [Figure 14E] Shows the results of transfection experiments for introducing mRNA into T cells using PbAE as described herein. [Figure 15A] Shows the results of transfection experiments for introducing mRNA into T cells in the presence and absence of electroporation as described herein. [Figure 15B]As described herein, the results of transfection experiments for introducing mRNA into T cells in the presence and absence of electroporation are shown. [Figure 16A] The results of transfection experiments using the methods and devices described herein with adipose-derived mesenchymal stem cells (AD-MSCs) and corresponding parameters are shown. [Figure 16B] The results of transfection experiments using the methods and devices described herein with adipose-derived mesenchymal stem cells (AD-MSCs) and corresponding parameters are shown. [Figure 16C] The results of transfection experiments using the methods and devices described herein with adipose-derived mesenchymal stem cells (AD-MSCs) and corresponding parameters are shown. [Figure 16D] The results of transfection experiments using the methods and devices described herein with adipose-derived mesenchymal stem cells (AD-MSCs) and corresponding parameters are shown. [Figure 17] This is a block diagram of an electroporation apparatus according to another embodiment of the present disclosure. [Figure 18] Figure 17 shows an exploded view of an electroporation channel that may be used in the electroporation apparatus according to another embodiment of the present disclosure. [Figure 19] Figure 18 is an isometric view of the electroporation chamber with the layers assembled together. [Figure 20] Figure 18 is a front view of the fluid channel layer of the electroporation chamber. [Figure 21] This is a front view of a fluid channel layer having a different fluid volume from the fluid channel layer of Figure 20, according to another embodiment of the present disclosure. [Figure 22] This is a front view of a fluid channel layer having a linear fluid channel according to another embodiment of the present disclosure. [Figure 23] Figure 20 is a front view of the fluid channel layer, showing an example of the dimensions of the fluid channel.

Claims

1. A chip assembly that electroporates cells together with cargo, First electrode and The second electrode and At least one fluid channel layer located between the first electrode and the second electrode, defining a fluid channel that allows the flow of fluid including the cells and the cargo during the electroporation process, and comprising at least one of a pressure-sensitive adhesive or polyethylene terephthalate (PET) material, First enclosure plate and Second enclosure plate, A first support layer located between the first housing plate and the first electrode, A second support layer located between the second electrode and the second housing plate, A chip assembly equipped with the following features.

2. The chip assembly according to claim 1, wherein the height of the fluid channel is uniform from the inlet to the outlet of the fluid channel, and a uniform electric field is applied to the fluid in the fluid channel during the electroporation process.

3. The chip assembly according to claim 2, wherein the height of the fluid channel is 300 micrometers.

4. The chip assembly according to claim 1, wherein the width of the fluid channel is uniform from the inlet to the outlet of the fluid channel, thereby promoting a constant velocity of cell flow through the fluid channel.

5. The chip assembly according to claim 4, wherein the width of the fluid channel is 3 millimeters.

6. The chip assembly according to claim 1, wherein the fluid channel has an S-shaped curve configuration between the inlet and outlet of the fluid channel.

7. The chip assembly according to claim 6, wherein the S-shaped curve configuration includes eight curved portions.

8. The chip assembly according to claim 1, wherein the fluid channel has a volume of 500 microliters.

9. The chip assembly according to claim 1, wherein the fluid channel has a volume of 250 microliters.

10. The chip assembly according to claim 1, wherein the fluid channel is a straight path from the inlet to the outlet of the fluid channel.

11. The chip assembly according to claim 1, wherein the fluid channel has a volume of 50 microliters.

12. The chip assembly according to claim 1, wherein the first electrode and the second electrode each include stainless steel.

13. The chip assembly according to claim 1, wherein the first support layer and the second support layer each contain a pressure-sensitive adhesive (PSA) material.

14. The chip assembly according to claim 1, wherein the first housing plate and the second housing plate each include an acrylic material.

15. The chip assembly according to claim 1, wherein the input and output openings of the first housing plate are configured to receive Luer connectors for connecting pipes and facilitating the flow of fluid to and from the fluid channels.

16. The at least one fluid channel layer, The first fluid channel layer, A second fluid channel layer located between the first fluid channel layer and the first electrode, wherein the second fluid channel layer defines a fluid channel having the same shape as the fluid channel of the first fluid channel layer, A third fluid channel layer located between the first fluid channel layer and the second electrode, wherein the second fluid channel layer defines a fluid channel having the same shape as the fluid channel of the first fluid channel layer, The chip assembly according to claim 1, comprising:

17. The chip assembly according to claim 16, wherein the fluid channels of the first fluid channel layer, the second fluid channel layer, and the third fluid channel layer are aligned with one another.

18. The chip assembly according to claim 16, wherein the second fluid channel layer and the third fluid channel layer each contain a pressure-sensitive adhesive.

19. The chip assembly according to claim 18, wherein the first fluid channel layer comprises polyethylene terephthalate (PET) material.

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