MICROFLOW ELECTROPORATION DEVICES AND METHODS FOR CELL TRANSFECTION

Abstract

Systems and methods for transfecting cells, such as mammalian and non-mammalian cells, using an electroporation device with an electroporation chamber comprising an upper micromesh electrode, a lower micromesh electrode, and a pathway defined within the electroporation chamber are provided. The electroporation device includes a first inlet that allows cells to pass into the electroporation chamber and a first outlet that allows electroporated cells to pass out of the electroporation chamber. The first inlet and the first outlet are spaced apart by an offset distance.

Description

Cross-Reference to Related ApplicationsThe present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Serial No. 62 / 695,436, filed July 9, 2018, the entire contents of which are hereby incorporated by reference.Field of the InventionThe field of the invention relates to transfection systems and methods, and more particularly to electroporation systems and methods.BACKGROUND OF THE INVENTIONThe background description includes information that may be useful for understanding the methods and techniques presented herein. It is not an admission that the information presented here is the prior art or is relevant to the subject matter presented here, or that a publication mentioned specifically or implicitly is the prior art.By means of transfection, nucleic acids can be introduced into cells to form 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).In electroporation, a cell is exposed to a controlled direct current (DC) pulse of relatively short duration. It is believed that the electrical impulse induces a transmembrane potential that results in a reversible breakdown of the ordered structure of a cell membrane, resulting in the formation of pores in the membrane. Molecules of interest may then enter the cell through the pores until the pores close, typically within milliseconds to seconds. The formation of pores can be controlled by adjusting various parameters, in particular the gap width (e.g. the distance between parallel electrode plates), the waveform of the electric pulse, the electric field strength, the temperature and the length of the electric pulse.Electroporation is usually used on a laboratory scale (e.g., using small cuvettes of about 0.5 ml capacity), but such laboratory procedures are not suitable for large scale clinical quality production due to lack of efficiency and high cost. Other transfection methods, such as lipid-based methods with lipofectamine 2000™ are also frequently used, but have similar disadvantages due to high costs. Moreover, lipid-based methods are not well suited for some cell types of clinical interest, e.g., NK cells such as haNK cells. NK cells were transfected with mRNA and electroporation, for example, in order to genetically manipulate primary NK cells in such a way that they express chimeric antigen receptors (CARs-see Leuk Res. (2009) 33:1255-9) or express cytokines for autocrine growth stimulation (Cytotherapy (2008) 10:265-74). There are commercial products such as Maxyte for large scale transfection, e.g., reaction rates greater than 1 ml, however, these products are also expensive and / or do not have high throughput capability. Similarly, these commercial products are not optimized for transfection of NK cells, often resulting in poor yields and / or problems with low cell viability. Typical electroporation rates can provide 2-5% DNA transfection efficiency with about 10-20% cell viability.In conventional techniques, as shown in Fig. 1A, electroporation is performed with two parallel plate electrodes. In this configuration, the cells typically flow between the two parallel plates. However, the electric field is non-uniform, with a more uniform electric field acting on cells in the middle of the chamber, shown as (a), while a non-uniform electric field acting on cells in the vicinity of an electrode, shown as (b), and in particular a peak of the electric field present at the boundary of the electrode acts. Other techniques employ parallel plates of micromesh electrodes 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 the cells flow between an upper micromesh electrode and a lower micromesh electrode. This technique provides a more uniform electric field than the passage between two parallel electrode plates, as shown by the similar cell lines in the middle of chamber (a) and near the rim of chamber (b), but the chamber width or distance between the upper micromesh electrode and the lower micromesh electrode is limited, resulting in low transfection efficiency. In the configurations shown in FIGS. 1A and 1B, an electric field strength of about 1-1.3 kV / cm (e.g., 200 V / 2 mm for parallel plates, 40 V / 400 μm for parallel micromesh electrodes) is used.In other aspects, single cell electroporation chips were used for transfection. In this technique, a silicon chip may comprise a plurality of linear channels, with opposing electrodes placed in each channel on opposite sides of the channel. The cells flow through the channel and are subjected to an electric field. In this type of technique, DNA transfection efficiencies of about 68% were achieved with cell viabilitys of about 79%.However, while there are various transfection systems and methods for mammalian cells, such techniques have one or more disadvantages. Accordingly, there is still a need to provide improved electroporation systems and methods, particularly for large-scale, high-throughput manufacturing methods.SummaryThe methods presented herein relate to various apparatus, systems and methods for electroporation of cells, such as mammalian cells and non-mammalian cells, for large scale continuous manufacturing processes. In particular, systems and methods for electroporation of cells using an electroporation device having a first inlet spaced by an offset distance from a first outlet can provide high efficiency and viability.In some aspects, a method of electroporation of cells with a charge is provided. In the method, the cells with the charge flow into an electroporation chamber. The electroporation chamber comprises an upper micromesh electrode, a lower micromesh electrode, and a path defined between the upper micromesh electrode and the lower micromesh electrode, on which the cells and the charge can flow. The upper micromesh electrode and the lower micromesh electrode each have a porosity. In addition, the upper micromesh electrode is bounded by a first material and the lower micromesh electrode is bounded by a second material. The first material includes a first inlet that allows passage of cells into the electroporation chamber and the second material includes a first outlet that allows passage of electroporated cells from the electroporation chamber. The first inlet and the first outlet are spaced by an offset distance. Furthermore, the cells are suspended in an electroporation medium.In another aspect, an apparatus for electroporating cells with a charge is provided. The device comprises one or more electroporation chambers. Each electroporation chamber includes an upper micromesh electrode, a lower micromesh electrode, and a path defined between the upper micromesh electrode and the lower micromesh electrode, along which the cells and charge can flow. The upper micromesh electrode and the lower micromesh electrode each have a porosity. In addition, the upper micromesh electrode is bounded by a first material and the lower micromesh electrode is bounded by a second material. The first material includes a first inlet that allows passage of cells into the electroporation chamber and the second material includes a first outlet that allows passage of electroporated cells from the electroporation chamber. The first inlet and the first outlet are spaced by an offset distance.In another aspect, a kit for cell transfection is provided. The kit comprises the apparatus for electroporation as described herein and optionally reagents selected from the group consisting of cells for transfection, an electroporation medium, or a combination thereof.In some aspects, an electroporation protocol involves subjecting cells to multiple electrical pulses, a staged fluid flow, an electroporation buffer having low conductivity and low osmolarity, relatively moderate capacity, and / or a relatively moderate time constant.Various objects, features, aspects, and advantages of the subject matter described herein will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings, in which like numerals designate like components.Brief Description of the DrawingsThe patent or the application files contain at least one drawing that is executed in color. Copies of this patent or patent application publication with colored drawing / drawings are provided by the office upon request and payment of the charge incurred.In Figures 1A and 1B, conventional electroporation devices with parallel plate electrodes (as shown in Figure 1A) and micromesh electrodes without offset between the inlet and the outlet (as shown in Figure 1B) are shown.Figures 2A-2D show aspects of an apparatus for electroporation comprising an electroporation apparatus according to some of the embodiments provided herein. In Figure 2A, an illustration of the structure of the inlet-outlet chamber offset electroporation apparatus is shown. In Figure 2B, an illustration of the electric field of the inlet-outlet chamber offset electroporation apparatus is shown. In Figure 2C, the electric field strength is shown as a function of the distance of travel of the cells through the inlet-outlet chamber offset electroporation apparatus. Referring now to FIG. 2D, an alternative configuration of an electroporation device according to some of the embodiments provided herein is shown.Figures 2E-2G show aspects of a path in an electroporation chamber according to some of the embodiments provided herein.Figures 2H-2J show alternative configurations of an electroporation device according to some of the embodiments provided herein.In FIGS. 2K-2L, exemplary microfluidic compartments for sorting cells according to some of the embodiments provided herein are shown. FIGS. 2M-2S are illustrations showing various aspects of the geometry and positioning of posts within the chamber according to some of the embodiments provided herein.Referring now to FIG. 2T, an example microfluidic chamber for buffer exchange is shown, in accordance with some of the embodiments provided herein.Referring now to FIG. 2U, an exemplary microfluidic device is shown having two separate chambers - one chamber for cell sorting and another chamber for buffer exchange - in accordance with some of the embodiments provided herein.Figures 2V and 2W show alternative configurations of an electroporation device according to some of the embodiments provided herein.Referring now to FIG. 3, a table of parameters associated with the chamber offset electroporation apparatus according to some of the embodiments provided herein is shown.In FIG. 4, examples of fluid flow waveforms (pump) and electrical waveforms (stim) to which cells flowing through the chamber offset electroporation apparatus may be exposed are shown, in accordance with some of the embodiments provided herein.Referring now to FIG. 5, various examples of micromesh formed from various materials suitable for use with the chamber offset electroporation apparatus are shown in accordance with some of the embodiments provided herein.Figures 6A-6E show results of electroporation experiments with haNK cells and corresponding parameters using the methods and apparatus described herein.Figures 7A-7D show further results of electroporation experiments with haNK cells and corresponding parameters using the methods and apparatus described herein.Figures 8A-8D show results of electroporation experiments with EC7 cells and corresponding parameters using the methods and apparatus described herein.Figures 9A-9E show further results of electroporation experiments with EC7 cells and corresponding parameters using the methods and apparatus described herein.In Figures 10A-10C, microscopic images of electroporated EC7 cells are shown.Figures 11A-11B show further results of transfection of EC7 cells using the methods and apparatus described herein.Figures 12A-12D show different transfection efficiencies for different cell lines using the methods and apparatus described herein.Figures 13A-13E show results of transfection experiments for introducing mRNA into T cells using PbAE as described herein.Figures 14A and 14B show results of transfection experiments for introducing mRNA into T cells with and without electroporation as described herein.Figures 15A-15D show results of transfection experiments with adipose-derived mesenchymal stem cells (AD-MSC) and corresponding parameters using the methods and apparatus described herein.DETAILED DESCRIPTIONProvided are systems and methods for electroporation of cells, for example mammalian cells (e.g., NK cells, EC-7 cells, T cells, etc.) and non-mammalian cells, using an electroporation device having a first inlet spaced by an offset distance from a first outlet. In particular, systems and methods are provided for electroporation of mammalian cells (e.g., NK cells, EC-7 cells, T cells, etc.) and non-mammalian cells, along with corresponding electroporation protocols that provide high efficiency and viability. In some aspects, the electroporation protocol subjects the cells to multiple electrical pulses, a staged fluid flow, an electroporation buffer with low conductivity and low osmolarity, relatively moderate capacity, and / or a relatively moderate time constant.According to various aspects, an apparatus for electroporating mammalian cells with a charge is provided. The device may comprise one or more electroporation chambers, each electroporation chamber comprising an upper micromesh electrode, a lower micromesh electrode, and a path defined between the upper micromesh electrode and the lower micromesh electrode, along which the cells and the charge may flow. The upper micromesh electrode and the lower micromesh electrode each have a porosity, for example to allow passage of cells through the micromesh electrodes either into or out of the electroporation chamber. For example, an upper micromesh electrode and a lower micromesh electrode may each have an open area porosity of about 30% to about 50%. A width of a pore opening of an upper micromesh electrode and a lower micromesh electrode may be about 70 μm to about 140 μm, respectively. The upper micromesh electrode may be bounded by a first material and the lower micromesh electrode may be bounded by a second material. The first material includes a first inlet that allows passage of cells into the electroporation chamber and the second material includes a first outlet that allows passage of electroporated cells from the electroporation chamber. As used herein, "electroporated cells" include transfected cells, dead cells, and untransfected cells. The first inlet and the first outlet are spaced by an offset distance. Additionally or alternatively, rather than the cells and / or charge flowing through the pores of the micromesh upper electrode to enter the electroporation chamber, the micromesh upper electrode may optionally include a second inlet that allows passage of cells and / or charge into the electroporation chamber. The second inlet may be substantially aligned with the first inlet in the first material. Additionally or alternatively, rather than the electroporated cells flowing through the pores of the bottom micromesh electrode to exit the electroporation chamber, the bottom micromesh electrode may include a second outlet that allows passage of electroporation from the electroporation chamber. The second outlet may be substantially aligned with the first outlet in the second material.For example, FIG. 2A illustrates an exemplary apparatus for electroporating cells with charge that includes an IOCO electroporation device 200. The IOCO electroporation apparatus 200 includes two micromesh electrodes, an upper micromesh electrode 240( 1) and a lower micromesh electrode 240( 2), shown aligned with each other here. The top surface of the upper micromesh electrode 240( 1) may be bounded by a first material, and the bottom surface of the lower micromesh electrode 240( 2) may be bounded by a second material. The first material and the second material may be, for example, a nonporous material such as polydimethylsiloxane (PDMS), acrylic, polyethylene, polypropylene, thereby holding the cells in the interior chamber of the electroporation device, and particularly in the chamber 220 between the two parallel micromesh electrodes. A first inlet 210 (a region without PDMS) is formed on the upper micromesh electrode 240( 1), and a first outlet 230 (a region without PDMS) is formed on the lower micromesh electrode 240( 2). Thus, in this example, the upper and lower micromesh electrodes 240( 1) and 240( 2) span the length of the electroporation chamber 220 bounded by a first material (an upper PDMS layer) and a second material (a lower PDMS layer), thereby directing the flow of cells along a lateral path of the chamber. An opening (first inlet 210) in the first material (upper PDMS layer) and an opening (first outlet 230) in the second material (lower PDMS layer) are spaced apart by an offset distance (d), which allows lateral flow of cells through the electroporation chamber 220.According to any embodiment, an electroporation chamber may have any desired shape, including, but not limited to, circular, oval, square, rectangular, or polygonal.According to any embodiment, the width (w) of an electroporation chamber may be in the range of about 0.01 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.1 mm to about 5 mm, about 1 mm to about 5 mm, about 2 mm to about 5 mm, about 0.01 mm to about 4 mm, about 0.1 mm to about 4 mm, about 1 mm to about 4 mm, about 0.01 mm to about 2 mm, about 0.05 mm to about 1 mm, about 0.1 mm to about 0.5 mm, or about 0.2 mm to about 0.4 mm. In some aspects, the width (w) of the chamber is about 0.3 mm.In some aspects, an offset distance (d) may be in the range of about 0.1 cm to about 10 cm, from about 0.1 cm to about 5 cm, from about 0.1 cm to about 4 cm, from about 1 cm to about 3 cm. In some aspects, the offset distance (d) is about 2 cm. In some aspects, there is no overlap between the inlet and the outlet.The first inlet and / or the second inlet are in fluid communication with the electroporation chamber and the electroporation chamber is in fluid communication with the first outlet and / or the second outlet. Inlet / inlets, outlet / outlets and electroporation chamber may further comprise one or more valves, regulators, pumps or any other microfluidic components for controlling the flow of cells through the electroporation chamber.In some aspects, both the first inlet and the first outlet in the first material may be positioned at the upper micromesh electrode, provided that an appropriate offset distance is maintained between the first inlet and the first outlet. Additionally or alternatively, both the second inlet and the second outlet may be in the upper micromesh electrode. In other aspects, both the first inlet and the first outlet may be positioned in the second material on the bottom micromesh electrode, provided that an appropriate offset distance is maintained between the first inlet and the first outlet. Additionally or alternatively, both the second inlet and the second outlet may be in the bottom micromesh electrode.According to some embodiments, the cells may enter through the first inlet, flowing in a first direction. After passing through the upper micromesh electrode, the flow of cells may change direction and flow laterally in a path along the length of the electroporation chamber in a direction perpendicular or substantially perpendicular to the first direction. Upon reaching the first outlet, the flow of cells may again change direction and exit the electroporation chamber in a second direction parallel to the first direction.In some aspects, a voltage is applied across the parallel micromesh electrodes that is capable of producing an electric field in the range of about 0.3 kV / cm to about 3 kV / cm. Typical electric fields range from about 1-1.3 kV / cm (e.g., 40 V / 300 μm) for haNK cells and about 0.3-1 kV / cm for EC-7 cells. In some aspects, 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 suitable range therein, or above.In some aspects, the diameters of the first inlet, the second inlet, the first outlet, and the second outlet may be about the same. In other aspects, the diameter of the first inlet may be greater or less than the diameter of the first outlet and / or the diameter of the second inlet may be greater or less than the diameter of the second outlet. For example, the diameter of the first inlet and / or the second inlet may be in the range of about 0.1 mm to about 10 mm, from about 1 mm to about 7 mm, from about 3 mm to about 5 mm, or may be about 4 mm. The diameter of the first outlet and / or the second outlet may be in the range of about 0.1 mm to about 10 mm, from about 1 mm to about 7 mm, from about 3 mm to about 5 mm, or may be about 4 mm.According to any embodiment, a length (l) of the electroporation chamber may be in the range of about 2 mm to about 100 mm, about 2 mm to about 80 mm, about 2 mm to about 60 mm, about 2 mm to about 40 mm, about 2 mm to about 20 mm, about 5 mm to about 15 mm, about 8 mm to about 12 mm, or about 10 mm.According to any embodiment, a 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.FIG. 2B is an illustration of the electric field in the IOCO electroporation apparatus 200. After entry of the cell into the electroporation chamber, the cell is exposed to a uniform electric field until it exits the electroporation chamber. Position (1) refers to a cell in the inlet (first inlet 210), position (2) refers to a cell in the electroporation chamber, and position (3) refers to a cell in the outlet (first outlet 230).FIG. 2C is a plot of electric field strength versus distance a cell has traveled through the IOCO electroporation device 200. Cells in the inlet (first inlet 210) shown at position (1) are not exposed to an electric field until they have entered the electroporation chamber through the micromesh electrode. Within the electroporation chamber, shown as position (2), the cells are exposed to a uniform (maximum strength) electric field as they flow along the length of the chamber. When in the outlet (first outlet 230) shown as position (3), the cells are not exposed to an electric field.Figure 2D shows an alternative configuration of an electroporation device illustrated herein that includes an electroporation device 310-330. In this embodiment, rather than following a linear (horizontal) flow path between the upper and lower meshes, the cells may follow a curved or circular (horizontal) flow path between the upper and lower meshes. The linear flow path and the curved flow path may each include at least one segment extending parallel to a plane defined by a surface of at least one of the upper and / or lower meshes.In this example, the various layers of an electroporation apparatus 310-330 are shown in Figure 2D. Solid outer plates 310( 1) and 310( 2) (e.g., plastic, metal, or any other suitable material) encapsulate the device. An adhesive layer or other suitable material (e.g., double-sided tape, pressure sensitive adhesive material, etc.) 320( 1) (first material) is placed between the upper fixed 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 material, etc.) 330( 2) (second material) is placed between the lower outer plate 310( 2) and the lower mesh 330( 2). Another adhesive layer (e.g., pressure sensitive adhesive or double-sided tape, etc.) (340) comprising a curved channel 335 over which the cells and charge can flow is positioned between the upper and lower meshes 330(1) and 330(2), respectively. Fluid may be forced through electroporation apparatus 310-330 by syringe 350. Electroporation apparatus 310-330 may also be connected to an electroporator pulser 360. According to any embodiment, electroporation device 310-330 may be a continuous electroporation device wherein cells continuously flow through the device.A path is defined in the electroporation chamber for cells and charge to flow through the electroporation chamber, for example, between the upper micromesh electrode and the lower micromesh electrode. It should be understood that any suitable path is contemplated by the embodiments provided herein, provided that the path includes at least one horizontal flow segment parallel to at least one of the upper mesh and the lower mesh. The horizontal flow segment may form more or less than any of 1 / 10, 1 / 4, 1 / 2, 3 / 4, 7 / 8, or 9 / 10 of the length of the path extending between the upper and lower micromesh electrodes. In some cases, the path may be linear, curved, branched, encircling, convoluted, or any combination thereof. Examples of curved paths include, but are not limited to, a circular path, a helical path, a conical path, or any combinations thereof. For example, as illustrated in FIG. 2B, a path is linear, with arrows illustrating the flow of cells. As illustrated in FIGS. 2D and 2E, a path is curved, with arrows illustrating the flow of cells. As illustrated in FIG. 2F, a path may be linear with one or more directional changes or serpentines, with arrows illustrating the flow of cells. As illustrated in FIG. 2G, a path may be circular or have one or more branches. As illustrated in FIGS. 2E-2G, the paths may include an inlet 380 (protruding out of the plane) that may correspond to or be substantially aligned with the first inlet and one or more outlets 390 (protruding into the plane) that may correspond to or be substantially aligned with the first outlet. The length of the path may be adjusted, e.g., shortened or extended, depending on the particular characteristics of the electroporation method.According to any embodiment, the electroporation devices described herein can comprise means for creating a flow of cells from a sample tube into an electroporation chamber of an electroporation apparatus and a flow through the apparatus into a collection tube. For example, as illustrated in Figure 2H, a means for creating a flow 520 is in fluid communication with a sample tube 510 containing cells and / or charge for transfection and an electroporation device 530. The means for creating a flow 520 may create a flow of cells and / or charge from the sample tube 510, through the electroporation device 530, and into the collection tube 550. The means for generating a flow may be any suitable pump, for example a peristaltic pump or a syringe pump. It is contemplated that more than one means for creating a flow 520 may be present in an electroporation device. For example, one or more means for creating a flow 520 may be present between and in fluid communication with the sample tube 510 and the electroporation device 530, and one or more means for creating a flow 520 may be present between and in fluid communication with the collection tube 550 and the electroporation device 530. Additionally or alternatively, the electroporation device 530 and / or the means for generating a flow 520 may also be connected to a control device 540, which comprises an electroporator pulse generator, for example.According to any embodiment, the electroporation devices described herein may also comprise a tube of priming buffer in fluid communication with the electroporation device to provide priming buffer as needed. Examples of suitable priming buffers include, but are not limited to, distilled water, demineralized water, isotonic buffer, and combinations thereof. For example, as illustrated in Figure 2I, a tube of priming buffer 560 is in fluid communication with electroporation device 530. A valve 570 may be opened to supply priming buffer to electroporation apparatus 530 via a means for creating flow 520, or valve 570 may be closed to terminate supply. A valve 575 may be opened to deliver cells and / or charge to electroporation device 530 via a flow generating means 520, or valve 575 may be closed to terminate delivery. In some embodiments, valves 570 and 757 may both be open for delivery of cells and priming buffer. According to some embodiments, priming buffer may be initially supplied, followed by the supply of cells and / or charge.According to any embodiment, the electroporation devices described herein may further comprise a first microfluidic device for sorting the cells prior to entering an electroporation chamber of an electroporation device. For example, as illustrated in Figure 2J, a first microfluidic device 580 may be in fluid communication with an electroporation device 530, a sample tube 510 containing cells for transfection, and optionally a buffer sorting tube 577. Flow generating means 520 may generate a flow of cells from sample tube 510 and / or buffer from sorting tube 577 through electroporation device 530 and into collection tube 550. The first microfluidic device 580 is capable of pre-sorting cells, for example based on the size of cells, from the sample tube 510 to the electroporation device 530. Additionally or alternatively, the first microfluidic device 580 is capable of performing a buffer change of the cells from the sample tube 510. Stream 590 contains sorted cells not supplied to electroporation apparatus 530, which may be either a waste stream or may be supplied to another electroporation apparatus with different parameters. Additionally or alternatively, a second microfluidic device 585 may be in fluid communication with the electroporation device 530 and collection tube 550. The second microfluidic device 585 is capable of sorting electroporated cells, for example, based on the size of the electroporated cells, prior to collection in the collection tube 550. Stream 595 contains the sorted electroporated cells that were not fed to collection tube 550.Although two microfluidic devices are shown in Figure 2J, it is contemplated herein that only one microfluidic device may be present in an electroporation device, for example, for sorting cells prior to entering an electroporation chamber or for sorting electroporated cells upon exiting an electroporation chamber.FIG. 2K is an illustration of an example microfluidic device (e.g., microfluidic device 580, microfluidic device 585). In this example, a solution 100 comprising cells (e.g., cells for transfection, electroporated cells, transfected cells) enters a chamber 105 at inlet mechanism 110. Cells are illustrated as circles. The cells pass through the chamber 105 which comprises a matrix of posts 115, shown here as rectangular structures, distributed along multiple lines with a gradient. As the cells pass the diagonally arranged rows of posts in the chamber, the cells are deflected laterally to a second exhaust mechanism 122. A side view of a portion of the chamber is shown in FIG. 2L, wherein the chamber includes a floor 160 and optionally a ceiling 150, which may be of the same material as the posts 115 or of a different material. Cells (black circles) passing through the array of posts are shown. It will be appreciated that the rows of posts may also be curved, which would also result in the cells being directed to one side of the chamber.In this example, two exhaust mechanisms for the chamber are shown. The solution exiting the chamber via a first outlet mechanism 120 (which may also be referred to as a third outlet mechanism if more than one microfluidic device is present) is depleted of cells, while the solution exiting the chamber via a second outlet mechanism 122 (which may also be referred to as a fourth outlet mechanism if more than one microfluidic device is present) is enriched in cells. According to any embodiment, the second outlet mechanism may be in fluid communication with an electroporation chamber, for example, with a first inlet. Depletion refers to a condition where the concentration of cells in the solution exiting the chamber via the outlet mechanism 120 has been reduced by 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% compared to the concentration of cells in the solution entering the chamber at the inlet mechanism 110. Enrichment or concentration refers to a state in which the concentration of cells in the solution exiting the chamber via the outlet mechanism 122 has been 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 at the inlet mechanism 110. In order for the cells to be directed to the outlet mechanism 122, the deflection point 130 of the chamber should be positioned so that the exiting cells are directed to the outlet mechanism 122 (and not to the outlet mechanism 120). Generally, the exhaust mechanism 122 is configured to have a larger cross-sectional area than the exhaust mechanism 120. Generally, the exhaust mechanisms will have a cross-sectional area large enough to allow fluid to flow at the flow rates described herein without the occurrence of high pressures that would damage the cells or the device.According to some embodiments, the width of the exhaust mechanism 120 is greater than the width of the exhaust mechanism 122. The width of the exhaust mechanism 120 is, for example, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times the width of the exhaust mechanism 122. According to other embodiments, the sum of the cross-sectional areas of the exhaust mechanisms may be equal to or greater than the sum of the cross-sectional areas of the intake mechanisms.In some embodiments, the microfluidic device is configured to connect a standard pump or syringe or the like to the microfluidic device such that a solution comprising cells is flowed through the device. Parameters that can affect the flow of solution through an appliance include the dimensions of the chamber 105, the dimensions of the posts 115 in and out of the plane, the spacing of the posts in a row (dx), the rotation of the posts (φ), the spacing between rows of posts (dy), the diameter of the inlet mechanism, and the diameters of the outlet mechanisms (see Figures 2M-2Q below). In some embodiments, the dimensions of these parameters are chosen to be compatible with the applied pressure of manual operation of syringes or standard pumps (e.g., peristaltic pumps, diaphragm pumps, syringe pumps, cam pumps, etc.) that drive the flow of solution through the device. A range of pressures is allowed provided that the pressure does not damage the microfluidic device or cells.Generally, cells may flow into the microfluidic device in series (one after the other and on a fixed path) or as multiples (multiple cells flowing into multiple paths).FIG. 2M is an illustration of a top view of a portion of the chamber 105, the portion comprising two rows of seven posts each. The flow of cells is shown along path (p). This illustration shows the positioning of the posts 115 in a portion of the chamber of the microfluidic device. Generally, the posts are arranged along lines with a slope defined by angle (θ) at a distance interval (dx), which may be fixed or variable, provided that the variation does not result in cells deviating from the path (p) between the posts.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 therebetween. The width of the individual paths (p) controls the characteristics of the streamlines. Depending on the width of the chamber, any number of paths, e.g., 1, 2, 4, 8, 12, 16, 32, etc., or any range therebetween, may be employed. In other embodiments, the flow rate of fluid through the chamber may be 1 mL / hr, 2 mL / hr, 3 mL / hr, 4 mL / hr, 5 mL / hr, 6 mL / hr, 7 mL / hr, 8 mL / hr, 9 mL / hr, 10 mL / hr, 15 mL / hr, 20 mL / hr, 25 mL / hr, 30 mL / hr, 40 mL / hr, 50 mL / hr, and so forth. The chamber is desirably rectangular, but may be circular, semicircular, or V-shaped, or other suitable shape.The angle (θ) is the angle between the horizontal axis with a zero slope and a line with a slope along which the posts are distributed (in this example, the angle provides a measure of the negative slope (tan (θ)=Δy / Δx) of a row of posts). It will be appreciated that the posts may have a positive or negative slope in this example, as the rows of posts are each arranged diagonally. Here, it is understood that diagonally may comprise any orientation that is not parallel or perpendicular to the chamber, e.g., an angle between 1 and 89 degrees, between 91 and 179 degrees, between 181 and 269 degrees, or between 271 and 359 degrees, clockwise or counter-clockwise. Other exemplary angular ranges may be 1 to 10 degrees, 11 to 20 degrees, 21 to 30 degrees, 31 to 40 degrees, 41 to 50 degrees, 51 to 60 degrees, 61 to 70 degrees, 71 to 80 degrees, 81 to 90 degrees, 91 to 100 degrees, 101 to 110 degrees, 111 to 120 degrees, 121 to 130 degrees, 131 to 140 degrees, 141 to 150 degrees, 151 to 160 degrees, 161 to 170 degrees, or 171 to 180 degrees.Generally, a plurality of rows of posts are present in a chamber, and each row has the same slope or substantially the same slope defined by angle (θ). When cells 100 enter the chamber 105 of the microfluidic device, the cells flow laterally through a plurality of paths (p) between the posts 115 to one side of the chamber 105 until they reach one side of the chamber. To concentrate the cells, the cells do not generally cross rows of posts, but rather the cells flow along a particular path (p). The cells then exit the microfluidic chamber via the outlet mechanism 122. According to other embodiments, the posts may have a curved component along the length of the chamber 105.As used herein, the term "post" refers to a structure in the chamber having an in-plane dimension, an out-of-plane dimension, a rotational angle, and a shape associated therewith. An in-plane dimension may refer to the post length (l) and width (w), while an out-of-plane dimension may refer to the post height (h). A tilt angle or angle of rotation (φ) refers to the rotation of the post with respect to the chamber.Shape refers to the 3-D characterization of the post, e.g., cylindrical, conical, pyramidal, cubic or cuboidal, etc. In general, a post will be positioned with its axis (out-of-plane dimension) perpendicular to the surface to which it is attached. According to some embodiments, all posts in the chamber have the same dimensions. According to other embodiments, the dimensions of the posts vary as a function of space, as a function of the position measured along the height (h) of the post. Generally, the post may have any shape and is not limited to any particular geometric shape illustrated herein. The shape of the post may be arbitrarily selected, as shown in FIG. 2R, represented by an axis length (l) and width (w) and a plurality of radii (r4-r12) associated with a curvature positioned along the axis length, e.g., at a repeated constant interval or a repeated variable interval. Any number of different geometries can be described by the plurality of radii. Moreover, referring to FIGS. 2R and 2S, one or more sides of the post may have the shape of a straight line. Accordingly, the posts may have any suitable shape formed from curves and / or straight lines.According to some embodiments, the posts are arranged at a distance (dx) along a line with a slope, the distance being regular or fixed. For example, every 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, and so forth, a post may be placed, including any values between these regions, depending on the size of the cell to be concentrated.According to other embodiments, the posts are arranged along a line with a slope, so that the distance is not fixed, but rather varies between any two successive posts. Any distance (dx) is allowed provided that the distance is small enough to prevent the cell from deviating from path (p). For example, with a variable distance, a first post may be placed at a particular location, the second post may be placed 5 μm from the first post, a third post may be placed 4 μm from the second post, and so forth. The distance can be selected according to the size of the cell.In some embodiments, the distance may be selected to be sufficiently large to allow concentration of a particular cell type while smaller cells may exit via the outlet mechanism 120. For example, a blood sample may comprise a variety of different cell types of relatively small size including red blood cells, neutrophilic cells (e.g., 12-14 μm in diameter), eosinophilic cells (e.g., 12-17 μm in diameter), basophilic cells (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 to 100 μm or more in diameter. In such cases, the posts may be arranged so that red and white blood cells can pass through while larger cells (e.g., normal cells, tumor cells, etc.) are concentrated. The configuration of the posts (spacing) is determined according to the type of cell to be insulated. Different shell-to-sample flow ratios may affect the size-based sorting performance.Each post has a length (l) associated therewith and a width (w), also referred to as in-plane dimensions, and a height (h), also referred to as out-of-plane dimensions (see also FIG. 2N ). According to 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, and so forth, or any value therebetween. 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, and so forth, or any value therebetween. Generally, the posts may have any shape (when viewed from above and with respect to dimensions w and l), including, but not limited to, circular, oval, square, rectangular, etc.In the examples herein, the posts are oval or rectangular in 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 2-3 times greater than the width of the post. According to some embodiments, the length and the width may have a ratio greater than or less than 20 to 1, 18 to 1, 16 to 1, 14 to 1, 12 to 1, 10 to 1, 9 to 1, 8 to 1, 7 to 1, 6 to 1, 5 to 1, 4 to 1, 3 to 1, 2 to 1, 1.1 to 1, or smaller or larger ratios.According to some embodiments, the cross section of the post may be defined by the intersection of the post with a plane parallel to a wall from which the post extends at height (h). The cross section may be symmetrical along one or both of the directions of the length (l) and the width (w).In still other embodiments, the length and width of each post in the matrix are the same. In still other embodiments, if the posts are circular, the radius of each post in the matrix is equal.Additional features include the rotation angle (φ) of the post, the height and dimension of the post out of plane, the spacing between the rows (dy), and the offset of the rows (xo), which are described in more detail in FIGS. 2N-2Q and in the application. As used herein, the term "angle of rotation" refers to the degree of rotation of a post with respect to its position in the chamber. In some embodiments, the post has a length greater than its width. According to this embodiment, a rotation angle of zero means that the length of the post is aligned with an axis perpendicular to one side of the chamber. To describe various rotational angles, the post may be rotated clockwise or counter-clockwise. For example, the posts in FIGS. 2M-Q are rotated about 35 degrees counterclockwise to result in the post geometry shown in these figures.Referring to FIG. 2N, each post has an out-of-plane height (h) associated therewith, such that the posts are sufficiently high to prevent the cells from exiting path (p) by flowing on an upward path (e.g., across the top ends of the posts into another path). For example, in some embodiments, the posts extend the height of the microfluidic chamber such that the posts are in contact with the bottom of the chamber 160 and the ceiling of the chamber 150. According to other embodiments, the out-of-plane height or dimension may extend over a fraction of the overall height of the microfluidic device. The posts may have, for example, 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, and so forth, or any value therebetween.Referring to FIG. 20, each post has an associated angle of rotation (φ) or inclination, as seen in plan view. In the present example, the rotation of the post is determined from 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 in a direction with the side of the chamber by rotating the post counterclockwise, e.g., about 35 degrees counterclockwise in this example, until the desired rotation is achieved. Any angle of rotation may be used which facilitates the flow of cells over path (p), and all such angles of rotation are contemplated herein. According to some embodiments, the angle of rotation is between 1 degree and 179 degrees, between 1 degree and 89 degrees, between 5 degrees and 85 degrees, between 10 degrees and 80 degrees, between 15 degrees and 75 degrees, between 20 degrees and 70 degrees, between 25 degrees and 65 degrees, between 30 degrees and 60 degrees, between 35 degrees and 55 degrees, between 40 degrees and 50 degrees, between 30 degrees and 40 degrees, between 32 degrees and 38 degrees, between 34 degrees and 36 degrees, or 35 degrees. According to other embodiments, the angle of rotation may be between 91 degrees and 179 degrees, between 95 degrees and 175 degrees, between 100 degrees and 170 degrees, between 105 degrees and 165 degrees, between 110 degrees and 160 degrees, between 115 degrees and 155 degrees, between 120 degrees and 150 degrees, between 125 degrees and 145 degrees, between 130 degrees and 140 degrees, or 135 degrees.As stated above, the posts are arranged along a line with a negative gradient. By orienting the cells along this line, the cells flowing via path (p) are directed laterally to one side of the chamber, while the solution in which the cells were originally suspended may flow (e.g., nearly horizontal or horizontal) to exit the microfluidic chamber via the outlet mechanism 120.Referring to FIG. 2P, the rows of posts are spaced apart (dy) according to still other embodiments. According to some embodiments, the posts are arranged at a distance (dy) along a vertical line, the distances being regular or fixed. For example, a post may be placed every 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, and so forth, including any values therebetween, depending on the size of the cell to be concentrated.According to other embodiments, the posts are arranged along a vertical line such that the distance (dy) is not fixed, but instead varies between two consecutive rows of posts. For example, a first row of posts may be placed at a particular location, the second row of posts may be placed 5 μm from the first row of posts, a third row of posts may be placed 4 μm from the second row of posts, and so forth, aligned with respect to a vertical line. The distance (dy) may be selected based on the size of the cell to be concentrated. All distances (dy) are allowed provided that the distance is sufficiently wide to allow flow of the solution through the chamber without requiring high pressures that could damage the microfluidic device. According to some embodiments, successive rows of posts may have an offset (xo) of zero.Referring to FIG. 2Q, successive rows of posts may have an offset (xo) with respect to the distance (dx). For example, a row (r) may be located at a particular location (e.g., using the Cartesian coordinate system x, y). The (r+1)th row may be offset to the right by an amount (xo). The (r+2)th row may be offset by an amount (2xo) to the right. The (r+3)th row may be offset by an amount (3xo) to the right, and so on up to the distance (dx). Offsets may be present in a fixed manner so that each additional row is offset by a fixed amount in the same direction. Alternatively, offsets may be present in a variable manner such that each additional row is offset by a variable amount (up to the amount of (dx)) in the same direction. According to other embodiments, offsets may be applied in an alternating direction such that, with respect to a row (r), the (r+1)th row has a fixed or variable offset in one direction and the (r+2)th row has a fixed or variable offset in the opposite direction (e.g., one offset is to the right and the next offset is to the left).The posts may be made of any suitable material, including poly(dimethyl siloxane) (PDMS), glass, plastic, elastomers, silicone, etc. PDMS may generally be made with a submicron resolution (e.g., features below 0.1 μm). In some embodiments, the chamber 105 and / or posts 115 are made of PDMS. According to other embodiments, the chamber may be made of glass and / or silicone and / or plastic and the posts may be made with PDMS (e.g., the bottom of the chamber may be glass or silicone and the ceiling of the chamber may be glass or plastic). To produce the chambers and posts as described herein, lithography well known in the art may be used. Materials having properties similar to PDMS may also be used to construct microfluidic devices described herein.Moreover, specific embodiments may include a number of additional features, including valves (e.g., between an inlet mechanism and the chamber, between the chamber and an outlet mechanism, between an outlet mechanism and another inlet mechanism or chamber, etc.), pumps, and mixers. In some embodiments, valves may be used in PDMS during curing.Various techniques can be used to produce a microfluidic device. The microfluidic device may be formed from one or more of the following materials: poly(methyl methacrylate) (PMMA), polycarbonate, polystyrene, polyethylene, polyolefins, silicones (e.g., poly(dimethylsiloxane) PDMS), silicone, and combinations thereof. Other materials are well known in the art.Methods for manufacturing a post chamber and microfluidic device using the materials listed herein are also 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), silicone micromachining, wet or dry chemical etching, etc.Glass materials can employ silicone manufacturing processes using photolithography followed by wet (KOH) or dry etching (plasma enhanced etching with fluorine or other reactive gas). For example, conventional photolithography can form a master of glass which then serves as a master template for molding techniques to produce a plastic or PDMS based device.A microfluidic device can be manufactured in one or more layers connected to one another, for example, by adhesives, clamps, heat, solvents, etc. Alternatively, the microfluidic device may be fabricated in a single piece, e.g., using stereolithography or other three-dimensional fabrication methods.In some embodiments, due to cell deformability, a gap distance may be chosen that is about 5 μm or less than the size (e.g., a diameter) of the cell that is concentrated. According to other embodiments, a gap distance may be selected that is about 5 μm or more than the size (e.g., a diameter) of the cell being sorted. For rigid cells or microspheres having the same or similar diameter as a deformable cell, the gap distance may vary from the gap distance for deformable cells of the same diameter (e.g., greater for rigid cells).FIG. 2T is an illustration of another exemplary microfluidic concentrator. In this example, a first cell solution 100 enters a chamber 105 at a first inlet mechanism 110. The cells are shown as circles. The cells pass through the chamber 105 which comprises a matrix of posts 115, here shown as rectangular structures distributed along lines of slope. As the cells pass through the diagonally oriented rows of posts in the chamber, the cells are deflected laterally to one side of the chamber. Similar to FIG. 2L, the chamber has a floor 160 (not shown) and optionally a ceiling 150 that may be of the same material as the posts 115 or of a different material.In the example, a second intake mechanism 112 is present in this system. A second solution (e.g., a buffer different from the first solution entering the chamber via the first inlet mechanism 110) enters the chamber via a second inlet mechanism 112, for example, buffers of buffer tubes 577. Thus, in some embodiments, there is no substantial mixing of the first solution and the second solution, and the upper region of chamber 155 comprises the first solution and, if any, only little of the second solution, while the lower region of chamber 165 comprises the second solution and, if any, only little of the first solution. Thus, as the cells deflect laterally and leave the chamber via the outlet mechanism 122, the solution leaving the chamber via the outlet mechanism 122 is the second solution buffer.In some aspects, buffer changes may occur sequentially, in a cascade design as shown in FIG. 2U. In this example, a first cell solution 100 enters a first chamber 105( 1) at a first inlet mechanism 110( 1). The cells are shown as circles. The cells pass through the chamber 105( 1) comprising a matrix of posts 115( 1), here shown as rectangular structures distributed along lines with a slope. As the cells pass through the diagonally oriented rows of posts in the chamber, the cells are deflected laterally. Similar to FIG. 2L, the chamber has a floor 160 (not shown) and optionally a ceiling (150), which may be of the same material as the posts 115 or of a different material. The cells (enriched) exit the chamber through outlet 122(1) which flows into inlet mechanism 110(2) where a second inlet mechanism 112 enters the system.A second solution (e.g., a buffer) different from the first solution entering the chamber via the first inlet mechanism 110( 1) enters the chamber via the second inlet mechanism ( 112). In some embodiments, there is no substantial mixing of the first solution and the second solution, and thus the upper region of the chamber (155) comprises the first solution and, if any, only little of the second solution, while the lower region of the chamber (165) comprises the second solution and, if any, only little of the first solution. The cells (enriched and buffered in inlet mechanism 112) exit chamber 105(2) through outlet mechanism 122(2).In other aspects, buffer changes may occur in parallel. For example, the flow path may be divided so that the sample flows into a plurality of chambers, each chamber having an inlet mechanism for a buffer. With this configuration, the same buffer can be supplied to each of the parallel chambers, so that the cells are concentrated in the same buffer. Alternatively, a different buffer may be supplied to each of the parallel chambers so that the cells are concentrated in different buffers, e.g., for different downstream assays.Generally, the inlet mechanism may be operated by a syringe pump or by manually depressing a syringe. In some aspects, one or more syringe pumps may be used with multiple inlets to automatically operate each inlet. In other aspects, with multiple inlets, manually depressing one or more syringes may operate the inlets manually, respectively.According to any embodiment, an electroporation device capable of providing various electroporation effects is described herein. For example, an electroporation apparatus 600 as illustrated in FIG. 2V is provided having three micromesh electrodes 640( 1), 640( 2), 640( 3). Electroporation device 600 includes two paths through which cells and charge can flow, a first path between micromesh electrode 640(1) and micromesh electrode 640(2), and a second path between micromesh electrode 640(2) and micromesh electrode 640(3), where the arrows indicate the flow of cells. The cells may enter an electroporation chamber at inlet 620 and leave it at outlet 630. Although not shown, it is contemplated herein that more than one outlet may be present; for example, the outlets may each collect different types of cells. In the electroporation apparatus 600, different electrode gap lengths (L1, L2, L3, L4) are present. The different electrode gap lengths and different paths result in different capacitance; therefore, different electric field strengths and electroporation effects can be achieved for the cells flowing along the respective paths. For example, L2may be greater than L4, and in such a case, a capacitance (C1) in the first path may be greater than a capacitance (C2) in the second path (C1>C2). According to any embodiment, cells (of the same or different types) may be presorted and / or selected to arrive at the first path or the second path using the microfluidic devices described above, for example.It is also contemplated herein that the electroporation devices described herein may be present as cartridges that may be in fluid communication with each other (e.g., connected, stacked) to provide a path of a desired length and geometry. For example, as illustrated in FIG. 2W, the cartridges 410 may each include an electroporation chamber that includes an upper micromesh electrode 440( 1) and a lower micromesh electrode 440( 2), where the cartridges may be stacked in series, with the arrows pointing into the flow of cells through the cartridges. For example, an outlet of the top cartridge 410 is in fluid communication with an inlet of the middle cartridge 410 and an outlet of the middle cartridge 410 is in fluid communication with an inlet of the bottom cartridge 410.The electroporation devices described herein have a number of advantages over mesh-based electroporation devices such as the configuration shown in Figure 1B. First, the distance through the electroporation chamber, measured from the first inlet to the first outlet of the electroporation chamber, can be increased without this leading to a corresponding increase in the electric field. The cells are thus exposed to a uniform electric field over a longer time than a strictly linear flow path, resulting in improved electroporation efficiency without a corresponding decrease in viability.Although the electroporation chamber width (w) between the upper mesh electrode and the lower mesh electrode in Figure 1B could be increased to provide a longer distance, a larger electroporation chamber width would have a larger impedance and a higher voltage would have to be applied to generate a suitable field strength. Moreover, a higher voltage would likely have a deleterious effect on cell viability. Furthermore, in parallel electrode plate configurations as in FIG. 1A, the electric field distribution would be non-uniform.Thus, according to embodiments, the electroporation devices described herein may have a shorter pair of electrodes due to the offset inlet-outlet design, thereby increasing the length of the electroporation chamber without increasing the pair-pair distance.In Figure 3, a table is shown illustrating differences between an electroporation apparatus as described herein and electroporation apparatuses from the prior art. Such differences include, but are not limited to: (1) the presence of the inlet-outlet offset distance, (2) the use of low conductivity, low osmolarity electroporation media instead of commercially available electroporation media such as BTX and RPMI, (3) exponentially discharging waveforms applied in a series of electrical pulses, and (4) a staged fluid flow scheme.While the example of FIGS. 2A-2C uses stainless steel micromesh electrodes, the electrodes are not limited to any particular material and may be formed from any suitable material. For example, and as shown in Figure 5, suitable material includes silicone, polyimide, and micromesh. Silicone and polyimide can be used to form suitable micromesh electrodes, however, such materials are typically processed in clean room equipment using copolymerization protocols known in the art. In contrast, stainless steel micromesh electrodes are easy to set up and do not require special equipment such as clean rooms.It will be appreciated that any suitable material may be used provided that the micromesh has pores that allow passage of cells (e.g., the micromesh pores are larger than the cells) and that the micromesh pores are compatible with the electrode assembly around the pore opening.Electroporation Methods and ParametersProvided herein are methods for electroporating cells with a charge, for example, with the electroporation devices as described above. The methods may comprise introducing the cells with the charge into an electroporation chamber. The electroporation chamber comprises an upper micromesh electrode as described herein, a lower micromesh electrode as described herein, and a path as described herein defined between the upper micromesh electrode and the lower micromesh electrode on which the cells and the charge can flow. The upper micromesh electrode and the lower micromesh electrode each have a porosity as described herein. Moreover, the upper micromesh electrode may be bounded by a first material as described herein. The first material may comprise a first inlet as described herein that allows passage of cells into the electroporation chamber. The bottom micromesh electrode may be bounded by a secondary material. The second material may comprise a first outlet as described herein that allows passage of electroporated cells from the electroporation chamber. The first inlet and the first outlet are spaced by an offset distance as described herein. The cells may be suspended in an electroporation medium. Optionally, the upper micromesh electrode may further comprise a second inlet as described herein, and / or the lower micromesh electrode may further comprise a second outlet as described herein.According to any embodiment, the flow of cells and charge may be step-wise. For example, a volume of fluid about half the total volume of the electroporation chamber can be pumped into the electroporation chamber at a fixed time interval. FIG. 4 shows an example of a series of electrical pulses (stim) and an example stepped fluid flow pattern (pump). In the stepped fluid flow scheme, the chamber volume is about 22 μl. Every second, 11 μl is pumped into the electroporation chamber, displacing half the volume. This may be repeated as long as necessary for processing the desired amount of cells.Any time interval may be used along with any suitable volume for the stepped fluid flow scheme. The time interval may range from about 0.1 seconds to about 10 seconds, from about 0.5 seconds to about 5 seconds, from about 1 second to about 2 seconds, or any period of time therebetween, for example, or may include longer intervals. In some aspects, the amount of fluid pumped into the electroporation chamber as a function of time may be three quarters of the volume of the inlet chamber, half 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.According to any embodiment, the cells in the electroporation chamber are exposed to a uniform or substantially uniform electric field. The cells may be exposed to multiple electrical pulses in the electroporation chamber, the individual electrical pulses being the same or different. According to some embodiments, a direct current (DC) electric pulse is applied. Additionally or alternatively, an alternating current (AC) electrical pulse is applied. According to any embodiment, the pulses may each have a form of an exponentially discharging waveform or a quadratic waveform. According to any embodiments, the multiple electrical pulses may include either an exponentially discharging waveform or a quadratic waveform.In the series of electrical pulses, a voltage may be applied to the electrodes every 100 ms to 5000 ms (e.g., every 100 ms, every 250 ms, every 500 ms, every 1000 ms, every 2000 ms, every 3000 ms, etc.) to establish an electric field of a given strength. This may be repeated as long as necessary for processing a desired amount of cells. Accordingly, it should be appreciated that while aspects may be referred to as continuous flow, continuous flow includes a stepped fluid flow scheme in which fluid is continuously pumped into the electroporation chamber at a defined time interval.Any suitable time intervals may be used along with any suitable duration of the electrical pulse for cells in the electroporation chamber. The time interval between pulses may range, for example, from about 0.1 seconds to about 5 seconds, from about 0.2 seconds to about 1 second, from about 0.4 seconds to about 0.6 seconds, or any period therebetween, and may include longer intervals. In some aspects, the duration of the electrical pulse may be in the range of about 10 ms to about 250 ms, from about 50 ms to about 150 ms, from about 75 ms to about 125 ms, or any range therebetween.Any suitable cells, for example mammalian cells, non-mammalian cells, or both, may be transfected with a charge. Examples of suitable mammalian cells include, but are not limited to, NK cells, EC-7 cells (a derivative of HEK 293 cells modified to produce adenovirus), T cells, embryonic cells, stem cells, epithelial cells, lymphocytes, macrophages, gamete cells, and fibroblasts. Examples of suitable non-mammalian cells include, but are not limited to, bacterial cells and yeast cells. Any suitable charge may be used, for example a nucleic acid. The nucleic acid may be an RNA, e.g., synthetic RNA, mRNA, in vitrotranscribed RNA, GFP mRNA, etc.) and / or a DNA (e.g., synthetic DNA, GFP-DNA, plasmid DNA, etc.). In particular, the IOCO electroporation apparatus described herein can be used to transfect NK cells, EC-7 cells (a derivative of HEK 293 cells modified to produce adenovirus) and T cells with RNA (e.g., synthetic RNA, mRNA, in vitrotranscribed RNA, GFP mRNA, etc.) and / or DNA (e.g., synthetic DNA, GFP DNA, plasmid DNA, etc.). It is also contemplated herein that the electroporation devices described herein can be used to perform in vitrofertilization. For example, an egg and sperm could flow through an electroporation apparatus as described herein to fertilize the egg.In further aspects contemplated, the medium or electroporation buffer in which the cells are transfected is a low conductivity, low osmolarity medium, optionally containing one or more nutrients. Suitable media thus include, but are not limited to, Cw100 (0.11 S / m, 0.12 osm / l) or Cw240. It is possible to use commercially available electroporation media, but this has proven to be suboptimal. Such commercial media include the following: RPMI (1.37 S / m, 0.28 osm / l) and BTX (8 mS / m, 0.27 osm / l). Suitable conductivities may range from about 0.05 mS / m to about 0.2 S / m. Suitable osmolaritys can range from about 0.05 osm / L to about 0.2 osm / L. The media are generally electrically conductive media which may also be sterile. Table 1. Composition of Electroporation Media Table 1. Composition of Electroporation MediaCw2402 mM5 mM1 mM200 mM15 mMCw1002 mM0 mM1 mM99 mM20 mMWith respect to suitable capacitances, it is contemplated that the capacitance may range from about 1 μF to about 150 μF. In some embodiments, the capacitance may range from about 1-100 μF, from about 5 to about 75 μF, from about 5 to about 50 μF, from about 10 to about 40 μF, or from about 10 to about 30 μF, or from about 10 to about 25 μF. According to other embodiments, the capacitance is about 10 μF.According to some embodiments, one may apply multiple electrical pulses together with a correspondingly short time constant. In some aspects, one may use a time constant of less than 30 ms, less than 20 ms, less than 10 ms, or less than 5 ms. In other aspects, a time constant may be in the range of about 0.5 to 30 ms, about 1 to 20 ms, and about 5 to 15 ms, or about 10 ms.In some aspects, the electric field strength for electroporation is between about 0.3-3 kV / cm. Lower electric field strengths (e.g., about 0.5-1 kV / cm) were found to be suitable for EC-7 cells, while higher electric field strengths (e.g., about 1-3 kV / cm) were found to be suitable for haNK cells. Generally, electric field strengths in the range of about 0.1 to about 5 kV / cm are contemplated for electroporation. The voltage may be chosen to generate suitable electric field strengths.Generally, the impedance (Rp) may range from about 200 Ω to infinity, or in other cases from about 200 Ω to about 1k.The concentration of charge added to the electroporation reaction, e.g., the material to be introduced into the cell, is about 50 μg / ml.In some aspects, GFP mRNA or GFP DNA was added to the electroporation reaction at a concentration of about 50 μg / ml, while in other embodiments dextran 500k was added to the electroporation reaction at a concentration of about 50 μg / ml.As for suitable pulse numbers and pulse-to-pulse intervals, at least two, three and four pulses may provide more desirable results than a single pulse or five or more pulses. It is therefore contemplated that pulses may range between 2-4 pulses. Most typically, the pulses are separated from subsequent pulses by a relatively short time interval, typically between 0.5 seconds and 15 seconds, although in some cases even longer intervals may be used.In some aspects, in a single chamber device, up to 7.33 μl / s can be achieved at 4×10 7 cells / ml (13.2 ml (5.28×10 8 cells) in 30 min. To achieve 20×10 9 cells in 30 min, the cell density can be increased by a factor of four and / or parallel processing can be carried out using a plurality of electroporation chambers.In some aspects and further to the examples below, electroporation electronics and parameters were specially developed and optimized for electroporation of haNK and EC7 cells.According to an embodiment, the methods described herein may further comprise a first cell sorting step and / or a second cell sorting step, for example using the microfluidics devices as described herein. In the first cell sorting step, the cells may be sorted prior to introduction into the electroporation chamber by applying pressure by which a first solution comprising the cells is caused to flow through a microfluidic chamber as described herein, comprising a plurality of rows of posts as described herein, and deflecting the cells through the rows of posts to one side of the chamber to de-enrich cells from the solution exiting a first outlet mechanism as described herein and to enrich cells in the solution exiting a second outlet mechanism as described herein. The cells exiting the second outlet mechanism can be loaded into the electroporation chamber. In the second cell sorting step, the electroporated cells may be sorted after exiting the electroporation chamber by applying pressure that causes a fourth solution comprising the electroporated cells to flow through a microfluidic chamber as described herein, comprising a plurality of rows of posts as described herein, and deflects the electroporated cells through the rows of posts to one side of the chamber to de-enrich electroporated cells from the solution exiting a third outlet mechanism as described herein and enrich electroporated cells in the solution exiting a fourth outlet mechanism as described herein.The techniques and methods provided herein may be incorporated into various devices or platforms as part of a workflow. For example, an electroporation device as described herein can be incorporated into an automated device, for example, in cartridge form, wherein the electroporation can be carried out on cells together with one or more other automated methods. Automated electroporation may be performed, for example, along with automated cell culturing and harvesting, as described in U.S. Patent Publication No. 2017 / 0037357, which is hereby incorporated by reference in its entirety.In other aspects, methods for in vivotransfection are provided wherein the methods described above are performed and the transfected cells can be administered, for example mixed with an isotonic buffer, to a patient for treating a disease or disorder. Examples of diseases include, but are not limited to, mitochondrial disorder, cardiac dysfunction, heart failure, autism, diabetes mellitus, and taubheite. It is also contemplated herein that the electroporation devices described herein can be adapted for application to target tissue of a subject to aid in the administration of a drug or formulation, for example, to aid in the administration of insulin or a drug for the treatment of diabetes. For example, electrodes for electroporation could be applied to the skin or a needle-like pair of electrodes administered subcutaneously.Kits are also provided herein. The kits may comprise, for example, an electroporation device as described herein and optionally suitable reagents. Suitable reagents include, but are not limited to, cells for transfection as described herein, an electroporation medium as described herein, and a combination thereof. Additionally or alternatively, a kit may further comprise electroporation chips, connector tubes, apparatus, tools for electroporation, electroporation buffers, additives, reagents, and combinations thereof.EXAMPLESExample 1. transfection of haNK cells.In some aspects, conditions for electroporation of haNK cells are shown in Table 2 below. Table 2. conditions for electroporation of haNK cells Table 2. conditions for electroporation of haNK cellsCharge ChargeDextran 500k (dx500k) at 50 μg / ml;Flow Rate4-6,8 Million cells / minGFP mRNA (mRNA) at 50 μg / mlCell density5×10 6~1×10 7 / mlEntire sample16 Millions of cellsMediaBTX: 8 mS / m, 0.27 osm / l;Frequency Frequency2 Hz HzCw100: 0.11 S / m, 0.12 osm / l;RPMI: 1.37 S / m, 0.28 osm / lResistance (Rp)200 Ω - infinityPulse number3-4 Pulses (Discharging or Monophasic Pulse Sequence)Electric field1-3 kV / cmQavg0,11 0.44 ml / minCapacitance Capacitance10 uF - 150 uFSample size (cuvette)0,45 ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml mlApparatus According to the Inventionø5mm, d=420um, reg;Time constant time constant10 - 30 msparametersø5mm, d=560um, reg;Ø10 mm, d=284 μm, offsetFigures 6A-E show the experimental results of electroporation reactions. FIG. 6A shows various experimental conditions with different efficiencies and viabilitys. It is noteworthy that conditions of 1.5 kv / cm, Rp1k, 10 μF using the IOCO electroporation apparatus provided greater than 80% efficiency with greater than 70% viability in GFP mRNA electroporation in haNK cells. The term "reg" refers to the original micromesh, while "offset" refers to the offset micromesh. Figure 6B shows a microscopy image of GFP mRNA electroporated into haNK cells. Figures 6C and 6D show results of cell sorting, wherein Figure 6C shows live cells and Figure 6D shows electroporated cells. Figure 6E shows a histogram corresponding to cell sorting results showing control cells and cells electroporated with GFP.Figures 7A-7D show another set of experimental results with varying experimental electroporation conditions, with an efficiency of greater than 50% and a viability of greater than 70%, in GFP mRNA electroporation in haNK cells. Figures 7B and 7C show results of cell sorting, wherein Figure 7B shows live cells and Figure 7C shows electroporated cells. Fig. 7D shows a histogram corresponding to the cell sorting results showing the control cells and cells electroporated with GFP. GFP expression in the living cells was counted (propidium iodide negative, PI). CTL stands for control experiments.Example 2. transfection of EC7 cells.Typical conditions for electroporation of EC7 cells are shown in Table 3 below. Table 3. Conditions for electroporation of EC7 cells Table 3. Conditions for electroporation of EC7 cellsCharge ChargeDextran 500k (dx500k) at 50 μg / ml;Flow Rate4 17.6 million cells / minGFP mRNA (mRNA) at 50 μg / ml;GFP DNA (DNA) at 50 μg / mlCell density5×10 6~4×10 7 / mlEntire sample16 Millions of cellsMediaBTX: 8 mS / m, 0.27 osm / l;Frequency Frequency2 Hz HzCw100: 0.11 S / m, 0.12 osm / l;Cw240: 0.2 S / m, 0.24 osm / l;Eppendorf: 0.35 S / m, 0.09 osm / l;WaterResistance (Rp)200 Ω to infinityPulse number3-4 Pulses (Discharging or Monophasic Pulse Sequence)Electric field Field0,3 - 3 kV / cmQavg0,44 ml / minCapacitance Capacitance10 - 75 uFSample size (cuvette)0,44 - 0.45 mlDevice Control System Apparatus According to the InventionØ5mm, d=420um, Reg;Time constant time constant5 - 20 msparameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameter parameterø5mm, d=560um, Reg;Ø10 mm, d=284 μm, offsetFigures 8A-D show the experimental results of electroporation reactions in EC-7 cells. FIG. 8A shows various experimental conditions with varying efficiencies and viabilitys. It is noteworthy that conditions of 1 kv / cm, Rp 200, 10 μF using the IOCO electroporation apparatus provided greater than 90% efficiency and greater than about 50% viability in GFP DNA electroporation in EC-7 cells. Figures 8B-8C show results of cell sorting, wherein Figure 8B shows live cells and Figure 8C shows electroporated cells. Fig. 8D is a histogram corresponding to the cell sorting results showing the control cells and cells electroporated with GFP.Figures 9A-9E show additional sets of experimental results in EC-7 cells under varying experimental electroporation conditions. Figs. 9B, 9C and 9D show results of cell sorting, wherein Fig. 9B shows living cells and Figs. 9C and 9D show electroporated living cells. Figure 9E shows a histogram corresponding to cell sorting results with control cells and cells electroporated with GFP. GFP expression in the living cells was counted (propidium iodide negative, PI). CTL stands for control experiments.Figures 10A-10C show images of GFP DNA successfully electroporated into EC-7 cells using the offset chamber electroporation apparatus of Figures 2A-C.Figure 10A was taken 20 hours after electroporation and shows electroporation of GFP DNA and GFP-AD5 (DNA encoding production of AD5 virus) into EC-7 cells. Figure 10B was taken 44 hours after electroporation and shows electroporation of GFP DNA and a DNA shuttle vector into EC-7 cells. Figure 10C was taken 6 days after electroporation and shows a comparison of electroporation results with micromesh versus cuvette.Figure 10C also shows comete formation during the early stages of AD5 virus production in EC7 cells, according to the embodiments depicted herein. In this series of images, the cells form a cluster or "comet" shaped plaque, with the shape of individual cells becoming more rounded.As shown by this series of images, the EC-7 cells were successfully electroporated with fluorescently tagged viral DNA (for AD5 virus production) and were capable of initiating virus production, indicating that the techniques and systems provided herein are suitable for adenovirus production (e.g., for use in viral vaccines and for production of viral proteins).Figures 11A and 11B show additional results of transfection of EC7 cells using the methods and apparatus described herein. Figure 11A shows a table of electroporation parameters in which the amount of charge was varied between electroporation runs. Figures 11B show images six days after electroporation. Fourteen days after electroporation, an efficiency of about 70-80% was observed. After transfection, several "comet" shaped clusters were observed. About 17-18 million cells / min can be transfected into EC7 cells, at a cell concentration of about 40 m / ml, in electroporation buffer cw100, which has a conductivity of about 240 mS / m.Example 3. Electroporation of Different Cell Types.In other aspects, the methods and systems provided herein may be used to transfect different different cell types. For example, the methods and systems provided herein allow up to 10 8 cells or more to be transfected. The methods and systems provided herein allow transfecting EC7 cells, haNK cells, CHO cells, and T cells. The methods and techniques presented herein may be used, for example, to transfect EC7 cells at a flow rate of 0.6 ml / min with a cell concentration of 4×10 7 cells / ml. In another example, the methods and techniques presented herein for transfecting haNK cells at a flow rate of 0.36 mL / min with a cell concentration of 3×10 7 cells / mL may be used. In yet another example, the methods and techniques provided herein may be used to transfect CHO cells at a flow rate of 0.45 mL / min with a cell concentration of 2.5×10 7 cells / mL. For T cells, the methods and techniques illustrated herein for transfecting T cells at a flow rate of 0.44 mL / min with a cell concentration of about 2×10 7 cells / mL may be used. These conditions can be varied further to achieve optimum conditions.Figures 12A-12D show different transfection efficiencies for these different cell lines using the methods and apparatus described herein. Figure 12A shows transfection efficiency greater than 95% with cell viability greater than 90% for haNK cells (transfected with GFP mRNA). Up to 0.127 billion cells / min were transfected. Figure 12B shows transfection efficiency greater than 90% with cell viability greater than 50% for CHO cells (transfected with GFP pmaxDNA). Up to 15 million cells / min were transfected. Figure 12C shows transfection efficiency greater than 95% with cell viability greater than 50% for EC7 cells (transfected with GFP adenovirus DNA). Up to 20 million cells / min or more were transfected. Figure 12D shows transfection efficiency greater than 90% with cell viability greater than 80% for T cells (transfected with GFP mRNA). Up to 8.8 million cells / min were transfected.Example 4. transfection of primary T cellsIn other aspects, the methods and systems presented herein have been used for electroporating primary T cells with mRNA bound to poly(β-amino ester). Certain poly(β-aminoester) polymers have been shown to act as effective RNA transfection agents.Nanoparticles comprising RNA and poly(β-amino ester) polymers were used to deliver mRNA to T cells (see, e.g., Moffett, et al., Nature Communications (2017) 8:389). For example, in accordance with some embodiments, nanoparticles having a polyglutamic acid (PGA)-based shell may be produced. To the PGA molecule, a targeting molecule (e.g., a binding domain of an antibody) may be attached to direct the nanoparticle to a corresponding target. For example, to direct the nanoparticle to T cells, one could attach to PGA molecules an anti-CD3 / anti-CD28 binding domain. For targeting, PGA or other suitable negatively charged molecule may be used. The nanoparticle can be incorporated by specific targeting or by cationic membrane association.The interior of the nanoparticle may comprise mRNA and a carrier molecule such as poly(β-amino ester). After uptake of the nanoparticle into the cell, the mRNA is released into the cell by degradation of the nanoparticle, by osmotic swelling, or by any other suitable process, and the mRNA is transcribed into its corresponding protein. In some cases, synthetic mRNA may be used to reduce mRNA degradation.The methods provided herein allow mRNA to be transfected into cells, e.g., using mRNA and a poly(β-amino ester)(PbAE) carrier, or using a nanoparticle in which mRNA and a poly(β-amino ester) are encapsulated. Figures 13A-13E show T cell transfections with PbAE complexed with mRNA (no free mRNA, and without electroporation). Figure 13A shows the structure of a poly(β-amino ester) suitable for use with the techniques provided herein. Figure 13B shows cell sorting results using flow cytometry of T cells transfected with mRNA using PbAE. Figure 13C is a graphical representation of normalized results for live cells based on various ratios of mRNA to carrier molecule. Figures 13D and 13E show cell viability results under various conditions for GFP mRNA transfection in stimulated T cells (13D) and unstimulated T cells (13E). In these instruments, a 60:1 PbAE:RNA ratio resulted in a high level of viable cells, providing a high GFP delivery rate.Figures 14A and 14B show T cell transfection with GFP mRNA with control cells (no electroporation) and electroporated cells. In particular, Figure 14A shows control cells (no electroporation) with mainly background fluorescence observed. Figure 14B shows electroporated cells, wherein in most cells GFP mRNA is detected as shown in the histogram.Example 5. transfection of adipose derived mesenchymal stem cells (AD-MSC)Adipose-derived mesenchymal stem cells (AD-MSC) are detached and harvested at a density of about 1×10^5 million per milliliter. Cells were washed with PBS and electroporation buffer and suspended in electroporation buffer at a density of 3.3 million cells per milliliter. Each condition was electroporated with 0.1 milliliter of final cell density with 30 μg of DNA. The electroporation conditions were as follows: pulse width=100 μs, pulse period=340 ms, the electric field strength was varied from 0.9 to 1.6 kV / cm, as shown in Table 4 below.Table 4 Table 4. Electroporation Field Strength Conditions11,621,531,441,351,261,171,080,9After electroporation, cells were seeded at a density of 30×10^3 cells per square centimeter. The efficiency of GFP expression was verified by flow cytometry 24 hours after electroporation. The results are shown in Figs. 15A-15D. The x-axis values of 1-8 in each of Figures 15A-15C correspond to the field strengths shown in Table 4 and "CTL" stands for control without electroporation. Figure 15A shows cell viability 24 h after electroporation. The lower the electric field strength, the more viable the cells. Figure 15B shows the expression of GFP (green fluorescent protein) read by flow cytometry in measuring cell transfection efficiency 24 hours after electroporation. The stronger the electric field, the higher the percentage of GFP-expressing living cells. FIG. 15C shows the median of GFP fluorescence indicating how much GFP illuminated in the flow cytometry measurement. The stronger the electric field, the brighter the measured GFP is illuminated. The results suggest that electric field strengths between 1.1 to 1.3 kv / cm provide the best performance for viability and efficiency. It should be noted that viability here includes both adherent cells and supernatant to reflect true overall viability. Figure 15D is a photograph of the MSC transfection result. The photograph on the left shows the morphology of the MSCs and the image on the right is a fluorescent image from the same optical field showing that most of the cells express green fluorescent protein (green).In some embodiments, the numbers expressing amounts of ingredients, properties such as concentration, reaction conditions, etc., used to describe and claim certain embodiments are to be understood to be modified by the term "about" in certain instances. Accordingly, in some embodiments, the numerical parameters recited in the specification and appended claims are approximations that may vary depending on the desired characteristics sought by a particular embodiment. In some embodiments, the numerical parameters should be designed in view of the number of significant locations indicated and by using conventional rounding techniques. Although the numerical ranges and parameters setting forth the broad scope of some embodiments are approximations, the numerical values given in the specific examples are given as accurately as possible. The numerical values listed in some embodiments may include certain errors that are constrained to result from the standard deviation found in their respective test measurements.As used in the present specification and the following claims, the meaning of "a" and "the" includes reference to the plural, unless the context clearly indicates otherwise. In addition, the meaning of "in" as used in the present specification includes "in" and "an" unless the context clearly indicates otherwise. Unless clearly indicated otherwise from context, all ranges listed herein, including their endpoints, should be interpreted and ranges without endpoints should be interpreted to include commercially practical values. Similarly, all enumerations of values should be considered to include intermediate values unless the context clearly indicates otherwise.It should also be apparent to those skilled in the art that many other modifications are possible in addition to those already described herein without departing from the inventive concepts described herein. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, meaning that the elements, components, or steps referred to may be present, or used, or combined with other elements, components, or steps that are not expressly pointed out. If the claims of the invention refer to at least one element selected from the group consisting of A, B, C..., and N, the text should be interpreted as requiring only one element from the group, and not A plus N, or B plus N, etc.

Claims

A method for electroporating cells having a charge, comprising: flushing the cells having the charge into an electroporation chamber, the electroporation chamber comprising: an upper electrode, a lower electrode, and a path defined between the upper electrode and the lower electrode along which the cells and the charge can flow, wherein the upper electrode and the lower electrode each have a porosity, wherein the upper electrode is bounded by a first material, wherein the first material comprises a first inlet that allows passage of cells into the electroporation chamber, wherein the lower electrode is bounded by a second material, wherein the second material comprises a first outlet that allows passage of electroporated cells from the electroporation chamber, wherein the porosity of the upper electrode allows passage of the cells and the charge from the first inlet through the upper electrode into the electroporation chamber, and / or the porosity of the lower electrode allows passage of the electroporated cells through the lower electrode and the first outlet from the electroporation chamber, wherein the first inlet and the first outlet are spaced apart by an offset distance and wherein the cells are suspended in an electroporation medium.The method of claim 1, wherein the flowing is gradual, wherein a volume of liquid of about half the total volume of the electroporation chamber is pumped into the electroporation chamber at a fixed time interval.The method according to claim 1 or claim 2, wherein the offset distance is 2 mm or more.The method of any preceding claim, wherein a width of the electroporation chamber is about 0.01 mm to about 2 mm.The method of any preceding claim, wherein the upper electrode further comprises a second inlet that allows passage of the cells and charge into the electroporation chamber, and / or the lower electrode further comprises a second outlet that allows passage of the electroporated cells from the electroporation chamber.The method of any preceding claim, wherein the path is linear, curved, branched, or a combination thereof.The method of any preceding claim, wherein the path is horizontal with respect to the top electrode and the bottom electrode.The method of any preceding claim, wherein the cells in the electroporation chamber flow laterally in the path along the length of the electroporation chamber.The method of any preceding claim, wherein the cells in the electroporation chamber are exposed to a uniform or substantially uniform electric field.The method of any preceding claim, further comprising applying a plurality of electrical pulses to the cells in the electroporation chamber, wherein the individual electrical pulses are the same or different.The method of claim 10, wherein the plurality of electrical pulses are applied at a field strength of about 0.3 kV / cm to about 3 kV / cm.The method of claim 10 or claim 11, wherein one or more of the following occurs: (a) each electrical pulse is in the form of an exponentially discharging waveform or a quadratic waveform, (b) a time interval between the individual electrical pulses is about 0.5 seconds to about 15 seconds, (c) a duration of each individual electrical pulse is about 0.1 seconds to about 5 seconds, (d) a time constant is about 10 ms to about 30 ms, (e) the electrical pulses are provided by a capacitor having a capacitance of about 5 μF to about 75 μF, and (f) a field strength is applied having a voltage of about 40 V.The method of any preceding claim, wherein the electroporation buffer has a conductivity of from 0.05 mS / m to about 0.2 S / m and an osmolarity of from about 0.05 osm / L to about 0.2 osm / L.The method of any preceding claim, wherein the cells are mammalian cells or non-mammalian cells.The method of claim 14, wherein the mammalian cells are selected from the group consisting of NK cells, EC-7 cells, T cells, embryonic cells, stem cells, epithelial cells, lymphocytes, macrophages, gamete cells and fibroblasts.The method of claim 14, wherein the non-mammalian cells are bacterial cells or yeast cells.The method of any preceding claim, wherein the charge is a nucleic acid.The method of claim 17, wherein the nucleic acid is an RNA or a DNA.The method of any preceding claim, wherein the electroporated cells are haNK cells having a viability of at least 70% and an efficiency of at least 80%.The method of any preceding claim, wherein the electroporated cells are EC-7 cells having a viability of at least 50% and an efficiency of at least 90%.The method of any preceding claim, further comprising a first cell sorting step of sorting the cells prior to introduction into the electroporation chamber, comprising: applying pressure with which a first solution comprising the cells is caused to flow through a microfluidic chamber, wherein the microfluidic chamber comprises a plurality of rows of posts, each row comprising a plurality of posts arranged along a line that is diagonally oriented with respect to the microfluidic chamber, and deflecting the cells to one side of the chamber by the rows of posts to de-enrich the cells from the solution exiting via a first outlet mechanism and to enrich the cells in the solution exiting via a second outlet mechanism, wherein the cells exiting via the second outlet mechanism are introduced with the charge into the electroporation chamber.The method of any preceding claim, further comprising a second cell sorting step of sorting the electroporated cells after exiting the electroporation chamber, comprising: applying pressure to cause a fourth solution comprising the electroporated cells to flow through a microfluidic chamber, wherein the microfluidic chamber comprises a plurality of rows of posts, each row comprising a plurality of posts arranged along a line that is diagonally oriented with respect to the microfluidic chamber, and deflecting the electroporated cells to one side of the chamber by the rows of posts to de-enrich the electroporated cells from the solution exiting via a third outlet mechanism and to enrich the electroporated cells in the solution exiting via a fourth outlet mechanism.An apparatus for electroporating cells with a charge, comprising: one or more electroporation chambers, each electroporation chamber comprising: an upper electrode, a lower electrode, and a path defined between the upper electrode and the lower electrode, along which the cells and the charge can flow, wherein the upper electrode and the lower electrode each have a porosity, wherein the upper electrode is bounded by a first material, wherein the first material comprises a first inlet that allows passage of cells into the electroporation chamber, wherein the lower electrode is bounded by a second material, wherein the second material comprises a first outlet that allows passage of electroporated cells from the electroporation chamber, wherein the porosity of the upper electrode is configured to:, allowing passage of the cells and charge from the first inlet through the top electrode into the electroporation chamber, and / or the porosity of the bottom electrode is configured to allow passage of the electroporated cells through the bottom electrode and the first outlet from the electroporation chamber, and wherein the first inlet and the first outlet are spaced apart by an offset distance.The apparatus of claim 23, wherein the offset distance is 2 mm or more.The device of claim 23 or claim 24, wherein the width of the electroporation chamber is about 0.01 mm to about 2 mm.The device of any one of claims 23 to 25, wherein the upper electrode and the lower electrode are each formed of stainless steel, polyimide, or silicone.The device of any one of claims 23 to 26, wherein the upper electrode further comprises a second inlet that allows passage of the cells and charge into the electroporation chamber, and / or the lower electrode further comprises a second outlet that allows passage of the electroporated cells from the electroporation chamber.The apparatus of any one of claims 23 to 27, wherein the path is linear, curved, branched, or a combination thereof.The apparatus of any one of claims 23 to 28, wherein the path is horizontal with respect to the top electrode and the bottom electrode.The device according to any of claims 23 to 29, wherein the electrical pulses are supplied by a capacitor having a capacitance of 5-75 μF.The device of any of claims 23 to 30, wherein the first material and the second material are each an adhesive material.The apparatus of claim 31, wherein the adhesive material is a pressure sensitive adhesive material.The device of any one of claims 23 to 32, wherein a length of the electroporation chamber may range from about 5 mm to about 100 mm.The apparatus of any one of claims 23 to 33, wherein a diameter of the first inlet, the second inlet, or both is about 2 mm to about 5 mm, respectively, and the diameter of the first outlet, the second outlet, or both is about 2 to about 5 mm, respectively.The apparatus of any one of claims 23 to 34, further comprising a first microfluidic device for sorting the cells prior to entering the electroporation chamber, the first microfluidic device comprising: an inlet mechanism for introducing a first solution comprising the cells; a microfluidic chamber in fluid communication with the inlet mechanism, the microfluidic chamber comprising a plurality of rows of posts, each row comprising a plurality of posts arranged along a line diagonally oriented with respect to the microfluidic chamber, and at least a first outlet mechanism and a second outlet mechanism, wherein the first outlet mechanism and the second outlet mechanism are in fluid communication with the microfluidic chamber and the second outlet mechanism is in further fluid communication with the first inlet of the electroporation chamber.The apparatus of any one of claims 23 to 35, further comprising a second microfluidic device for sorting the electroporated cells exiting the electroporation chamber, the second microfluidic device comprising: an inlet mechanism for introducing a fourth solution comprising the electroporated cells, the inlet mechanism being in fluid communication with the first outlet, a microfluidic chamber in fluid communication with the inlet mechanism, the microfluidic chamber comprising a plurality of rows of posts, each row comprising a plurality of posts arranged along a line diagonally oriented relative to the microfluidic chamber, and at least a third outlet mechanism and a fourth outlet mechanism.The device of any one of claims 23 to 36, wherein when there is more than one electroporation chamber, the electroporation chambers are connected in series.A kit for cell transfection, comprising: the device of any one of claims 23 to 37 and optionally reagents selected from the group consisting of cells for transfection, an electroporation medium, and a combination thereof.The method of any one of claims 1 to 22, wherein both the porosity of the top electrode allows passage of the cells and the charge from the first inlet through the top electrode into the electroporation chamber and the porosity of the bottom electrode allows passage of the electroporated cells through the bottom electrode and the first outlet from the electroporation chamber.The device of any one of claims 23 to 37, wherein both the porosity of the top electrode is configured to allow passage of the cells and charge from the first inlet through the top electrode into the electroporation chamber and the porosity of the bottom electrode is configured to allow passage of the electroporated cells through the bottom electrode and the first outlet from the electroporation chamber.A method for electroporating cells having a charge, comprising: flushing the cells having the charge into an electroporation chamber, the electroporation chamber comprising: an upper electrode, a lower electrode, and a path defined between the upper electrode and the lower electrode, along which the cells and the charge can flow, a first inlet that allows passage of cells into the electroporation chamber, and a first outlet that allows passage of electroporated cells from the electroporation chamber, wherein the upper electrode and the lower electrode each have a porosity, wherein the upper electrode is bounded by a first material, wherein the lower electrode is bounded by a second material, wherein the first inlet and the first outlet are present in the first material or the first inlet and the first outlet are present in the second material, wherein the first inlet and the first outlet are spaced by an offset distance, and wherein the cells are suspended in an electroporation medium.An apparatus for electroporating cells with a charge, comprising: one or more electroporation chambers, each electroporation chamber comprising: an upper electrode, a lower electrode, a path defined between the upper electrode and the lower electrode, along which the cells and the charge can flow, a first inlet that allows passage of cells into the electroporation chamber, and a first outlet that allows passage of electroporated cells from the electroporation chamber, wherein the upper electrode and the lower electrode each have a porosity, wherein the upper electrode is bounded by a first material, wherein the lower electrode is bounded by a second material, wherein the first inlet and the first outlet are present in the first material or the first inlet and the first outlet are present in the second material, and wherein the first inlet and the first outlet are spaced apart by an offset distance.

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