JP7817391B2 - Electroporation device and method for cell transfection - Google Patents

Description

Detailed Description of the Invention

[Technical Field]
CROSS-REFERENCE TO RELATED APPLICATIONS This application is a PCT international patent application claiming the benefit and priority of U.S. Patent Application No. 17/513,007, filed October 28, 2021, which is a continuation-in-part of U.S. Patent Application No. 16/923,606, filed July 8, 2020, which is a continuation-in-part of U.S. Patent Application No. 16/506,190, filed July 9, 2019, which claims the benefit of U.S. Provisional Patent Application No. 62/695,436, filed July 9, 2018. The entire disclosure of each of the above-cited applications is incorporated herein by reference.

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

[Background technology]
The background discussion includes information that may be useful in understanding the methods and techniques presented herein. No admission is made that any of the information provided herein is prior art or related to the subject matter presented herein, or that any publication referenced expressly or implicitly is prior art.

Transfection can be used to introduce nucleic acids into cells and create genetically modified cells. A variety of physical, chemical, and viral methods exist for transfecting cells, including photoperforation, polymer-based methods using calcium phosphate, microinjection, electroporation, viral transduction, and lipid-mediated methods (e.g., using liposome-DNA complexes).

Electroporation involves the application of controlled direct current (DC) electrical pulses of relatively short duration to cells. The electrical pulse is thought to induce a membrane potential that causes reversible disassembly of the ordered structure of the cell membrane, resulting in the formation of pores in the membrane. Molecules of interest can then enter the cell through the pores until they close, typically within milliseconds to seconds. Pore formation can be controlled by adjusting various parameters, particularly the gap width (e.g., the distance between the parallel electrode plates), the shape of the electrical pulse wave, the electric field strength, the temperature, and the electrical pulse length.

While electroporation is commonly used at the laboratory scale (e.g., using small cuvettes with a capacity of approximately 0.5 mL), such laboratory-based techniques are not suitable for large-scale clinical production due to their reduced efficiency and high cost. Other transfection methods, such as lipid-based techniques using Lipofectamine 2000™, are also frequently used but suffer from similar drawbacks due to their high cost. In addition, lipid-based methods do not work well for some cell types of clinical interest, such as NK cells (haNK). NK cells have been transfected using mRNA and electroporation to express chimeric antigen receptors (CAR-Leuk Res. (2009) 33:1255-9) or autocrine growth-stimulating cytokines (Cytotherapy (2008) 10:265-74), for example, by genetically engineering primary NK cells. While commercial products such as Maxcyte exist for large-scale transfection (e.g., reaction sizes greater than 1 mL), these products are also expensive and/or lack high-throughput capabilities. Similarly, these commercial products are not optimal for transfecting NK cells and often result in problems with low yields and/or low cell viability. Typical electroporation rates can result in DNA transfection efficiencies of 2-5%, along with cell viability of approximately 10-20%.

In a typical technique, electroporation is performed using two parallel-plate electrodes, as shown in Figure 1A. In this configuration, cells typically flow between the two parallel plates. However, the electric field is non-uniform; cells in the center of the chamber, shown in (a), experience a more uniform electric field, while cells near the electrodes, shown in (b), experience a non-uniform electric field, particularly field action potentials that arise at the electrode boundaries. Another technique utilizes parallel plates of micromesh electrodes, as shown in FIG. 1B (Selmeczi, D. et al., "Efficient large volume electroporation of dendritic cells through micrometer scale manipulation of flow in a disposable polymer chip," Biomed Microdevices, 13:383-392 (2011)), where cells pass through upper and lower micromesh electrodes. While this technique provides a more uniform electric field than passing between two parallel electrode plates, as shown by the similar trajectories of cells in the center of the chamber (a) and near the chamber border (b), the width of the chamber or the distance between the upper and lower micromesh electrodes is limited, resulting in lower transfection efficiency. The configuration shown in Figures 1A and 1B utilizes a field strength of approximately 1-1.3 kV/cm (e.g., 200 V/2 mm at the parallel plates and 40 V/400 μm at the parallel micromesh electrodes).

In another aspect, single-cell electroporation chips are used for transfection. In this technique, a silicon chip may contain multiple linear channels, each with a counter electrode positioned on the opposite side of the channel. Cells pass through the channels and are exposed to an electric field. With this type of technique, a DNA transfection efficiency of approximately 68% has been achieved, along with a cell viability of approximately 79%.

[Summary of the Invention]
[Problem to be solved by the invention]
Although a variety of transfection systems and methods exist for mammalian cells, such techniques suffer from one or more disadvantages. Thus, there remains a need to provide improved electroporation systems and methods, particularly for high-throughput, large-scale manufacturing processes.

[Means for solving the problems]
The technology presented herein is directed to various devices, systems, and methods for electroporation of cells, e.g., mammalian and non-mammalian cells, e.g., for large-scale continuous manufacturing processes. In particular, systems and methods for electroporation of cells using an electroporation apparatus including a first input separated by an offset distance from a first output can provide high efficiency and viability.

In some aspects, methods for electroporating cells with a cargo are provided. The methods include flowing the cells along with the cargo into an electroporation chamber. The electroporation chamber includes a first electrode, a second electrode, a first input that allows passage of the cells and cargo into the electroporation chamber, a first output that allows passage of the electroporated cells from the electroporation chamber, and a fluid channel region having a path defined therein between the first and second electrodes for the flow of the cells and cargo. Further, the cells are suspended in electroporation medium and may have a cell density of about 1 x ¹⁰ cells/ml to about 500 x ¹⁰ cells/ml. The method further includes applying a plurality of electric pulses to the cells in the electroporation chamber, wherein (i) each electric pulse has an exponential ejection waveform or a square waveform, (ii) the plurality of electric pulses are applied at a field strength of about 0.3 kV/cm to about 3 kV/cm; (iii) the field strength is applied at a voltage of about 15 V to about 100 V; (iv) the duration between each electric pulse is about 0.1 seconds to about 10 seconds; and (v) each electric pulse has a pulse width of about 10 μs to about 10,000 μs.

In another aspect, an apparatus for electroporating a cargo into cells is provided. The apparatus includes one or more electroporation chambers. Each electroporation chamber includes a first electrode, a second electrode, a first input that allows passage of cells and cargo into the electroporation chamber, a first output that allows passage of electroporated cells from the electroporation chamber, and a fluid channel region having a defined path therein between the first and second electrodes for the flow of cells and cargo.

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

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 numerals represent elements.

BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
BRIEF DESCRIPTION OF THE DRAWINGS

1A-1B show a typical electroporation device that includes parallel plate electrodes (as shown in FIG. 1A) and a micromesh electrode with no offset between the input and output (as shown in FIG. 1B).
2A-2B show aspects of an apparatus for electroporation according to some embodiments provided herein.
FIG. 2C illustrates aspects of a first electrode and a second electrode according to some embodiments provided herein.
FIG. 2D is a block diagram of an apparatus for generating and providing electrical pulses to cells within an electroporation device as described herein, according to some embodiments provided herein.
FIG. 2E is a block diagram of a further apparatus for generating and providing electrical pulses to cells within an electroporation device as described herein according to some embodiments provided herein.
3A-3D show aspects of an apparatus for electroporation, including an electroporation device, according to some embodiments provided herein. FIG. 3A shows a diagram of the structure of an input-output chamber offset electroporation device. FIG. 3B shows a diagram of the electric field of an input-output chamber offset electroporation device. FIG. 3C shows electric field strength as a function of cell migration distance through an input-output chamber offset electroporation device. FIG. 3D shows an alternative configuration of an electroporation device according to some embodiments provided herein.
3E-3G show aspects of pathways within an electroporation chamber according to some embodiments provided herein.
3H-3J show alternative configurations of an electroporation device according to some embodiments provided herein.
3K-3L illustrate example microfluidic chambers for sorting cells, according to some embodiments provided herein.
3M-3S are drawings illustrating various aspects of post geometry and positioning within a microfluidic chamber according to some embodiments provided herein.
FIG. 3T illustrates an example microfluidic chamber for changing buffer solutions, according to some embodiments provided herein.
FIG. 3U illustrates an example microfluidic chamber having two separate chambers, one for cell sorting and another for buffer change, according to some embodiments provided herein.
3V-3W show alternative configurations of an electroporation device according to some embodiments provided herein.
FIG. 3X illustrates a modular arrangement of an electroporation system including an electroporation device according to some embodiments provided herein.
FIG. 4 shows a table of parameters associated with a chamber offset electroporation apparatus, according to some embodiments provided herein.
FIG. 5 shows examples of fluid flow waveforms (pumps) and electrical waveforms (stimuli) that may be applied to cells flowing through a chamber offset electroporation device, according to some embodiments provided herein.
FIG. 6 illustrates various examples of micromeshes formed from different materials suitable for use in a chamber offset electroporation apparatus, according to some embodiments provided herein.
7A-7E show the results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein.
8A-8D show further results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein.
9A-9D show the results of electroporation experiments on EC7 cells and corresponding parameters using the methods and devices described herein.
10A-10E show further results of electroporation experiments on EC7 cells and corresponding parameters using the methods and devices described herein.
11A-11C show microscopic images of electroporated EC7 cells.
12A-12B show further results of transfected EC7 cells using the methods and devices described herein.
13A-13D show the various transfection efficiencies in different cell lines using the methods and devices described herein.
14A-14E show the results of transfection experiments using PbAE as described herein to introduce mRNA into T cells.
15A-15B show the results of transfection experiments to introduce mRNA into T cells in the presence and absence of electroporation, as described herein.
16A-16D show the results of transfection experiments in adipose-derived mesenchymal stem cells (AD-MSCs) and corresponding parameters using the methods and devices described herein.
FIG. 17 is a block diagram of an electroporation device according to another example embodiment of the present disclosure.
18 is an exploded view of an electroporation channel that can be used in the electroporation device of FIG. 17, according to another example embodiment of the present disclosure.
FIG. 19 is an isometric view of the electroporation chamber of FIG. 18 with the layers assembled together.
20 is a front view of the fluid channel layer of the electroporation chamber of FIG. 18.
21 is a front view of a fluid channel layer having a different fluid volume than the fluid channel layer of FIG. 20, according to another example embodiment of the present disclosure.
FIG. 22 is a front view of a fluid channel layer having straight fluid channels according to another example embodiment of the present disclosure.
FIG. 23 is a front view of the fluid channel layer of FIG. 20 with example dimensions of the fluid channels.

[Mode for Carrying Out the Invention]
Systems and methods for electroporation of cells, e.g., mammalian cells (e.g., NK cells, EC-7 cells, T cells, etc.) and non-mammalian cells, are provided along with corresponding electroporation protocols that provide high efficiency and viability. In some aspects, the electroporation protocols include subjecting the cells to multiple electric pulses, graded fluid flow, electroporation buffers of low conductivity and low osmolality, relatively moderate volumes, and/or relatively moderate time constants.

In various aspects, a device for electroporating cargo into mammalian cells is provided. The device may include one or more electroporation chambers, where each electroporation chamber includes a first electrode, a second electrode, and a fluid channel region having a pathway defined therein between the first and second electrodes. Each of the first and second electrodes may be a solid electrode or a mesh electrode, e.g., a micromesh electrode. The device also includes a first input that allows passage of cells and cargo into the electroporation chamber, and a first output that allows passage of electroporated cells from the electroporation chamber. As used herein, "electroporated cells" includes transfected cells, dead cells, and non-transfected cells. In some embodiments, the first input and first output may be separated by an offset distance. The first electrode may be surrounded by a first material, and the second electrode may be surrounded by a second material, which may be the same or different.

For example, FIG. 2A illustrates an exemplary apparatus for electroporating cargo into cells, including an electroporation apparatus 700a. Electroporation apparatus 700a includes an electroporation chamber 720 including two electrodes, e.g., a first electrode 740(1) and a second electrode 740(2), shown here in line with one another. Electroporation apparatus 700a further includes a first input 710 that allows passage of cells and cargo into electroporation chamber 720 and a first output 730 that allows passage of electroporated cells from electroporation chamber 720. First input 710 and first output 730 may be separated by an offset distance (d) to facilitate lateral flow of cells through electroporation chamber 720. A fluid channel region 725 resides within the electroporation chamber 720, and the fluid channel region 725 includes a pathway 735 defined therein between a first electrode 740(1) and a second electrode 740(2) for the flow of cells and cargo. Although not shown, the pathway 735 may have a pathway inlet corresponding to a first input for allowing the passage of cells and cargo into the pathway 735, and a pathway outlet corresponding to a first output 730 for allowing the passage of electroporated cells from the pathway 735.

In any embodiment, the first input may be present or defined on the first electrode, the second electrode, or the fluid channel region. Additionally, the first output may be present or defined on the first electrode, the second electrode, or the fluid channel region. Both the first input and the first output may be present on either the first electrode or the second electrode. For example, as shown in FIG. 2A, both the first input 710 and the first output 730 are present on the second electrode 740(2). Alternatively, the first input may be present on the first electrode and the first output may be present on the second electrode, or the first input may be present on the second electrode and the first output may be present on the first electrode. It is contemplated herein that in the case of a fluid channel region, the first input, the first output, or both may be present on, for example, a sidewall of the fluid channel region.

As further illustrated in FIG. 2B , within electroporation device 700b, the upper surface 706 of first electrode 740(1) may be surrounded by or connected to a first material 715(1), and the lower surface 711 of second electrode 740(2) may be surrounded by or connected to a second material 715(2). For example, the first and second materials may be non-porous materials such as polydimethylsiloxane (PDMS), acrylic, polyethylene, polypropylene, or acrylic glass to contain cells within the interior chamber of the electroporation device, particularly within the electroporation chamber between the parallel first and second electrodes. Additionally or alternatively, electroporation device 700b may further include a first external input 750 to further enable passage of cells and cargo into electroporation chamber 720, and a first external output 760 to further enable passage of electroporated cells from electroporation chamber 720. The first external input can be present or defined in a first material, and the first external output can be present or defined in a second material. Alternatively, the first external input can be present or defined in a second material, and the first external output can be present or defined in a first material. In further embodiments, both the first external input and the first external output can be present or defined in a first material or a second material. For example, as shown in FIG. 2B, both the first external input 750 and the first external output 760 are present or defined in a second material 715(2), with arrows indicating the flow direction of cells and cargo through the electroporation device and electroporated cells exiting the electroporation device. Other openings or holes shown in FIGS. 2A and 2B, but without numbers, represent openings for connecting various components of the electroporation device, for example, via screws and/or plugs, as known by those skilled in the art.

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

In any embodiment, any one of the electroporation devices disclosed herein may include components for providing an electrical pulse to cells during an electroporation process involving the electroporation parameters described herein. For example, FIG. 2D illustrates an apparatus 852 for generating and providing an electrical pulse to cells as described herein. Specifically, as shown in FIG. 2D, apparatus 852 includes a power supply 854 and control circuitry 858 coupled to power supply 854. Power supply 854 provides the electrical pulse to electroporation device 860 (e.g., any one of the example electroporation devices disclosed herein).

2D , the power supply 854 includes a power source 856, a power converter 862 coupled to the power source 856, and a pulse circuit 864 coupled between the power converter 862 and the electroporation device 860. The power converter 862 may include DC-DC power conversion circuitry and/or AC-DC power conversion circuitry, depending on, for example, the type of power provided by the power source 856. For example, the power converter 862 may receive DC power from the power source 856 (e.g., one or more batteries) and output a regulated DC voltage to the pulse circuit 864. In such an example, the power converter 862 may include a DC-DC power conversion circuitry having any suitable conversion topology (e.g., buck, boost, etc.). In other examples, the power converter 862 may receive AC power from the power source 856 (e.g., AC mains). In such an example, the power converter 862 may include an AC-DC power conversion circuitry having any suitable conversion topology (e.g., AC-DC rectifier, PFC boost, etc.).

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

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

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

In some examples, control circuitry 858 may receive one or more additional signals for controlling power conversion device 862 and/or pulse generator 868. For example, and as shown in FIG. 2D , pulse generator 868 within control circuitry 858 may optionally receive one or more signals 878a. Signal 878a may be a feedback signal from pulse circuitry 864, electroporation device 860, etc., and may be used to control pulse circuitry 864 to generate electrical pulses. Additionally, and as shown in FIG. 2D , controller 866 within control circuitry 858 may optionally receive one or more signals 880 representing feedback signals from pulse circuitry 864, electroporation device 860, etc., and/or one or more signals 882 representing user-defined commands from a user interface. In such examples, signals 880 and/or 882 may be used to control pulse circuitry 864 and/or power conversion device 862, monitor pulse timing, etc. In some examples, the device 852 may include one or more detectors (not shown) for monitoring tip voltage and/or tip current associated with the electroporation device 860. In such examples, the pulse generator 868 and/or the controller 866 may receive the sensed tip voltage and/or the sensed tip current via signals 878a, 880 and then control the pulse circuitry 864 and/or the power conversion device 862 based on the sensed tip voltage and/or current.

The control circuitry 858 may also generate one or more additional signals for control purposes. For example, the controller 866 may optionally generate one or more signals 884 to other components of the electroporation apparatus and/or components external to the electroporation apparatus. In such examples, the signals 884 may represent user feedback provided to a user interface, control signals for controlling motors (e.g., stepper motors) of liquid pumps, peristaltic pumps, pinch valves within the apparatus, etc. Additionally, the pulse circuitry 864 may optionally generate one or more signals 878b to other components of the electroporation apparatus and/or components external to the electroporation apparatus. For example, the pulse circuitry may generate signal 878b to a detector to instruct the detector to begin monitoring voltage and current.

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

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

The control circuits disclosed herein may include hardware components and/or programmed software components for performing various functions, including those disclosed herein. For example, either one of the control circuits 858, 890 of FIGS. 2D and 2E may include one or more sensors for monitoring parameters of the power supplies 854, 888 (as described herein), parameters associated with the electroporation device 860, etc. The parameters may include electrical parameters associated with the power supplies 854, 888 and/or the electroporation device 860, fluid parameters associated with the electroporation device 860, etc. In such examples, the control circuits 858, 890 may control the power supplies 854, 888 and/or other components of the devices 852, 886 (e.g., liquid pumps, etc.) based on the parameters. It is contemplated herein that any one of the power supplies 854, 888 and any one of the control circuits 858, 890 described herein can provide electrical pulses and electroporation parameters as further described below, such as waveform, field strength, voltage, duration between each electrical pulse, pulse width, duration of each electrical pulse, etc. For example, any one of the control circuits 858, 890 can provide one or more of the following: (i) each electrical pulse has the form of an exponential ejection waveform or a square waveform; (ii) the multiple electrical pulses are applied at a field strength of about 0.3 kV/cm to about 3 kV/cm; (iii) the field strength is applied at a voltage of about 15 V to about 100 V; (iv) the duration between each electrical pulse is about 0.1 seconds to about 10 seconds; and (v) each electrical pulse has a pulse width of about 10 μs to about 10,000 μs.

In some embodiments, the first electrode may be an upper micromesh electrode, and the second electrode may be a lower micromesh electrode. In such embodiments, the electroporation chamber may include an upper micromesh electrode, a lower micromesh electrode, and a defined pathway between the upper and lower micromesh electrodes for cell and cargo flow. Each of the upper and lower micromesh electrodes has a porosity, e.g., to allow passage of cells through the micromesh electrodes either into or out of the electroporation chamber. For example, each of the upper and lower micromesh electrodes may have a porosity of about 30% to about 50% open area. The width of the pore openings in the upper and lower micromesh electrodes may be about 70 μm to about 140 μm. The upper micromesh electrode may be surrounded by a first material, and the lower micromesh electrode may be surrounded by a second material. The first material includes a first input that allows passage of cells into the electroporation chamber, and the second material includes a first output that allows passage of electroporated cells from the electroporation chamber. Additionally or alternatively, rather than cells and/or cargo migrating through the pores of the upper micromesh electrode and entering the electroporation chamber, the upper micromesh electrode may optionally include a second input for allowing passage of cells and/or cargo into the electroporation chamber. The second input may be substantially aligned with the first input in the first material. Additionally or alternatively, rather than electroporated cells migrating through the pores of the lower micromesh electrode and exiting the electroporation chamber, the lower micromesh electrode may include a second output for allowing passage of electroporated cells out of the electroporation chamber. The second output may be substantially aligned with the first output in the second material.

For example, FIG. 3A illustrates an exemplary device for electroporating cargo into cells, including an IOCO electroporation device 200 that includes micromesh electrodes. The IOCO electroporation device 200 includes two micromesh electrodes, shown here aligned with one another, upper micromesh electrode 240(1) and lower micromesh electrode 240(2). The upper surface of upper micromesh electrode 240(1) may be surrounded by a first material, and the lower surface of lower micromesh electrode 240(2) may be surrounded by a second material. For example, the first and second materials may be non-porous materials, such as polydimethylsiloxane (PDMS), acrylic, polyethylene, polypropylene, etc., to contain cells within the interior chamber of the electroporation device, particularly within chamber 220 between the two parallel micromesh electrodes. The first input 210 is formed on the upper micromesh electrode 240(1) (area without PDMS), and the first output 230 is formed on the lower micromesh electrode 240(2) (area without PDMS). Thus, in this example, the upper micromesh electrode 240(1) and the lower micromesh electrode 240(2) span the length of the electroporation chamber 220, surrounded by the first material (upper PDMS layer) and the second material (lower PDMS layer), to direct the flow of cells along the lateral distance of the chamber. The opening (first input 210) in the first material (upper PDMS layer) and the opening (first output 230) in the second material (lower PDMS layer) are separated by an offset distance (d) to facilitate the flow of cells through the electroporation chamber 220 laterally.

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

In any embodiment, the electroporation chamber width (w) may be in the range of about 0.01 mm to about 15 mm, 0.01 mm to about 10 mm, 0.01 mm to about 7.5 mm, 0.01 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.1 mm to about 15 mm, 0.1 mm to about 10 mm, 0.1 mm to about 7.5 mm, about 0.1 mm to about 5 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 7.5 mm, about 1 mm to about 5 mm, about 2 mm to about 10 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 embodiments, the chamber width (w) is about 0.3 mm or about 4 mm.

In some embodiments, the offset distance (d) between the first input and the first output may range from 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, or from about 1 cm to about 3 cm. In some embodiments, the offset distance (d) is about 2 cm. In some embodiments, there is no overlap between the input and the output.

The first input and/or the second input are in fluid communication with the electroporation chamber, and the electroporation chamber is in fluid communication with the first output and/or the second output. The input, output, and electroporation chamber may additionally include one or more valves, regulators, pumps, or any other microfluidic components to control the flow of cells through the electroporation chamber.

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

In some embodiments, cells may enter the first input and flow in a first direction. Once past the first or second 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. Once at the first output, the flow of cells may change direction again and exit the electroporation chamber in a second direction parallel to the first direction.

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

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

In any embodiment, the 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 100 mm, about 5 mm to about 80 mm, about 5 mm to about 60 mm, about 5 mm to about 40 mm, about 5 mm to about 20 mm, about 5 mm to about 15 mm, about 8 mm to about 12 mm, or may be about 10 mm.

In any embodiment, the height (h) of the electroporation chamber may range from about 100 μm to about 1000 μm, about 100 μm to about 750 μm, about 100 μm to about 500 μm, about 100 μm to about 300 μm, about 100 μm to about 200 μm, about 200 μm to about 1000 μm, about 200 μm to about 750 μm, about 200 μm to about 500 μm, or about 200 μm to about 300 μm. In some aspects, the chamber height (h) is about 284 μm.

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

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

Figure 3C is a plot of electric field strength versus cell migration distance through the IOCO electroporation device 200. Cells at the input (first input 210), designated position (1), are not exposed to an electric field until they pass through the micromesh electrode and enter the electroporation chamber. Within the electroporation chamber, designated position (2), the cells experience a uniform (maximum strength) electric field while migrating along the length of the chamber. Once they enter the output (first output 230), designated position (3), the cells are not exposed to an electric field.

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

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

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

In any embodiment, the electroporation devices described herein may include a means for generating a flow of cells from a sample tube into the electroporation chamber of the electroporation device, through the device, and into a collection tube. For example, as illustrated in FIG. 3H, a flow generating means 520 is in fluid communication, e.g., via tubing, with a sample tube 510 containing cells and/or cargo to be transfected and an electroporation device 530. The flow generating means 520 may generate a flow of cells and/or cargo from the sample tube 510, through the tubing, through the electroporation device 530, and into a collection tube 550. Although not shown, an air filter device may be in fluid communication with the contents of the sample tube 510, the collection tube 550, or both. The flow generating means may be any suitable pump, e.g., a peristaltic pump or a syringe pump. It is contemplated herein that more than one flow generating means 520 may be present in an electroporation device. For example, one or more flow generating means 520 may be present in fluid communication between the sample tube 510 and the electroporation device 530, and one or more flow generating means 520 may be present in fluid communication between the collection tube 550 and the electroporation device 530. Additionally or alternatively, the electroporation device 530 and/or the flow generating means 520 may also interface with a controller 540, which may include, for example, an electroporator pulser.

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

In any embodiment, the electroporation apparatus described herein may also include a first microfluidic device for sorting cells before they enter the electroporation chamber of the electroporation apparatus. For example, as illustrated in FIG. 3J, a first microfluidic device 580 may be in fluid communication with the electroporation apparatus 530, a sample tube 510 containing cells to be transfected, and, optionally, a buffer sorting tube 577. A flow generating means 520 may generate a flow of cells from the sample tube 510 and/or buffer from the sorting tube 577 through the electroporation apparatus 530 and into a collection tube 550. The first microfluidic device 580 may be capable of pre-sorting cells from the sample tube 510 to the electroporation apparatus 530, e.g., based on cell size. Additionally or alternatively, the first microfluidic device 580 may be capable of effecting a buffer exchange of the cells from the sample tube 510. Stream 590 contains cells that are sorted out and not delivered to electroporation device 530 and can either be a waste stream or can be delivered to another electroporation device using different parameters. Additionally or alternatively, a second microfluidic device 585 can be in fluid communication with electroporation device 530 and collection tube 550. Second microfluidic device 585 is capable of sorting electroporated cells, for example, based on the size of the electroporated cells, prior to collection in collection tube 550. Stream 595 contains electroporated cells that are sorted out and not delivered to collection tube 550.

Although two microfluidic devices are shown in FIG. 3J, it is contemplated herein that only one microfluidic device may be present in the electroporation apparatus, for example, to sort cells before they enter the electroporation chamber or to sort electroporated cells exiting the electroporation chamber.

FIG. 3K is a diagram of an example microfluidic device (e.g., microfluidic device 580, microfluidic device 585). In this example, a solution containing cells 100 (e.g., cells to be transfected, electroporated cells, transfected cells) enters chamber 105 at input mechanism 110. Cells are depicted as circles. The cells pass through chamber 105, which contains a matrix of posts 115, shown here as rectangular structures distributed along multiple sloped lines. As the cells pass between the diagonally oriented post rows in the chamber, they are deflected laterally toward second output mechanism 122. A side view of a portion of the chamber is shown in FIG. 3L, where the chamber has a floor 160 and, optionally, a ceiling 150, which may be made of the same or a different material as posts 115. Cells (black circles) are shown flowing through the matrix of posts. It is understood that the post rows may also be arranged in a curved fashion, resulting in cells being directed toward the sides of the chamber.

In this example, two output mechanisms are shown for the chamber. The solution exiting the chamber via the first output mechanism 120 (which may also be referred to as the third output mechanism when more than one microfluidic device is present) is depleted of cells, while the solution exiting the chamber via the second output mechanism 122 (which may also be referred to as the fourth output mechanism when more than one microfluidic device is present) is enriched in cells. In any embodiment, the second output mechanism may be in fluid communication with the electroporation chamber, e.g., the first input. Depletion refers to a condition in which the concentration of cells in the solution exiting the chamber via output mechanism 120 is 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 input mechanism 110. Enrichment or concentration refers to a condition in which the concentration of cells in the solution exiting the chamber via output mechanism 122 is increased by 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more compared to the concentration of cells in the solution entering the chamber at input mechanism 110. For cells to be directed to output mechanism 122, deflection point 130 of the chamber must be positioned so that any cells present are directed to output mechanism 122 (and not output mechanism 120). Generally, output mechanism 122 is configured to have a larger cross-sectional area than output mechanism 120. Generally, the output mechanism has a cross-sectional area large enough for fluid flow at the flow rates described herein to avoid creating high pressures that may damage the cells or the device.

In some embodiments, the width of output mechanism 120 is greater than the width of output mechanism 122. For example, the width of output mechanism 120 is 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, or 9.5 times the width of output mechanism 122. In other embodiments, the total cross-sectional area of the output mechanisms may be greater than or equal to the total cross-sectional area of the input mechanisms.

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

In general, cells may flow through a microfluidic device serially (one at a time in a particular pathway) or in multiples (multiple cells flowing in multiple pathways).

Figure 3M is a drawing of a top-down view of a cross section of chamber 105, where each cross section contains two rows of seven posts. Cell flow is shown along a path (p). This drawing shows the positioning of posts 115 within the cross section of the chamber of the microfluidic device. Generally, the posts are aligned along a line having a slope as defined by an angle (θ) at a spatial interval (dx), which may be fixed or variable, provided that the variation does not result in cells avoiding 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 in between. The width of each path (p) controls the characteristics of the flow line. For example, any number of paths may be utilized depending on the width of the chamber, such as 1, 2, 4, 8, 12, 16, 32, etc., or any range therebetween. In other embodiments, the flow rate of the 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, etc. The chamber is preferably rectangular, but may also be circular, semicircular, V-shaped, or any other suitable shape.

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

Typically, multiple rows of posts are present within the chamber, each row having the same or substantially the same slope as defined by the angle (θ). When cells 100 enter the chamber 105 of the microfluidic device, they flow laterally toward the side of the chamber 105, following multiple paths (p) between the posts 115 until they reach the side of the chamber. When concentrating cells, the cells generally do not traverse the rows of posts; rather, the cells typically travel along specific paths (p). The cells then exit the microfluidic chamber via the output mechanism 122. In other embodiments, the posts may have curved components along the length of the chamber 105.

As used herein, the term "post" refers to a structure within a chamber having associated planar dimensions, out-of-plane dimensions, rotation angle, and shape. In-plane dimensions may refer to the length (l) and width (w) of the post, while out-of-plane dimensions may refer to the height (h) of the post. The tilt angle or rotation angle (Φ) refers to the rotation of the post relative to the chamber.

Shape refers to the three-dimensional characterization of the post, such as cylindrical, conical, pyramidal, cubic, or cuboid. Generally, the post is positioned so that its axis (out-of-plane dimension) is perpendicular to the surface to which it is attached. In some embodiments, all posts within the chamber have the same dimensions. In other embodiments, the dimensions of the posts vary as a function of space depending on their position, for example, as measured along the height (h) of the post. In general, the posts may be any shape and are not limited to the specific geometric shapes presented herein. The post shape may be arbitrary, as shown in FIG. 3R, which is represented by an axial length (l) and width (w) as well as multiple radii of curvature (r4-r12) located at, for example, repeating regular intervals or repeating variable intervals along the axial length. Several different shapes may be described by the multiple radii. Additionally, with reference to FIGS. 3R and 3S, one or more sides of the post may be rectilinear. Thus, the post may have any suitable shape consisting of curves and/or straight lines.

In some embodiments, the posts are arranged at intervals (dx) along a gradient line, where the intervals are regularly spaced or fixed. For example, the posts may be positioned, e.g., 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, or 100 μm (including any value between these ranges), depending on the size of the cells to be enriched.

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

In some embodiments, the spacing can be selected to be large enough to allow for enrichment of certain types of cells while allowing smaller cells to exit via the output mechanism 120. For example, a blood sample may contain several different cell types that are relatively small in size, such as red blood cells, neutrophils (e.g., 12-14 μm in diameter), eosinophils (e.g., 12-17 μm in diameter), basophils (e.g., 14-16 μm in diameter), lymphocytes (e.g., 10-14 μm in diameter), and monocytes (e.g., 20 μm in diameter). Other types of cells in the human body are much larger, e.g., in the 40-100 μm range or larger in diameter. In such cases, the posts may be spaced apart to allow red blood cells and white blood cells to pass through while enriching larger cells (e.g., normal cells, tumor cells, etc.). The placement (spacing) of the posts is determined based on the type of cell being isolated. Different sheath flow-to-sample flow ratios can affect size-based sorting performance.

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

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

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

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

Additional features include the post rotation angle (Φ), post height or out-of-plane dimension, row-to-row spacing (dy), and row offset (xo), which are described in more detail in Figures 3N-3Q and throughout this application. As used herein, the term "rotation angle" refers to the degree of rotation of the post relative to its position within the chamber. In some embodiments, the post has a length greater than its width. In this embodiment, a rotation angle of 0 means that the post's length is aligned with an axis perpendicular to the side of the chamber. To describe different rotation angles, the post may be rotated in a clockwise or counterclockwise direction. By way of example, the post in Figures 3M-3Q is rotated approximately 35° in a counterclockwise motion to reach the post geometry shown in these figures.

Referring to FIG. 3N, each post has a height (h) or out-of-plane dimension associated with the post being sufficiently tall to prevent cells from escaping pathway (p) by flowing up the pathway (e.g., over the top of the post into a different pathway). For example, in some embodiments, the post will span the height of the microfluidic chamber, such as being in contact with the chamber floor 160 and the chamber ceiling 150. In other embodiments, the height or out-of-plane dimension of the post may span a portion of the entire height of the microfluidic device. For example, the post may have a height of 1 μm, 3 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 85 μm, 90 μm, 95 μm, 100 μm, etc., or any value therebetween.

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

As previously shown, the posts are distributed along a line with a negative slope. By directing the cells along this line, cells flowing through path (p) are directed laterally toward the side of the chamber, while the solution in which the cells were initially suspended flows in a manner (e.g., approximately horizontally or horizontally) and can exit the microfluidic chamber via output mechanism 120.

Referring to FIG. 3P, in yet other embodiments, the rows of posts are spaced apart by a distance (dy). In some embodiments, the posts are arranged along a vertical line at a distance (dy), where the distance is regularly spaced or fixed. For example, the posts may be positioned 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, etc. (including any value therebetween), depending on the size of the cells to be enriched.

In other embodiments, the posts are distributed along a vertical line such that the spacing (dy) is not fixed but instead varies between any two consecutive post rows. For example, a first post row may be positioned at a specific location, a second post row may be positioned 5 μm from the first post row, a third post row may be positioned 4 μm from the second post row, and so on, all aligned with a vertical line. The spacing (dy) may be selected based on the size of the cells to be enriched. Any spacing (dy) is acceptable, provided that the spacing is wide enough to allow solution flow through the chamber without requiring high pressures that could damage the microfluidic device. In some embodiments, consecutive post rows may have a zero offset (xo).

Referring to FIG. 3Q, successive post rows may have an offset (xo) relative to the spacing (dx). For example, row (r) may be defined at a particular location (e.g., using a Cartesian coordinate system x, y). The (r+1)th row may be displaced to the right by an amount (xo). The (r+2)th row may be displaced to the right by an amount (2xo). The (r+3)th row may be displaced to the right by an amount (3xo), and so on, up to the spacing (dx). The offset may be provided in a fixed manner, such that each successive row is displaced in the same direction by a fixed amount. Alternatively, the offset may be provided in a variable manner, such that each successive row is displaced in the same direction by a variable amount (up to the amount (dx)). In other embodiments, the offsets may be applied in alternating directions relative to the columns (r), such that the (r+1)th column has a fixed or variable offset in one direction and the (r+2)th column has a fixed or variable offset in the opposite direction (e.g., one offset to the right and the next offset to the left).

The posts may be made of any suitable material, such as poly(dimethylsiloxane) (PDMS), glass, plastic, elastomer, silicon, etc. PDMS can generally be fabricated with submicron resolution (e.g., features less than 0.1 μm). In some embodiments, the chamber 105 and/or the posts 115 may be fabricated from PDMS. In other embodiments, the chamber may be fabricated from glass and/or silicon and/or plastic, and the posts may be fabricated using PDMS (e.g., the bottom of the chamber may be glass or silicon, and the top of the chamber may be glass or plastic). Lithography techniques known in the art may be used to fabricate the chambers and posts as described herein. Materials with properties similar to PDMS may also be used to construct the microfluidic devices shown herein.

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

Various techniques are available for fabricating microfluidic devices. Microfluidic devices 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)), silicon, and combinations thereof. Other materials are known in the art.

Methods for fabricating chambers and microfluidic devices with posts using the materials referenced 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), silicon micromachining, wet or dry chemical etching, and the like.

Silicon fabrication techniques using photolithography followed by wet (KOH) or dry etching (reactive ion etching with fluorine or other reactive gases) can be used for glass materials. For example, a glass master can be created by conventional photolithography and serve as a master mold in molding techniques to create plastic or PDMS-based devices.

Microfluidic devices may be fabricated in one or more layers joined together, for example, by adhesives, clamps, heat, solvents, etc. Alternatively, microfluidic devices may be fabricated as a single piece, for example, using stereolithography or other three-dimensional fabrication techniques.

In some embodiments, due to the deformability of the cells, the gap distance may be selected to be about 5 μm or less relative to the size (e.g., diameter) of the cells to be enriched. In other embodiments, the gap distance may be selected to be about 5 μm or more relative to the size (e.g., diameter) of the cells to be sorted. For rigid cells or microparticles having the same or similar diameter as the deformable cells, the gap distance may be different (e.g., larger for rigid cells) compared to the gap distance for deformable cells of the same diameter.

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

In this example, a second input mechanism 112 is present in the system. A second solution (e.g., a different buffer solution than the first solution entering the chamber through the first input mechanism 110), such as a buffer solution from buffer tube 577, enters the chamber through the second input mechanism 112. In some embodiments, the first and second solutions do not substantially mix, such that the upper region 155 of the chamber contains the first solution and little, if any, of the second solution, while the lower region 165 of the chamber contains the second solution and little, if any, of the first solution. Thus, when the cells are deflected laterally and exit the chamber through output mechanism 122, the solution exiting the chamber via output mechanism 122 is in the buffer solution of the second solution.

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

A second solution (e.g., a buffer solution) enters the chamber through a second input mechanism (112), distinct from the first solution, which enters the chamber through the first input mechanism 110(1). In some embodiments, the first and second solutions do not substantially mix, such that the upper region (155) of the chamber contains the first solution and little, if any, of the second solution, while the lower region (165) of the chamber contains the second solution and little, if any, of the first solution. The cells (concentrated and present in the buffer solution from the input mechanism 112) exit chamber 105(2) through output mechanism 122(2).

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

Generally, the input mechanism may be actuated by a syringe pump or by manual depression of a syringe. In some embodiments, multiple inputs may use one or more syringe pumps to automatically actuate each input. In other embodiments, multiple inputs may use manual depression of one or more syringes to manually actuate each input.

In any embodiment, electroporation devices capable of providing different electroporation effects are described herein. For example, as illustrated in FIG. 3V, an electroporation device 600 is provided having three electrodes 640(1), 640(2), and 640(3). The electroporation device 600 includes two paths for cell and cargo movement: a first path between electrodes 640(1) and 640(2) and a second path between electrodes 640(2) and 640(3), where arrows indicate cell flow. Cells can enter the electroporation chamber at inlet 620 and exit via outlet 630. While not shown, it is contemplated herein that more than one outlet may be present, e.g., each outlet may collect a different type of cell. Different electrode gap lengths (L1, L2, L3, L4) are present within the electroporation device 600. Different electrode gap lengths and different pathways result in different capacitances; therefore, different electric field strengths and electroporation effects can be achieved in cells moving through each pathway. For example, L2 can be greater than L4, and in such an example, the capacitance (C1) in the first pathway can be greater than the capacitance (C2) in the second pathway (C1 > C2). In any embodiment, cells (of the same or different types) can be pre-sorted and/or selected, for example, using the microfluidic device described above, before entering the first or second pathway.

It is also contemplated herein that the electroporation devices described herein may exist as cartridges that may be in fluid communication (e.g., connected, stacked) with one another to provide a pathway of a desired length and shape. For example, as illustrated in FIG. 3W, each of the cartridges 410 includes an electroporation chamber that includes a first electrode 440(1) and a second electrode 440(2), where the cartridges may be stacked sequentially and the arrows indicate the flow of cells through the cartridges. For example, the outlet of the top cartridge 410 is in fluid communication with the inlet of the middle cartridge 410, and the outlet of the middle cartridge 410 is in fluid communication with the inlet of the bottom cartridge 410.

In a further embodiment, a modular system is provided that includes the electroporation apparatus described herein. For example, as illustrated in FIG. 3X, modular system 800 includes three modules configured to be in fluid communication or connection with one another: first module 805, second module 815, and third module 825. First module 805 includes a first container 807 (e.g., a sample tube) for containing cells and/or cargo to be transfected, and tubing 810 for fluidly connecting first container 807 with second module 815. First module 805 may also, optionally, include an air filter device 809 and tubing 810 for fluidly connecting air filter device 809 to first container 807. The second module 815 includes an electroporation device 820 and tubing 810 for fluidly connecting the electroporation device to the first module 805, e.g., the first container 807, and the second module 825, as well as tubing 810 for fluidly connecting a flow generating means (e.g., a peristaltic pump) as described herein to the electroporation device 820 and the first module 805, e.g., the first container 807. The third module 825 includes a second container 830 (e.g., a collection tube) for collecting electroporated cells, and tubing 810 for fluidly connecting the second container 830 to the second module 815, e.g., the electroporation device 820. The third module 825 may also optionally include an air filter device 809 and tubing 810 for fluidly connecting the air filter device 809 to the second container 830. Optionally, connectors 850, e.g., luer locks, may connect the modules together, e.g., first module 805 with second module 815 and second module 815 with third module 825. It is contemplated herein that each of first module 805, second module 815, and third module 825 may be packaged and sterilized separately.

The electroporation devices described herein have several advantages over mesh-based electroporation devices, such as the configuration shown in FIG. 1B. First, the increased migration distance through the electroporation chamber, as measured from the first input to the first output of the electroporation chamber, does not result in a corresponding increase in the electric field. Thus, cells are exposed to a uniform electric field for a longer period than in a strictly straight channel, resulting in improved electroporation efficiency without a corresponding decrease in viability.

The electroporation chamber width (w) between the upper and lower mesh electrodes may be increased in Figure 1B, resulting in a longer migration distance; however, a larger electroporation chamber width will have a larger impedance, and a higher voltage will need to be applied to generate a suitable electric field. Additionally, a higher voltage is more likely to have a detrimental effect on cell viability. Furthermore, in parallel plate electrode configurations, such as those in Figure 1A, the electric field distribution will be less uniform.

Thus, according to embodiments, the electroporation devices described herein can have shorter electrode pairs due to the offset input/output design, thereby extending the length of the electroporation chamber without increasing the electrode pair distance.

Figure 4 presents a table illustrating differences between the electroporation device as described herein and prior art electroporation devices. Such differences include, but are not limited to, (1) the presence of an input-output offset distance, (2) the use of a low-conductance, low-osmolality electroporation medium instead of commercially available electroporation media such as BTX and RPMI, (3) an exponential ejection waveform applied in a series of electrical pulses, and (4) a step-wise fluid flow scheme.

While the examples in Figures 3A-3C utilize stainless steel micromesh electrodes, the electrodes are not limited to any particular material and may be formed of any suitable material. For example, as shown in Figure 6, suitable materials include silicon and polyimide. While silicon and polyimide may be used to form suitable micromesh electrodes, such materials are typically processed in cleanroom facilities using microfabrication protocols known in the art. In contrast, stainless steel micromesh electrodes are easy to set up and do not require specialized facilities, such as cleanrooms.

It will be appreciated that any suitable material may be used, provided the micromesh has pores that allow cells to pass through (e.g., the micromesh pores are larger than the cells) and the micromesh pores are compatible with the electrode patterning around the pore openings.

Electroporation Methods and Parameters [0013] A method for electroporating a cargo into cells is provided herein, along with an electroporation device, e.g., as described above. The method can include flowing the cells along with the cargo into an electroporation chamber. The electroporation chamber includes a first electrode (e.g., a solid electrode), a second electrode (e.g., a solid electrode), and a fluid channel region having a defined path therein between the first and second electrodes (all as described herein). The device also includes a first input that allows passage of the cells and cargo into the electroporation chamber as described herein, and a first output that allows passage of the electroporated cells from the electroporation chamber as described herein. In some embodiments, the first input and the first output can be separated by an offset distance. The cells can be suspended in an electroporation medium. The first electrode can be surrounded by a first material as described herein, and the second electrode can be surrounded by a second material as described herein, which can be the same or different. The first material and the second material can also include a first external input as described herein and a first external output as described herein.

In some embodiments, the electroporation chamber includes an upper micromesh electrode as described herein, a lower micromesh electrode as described herein, and a defined pathway between the upper and lower micromesh electrodes for cell and cargo flow as described herein. Each of the upper and lower micromesh electrodes has a porosity as described herein. Additionally, the upper micromesh electrode may be surrounded by a first material as described herein. The first material may include a first input as described herein that allows passage of cells into the electroporation chamber. The lower micromesh electrode may be surrounded by a second material. The second material may include a first output as described herein that allows passage of electroporated cells from the electroporation chamber. The first input and first output are separated by an offset distance as described herein. The cells may be suspended in electroporation medium. Optionally, the upper micromesh electrode may further include a second input as described herein, and/or the lower micromesh electrode may further include a second output as described herein.

In any embodiment, the flow of cells and cargo may be performed in a stepwise manner. For example, a flow rate of approximately half or more of the total volume of the electroporation chamber may be pumped into the electroporation chamber at a specified time interval. In some embodiments, a flow rate approximately equal to the total volume of the electroporation chamber may be pumped into the electroporation chamber. Figure 5 shows an example series of electrical pulses (stimuli) and an example of a stepwise fluid flow scheme (pump). For the stepwise fluid flow scheme, the chamber volume is approximately 22 μL. Every second, 11 μL is pumped into the electroporation chamber, replacing half of the flow rate. This may be repeated for as long as necessary to process the desired amount of cells.

Additionally or alternatively, any suitable time interval may be used with any amount suitable for a stepwise fluid flow scheme. For example, the time interval may 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 time therebetween, 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 the volume of the input chamber, one-half the volume of the electroporation chamber, one-quarter the volume of the electroporation chamber, one-eighth the volume of the electroporation chamber, or less. Any suitable flow rate of cells and cargo may be used with the parameters described herein. For example, the flow rate of the cells and cargo in the electroporation medium may be about 0.1 ml/min or more, about 0.5 ml/min or more, about 1 ml/min or more, about 2.5 ml/min or more, about 5 ml/min or more, about 7.5 ml/min or more, about 10 ml/min or more, about 12.5 ml/min or more, or about 15 ml/min or more, or about 0.1 ml/min to about 15 ml/min, about 0.5 ml/min to about 15 ml/min, about 1 ml/min to about 15 ml/min, about 1 ml/min to about 12.5 ml/min, about 1 ml/min to about 10 ml/min, about 1 ml/min to about 7.5 ml/min, about 1 ml/min to about 5 ml/min, or about 1 ml/min to about 2.5 ml/min.

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

For a series of electrical pulses, voltage may be applied to the electrodes every 100 ms to 5000 ms (e.g., every 100 ms, 250 ms, 500 ms, 1000 ms, 2000 ms, 3000 ms, etc.) to generate an electric field of a given strength. This may be repeated for as long as necessary to process the desired amount of cells. Thus, while an embodiment may be referred to as continuous flow, it is understood that continuous flow includes a step-wise fluid flow scheme in which fluid is continuously pumped into the electroporation chamber according to predetermined time intervals.

Additionally or alternatively, any suitable duration between electrical pulses may be used with any suitable duration of electrical pulses to the cells in the electroporation chamber. For example, the duration between each pulse may range from about 0.1 seconds to about 15 seconds, from about 0.1 seconds to about 10 seconds, 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 time period therebetween, including longer intervals. In some embodiments, the duration of the electrical pulse may be in the range of about 10 ms to about 10 s, about 10 ms to about 5 s, about 10 ms to about 1 s, about 10 ms to about 500 ms, about 10 ms to about 250 ms, about 50 ms to about 150 ms, about 75 ms to about 125 ms, about 100 ms to about 10 s, about 100 ms to about 5, about 100 ms to about 1 s, about 100 ms to about 500 ms, about 100 ms to about 250 ms, or any range therebetween.

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

In any embodiment, the voltage is from about 0.1 kV/cm to about 5 kV/cm, from about 0.1 kV/cm to about 3 kV/cm, from about 0.1 kV/cm to about 2 kV/cm, from about 0.1 kV/cm to about 1 kV/cm, from about 0.1 kV/cm to about 0.5 kV/cm, from about 0.3 kV/cm to about 5 kV/cm, from about 0.3 kV/cm to about 3 kV/cm, from about 0.3 kV/cm to about 2 kV/cm, from about 0.3 kV/cm to about 1 kV/cm, or from about 0.3 kV/cm to about 0 Multiple electrical pulses may be applied at voltages suitable to generate a field strength of about 0.5 kV/cm, about 0.5 kV/cm to about 3 kV/cm, about 0.5 kV/cm to about 2 kV/cm, about 0.5 kV/cm to about 1 kV/cm, about 0.8 kV/cm to about 3 kV/cm, about 0.8 kV/cm to about 2 kV/cm, about 0.8 kV/cm to about 1 kV/cm, about 1 kV/cm to about 3 kV/cm, or about 1 kV/cm to about 2 kV/cm. Suitable voltages may be about 10V, about 15V, about 20V, about 30V, about 40V, about 50V, about 75V, about 100V, or about 200V, or about 10V to about 200V, about 10V to about 100V, about 10V to about 75V, about 10V to about 50, about 10V to about 40V, about 10V to about 30V, about 15V to about 200V, about 15V to about 100V, about 15V to about 75V, about 15V to about 50, about 15V to about 40V, about 15V to about 30V, about 20V to about 50, about 20V to about 40V, about 20V to about 30V, about 30V to about 50V, about 30V to about 40V, or any suitable range therein.

Additionally or alternatively, each electrical pulse may be about 10 μs or more, about 50 μs or more, about 75 μs or more, about 100 μs or more, about 250 μs or more, about 500 μs or more, about 750 μs or more, about 1,000 μs or more, about 5,000 μs or more, or about 10,000 μs; or about 10 μs to about 10,000 μs, about 10 μs to about 5,000 μs, about 10 μs to about 1,000 μs, about 10 μs to about 750 μs, about 10 μs to about 50 The pulse width may be 0 μs, about 10 μs to about 250 μs, about 10 μs to about 100 μs, about 10 μs to about 50 μs, about 100 μs to about 1000 μs, about 100 μs to about 750 μs, about 100 μs to about 500 μs, about 100 μs to about 250 μs, about 250 μs to about 1000 μs, about 250 μs to about 750 μs, about 250 μs to about 500 μs, about 500 μs to about 1000 μs, or about 500 μs to about 750 μs.

Any suitable cells, e.g., mammalian cells, non-mammalian cells, or both, may be transfected with a cargo. Examples of suitable mammalian cells include, but are not limited to, NK cells (e.g., haNK cells, primary NK cells, activated (aNK) cells), EC-7 cells (a derivative of HEK293 cells modified to produce adenovirus), T cells (e.g., primary CD4/CD8 T cells), CHO-S cells, dendritic cells, embryonic cells, stem cells (e.g., adipose-derived mesenchymal stem cells (AD-MSCs)), epithelial cells, lymphocytes, macrophages, gametocytes, and fibroblasts. Examples of suitable non-mammalian cells include, but are not limited to, bacterial cells and yeast cells. Any suitable cargo, e.g., nucleic acids, may be used. The nucleic acid may be RNA (e.g., synthetic RNA, mRNA, in vitro transcribed RNA, GFP-mRNA, etc.) and/or DNA (e.g., synthetic DNA, GFP-DNA, plasmid DNA, etc.). In particular, NK cells, EC-7 cells (a derivative of HEK293 cells modified to produce adenovirus), and T cells can be transfected with RNA (e.g., synthetic RNA, mRNA, in vitro transcribed RNA, GFP-mRNA, etc.) and/or DNA (e.g., synthetic DNA, GFP-DNA, plasmid DNA, etc.) using the electroporation device described herein. It is further contemplated herein that the electroporation device described herein can be used to perform in vitro fertilization. For example, eggs and sperm can be passed through an electroporation device such as that described herein to achieve fertilization of the eggs.

In further contemplated aspects, the medium or electroporation buffer in which cells are transfected is a low conductivity and low osmolality medium, optionally containing one or more nutrients. In any embodiment, the electroporation medium has a conductivity of about 0.05 mS/m or more, about 1 mS/m or more, about 5 mS/m or more, about 10 mS/m or more, about 15 mS/m or more, about 25 mS/m or more, about 30 mS/m or more, about 50 mS/m or more, about 100 mS/m or more, about 150 mS/m or more, or about 200 mS/m or more. or greater; or from about 0.05 mS/m to about 200 mS/m; from about 1 mS/m to about 30 mS/m, from about 1 mS/m to about 20 mS/m, from about 1 mS/m to about 10 mS/m, from about 1 mS/m to about 5 mS/m, from about 3 mS/m to about 30 mS/m, from about 5 mS/m to about 20 mS/m, or from about 5 mS/m to about 15 mS/m. Additionally or alternatively, the electroporation medium has a pH of about 50 mOsm/l or more, about 100 mOsm/l or more, about 150 mOsm/l or more, about 200 mOsm/l or more, about 250 mOsm/l or more, about 300 mOsm/l or more, about 350 mOsm/l or more, or about 400 mOsm/l; or about 50 mOsm/l to about 400 mOsm/l, about 50 mOsm/l to about 300 mOsm/l. , about 50 mOsm/l to about 200 mOsm/l, about 100 mOsm/l to about 400 mOsm/l, about 100 mOsm/l to about 300 mOsm/l, about 200 mOsm/l to about 400 mOsm/l, about 200 mOsm/l to about 300 mOsm/l, about 250 mOsm/l to about 400 mOsm/l, or about 250 mOsm/l to about 300 mOsm/l.

In any embodiment, the electroporation medium may include one or more salts, sugars, and buffering agents. Suitable salts include, but are not limited to, metal halide salts, phosphate salts, metal sulfate salts, and combinations thereof. Examples of suitable metal halide salts include, but are not limited to, potassium chloride (KCl), sodium chloride (NaCl), lithium chloride (LiCl), calcium chloride (CaCl ₂ ), chromium chloride (CrCl ₃ ), potassium bromide (KBr), sodium bromide (NaBr), magnesium chloride (MgCl ₂ ), magnesium bromide (MgBr ₂ ), magnesium fluoride (MgF ₂ ), magnesium iodide (MgI ₂ ), lithium bromide (LiBr), potassium iodide (KI), sodium iodide (NaI), and lithium iodide (LiI). Examples of suitable phosphate salts include, but are not limited to, monosodium phosphate ( _NaH2PO4 ), _disodium phosphate ( _Na2HPO4 ), trisodium phosphate ( _Na3PO4 ), monomagnesium phosphate (Mg _{( H2PO4} ) ₂ ), dimagnesium phosphate ( _MgHPO4 ), trimagnesium phosphate ( _Mg3 ( _PO4 ) ₂ , monopotassium phosphate ( _KH2PO4 ), dipotassium phosphate ( _K2HPO4 ), tripotassium phosphate _{( K3PO4} ), monocalcium phosphate (Ca( _{H2PO4 ) 2 )} , dicalcium phosphate ( _CaHPO4 ), tricalcium phosphate ( _Ca3 ( _{PO4 )2 )} , and chromium phosphate ( _CrPO4 ). Examples of suitable metal sulfate salts include, but are not limited to, _sodium sulfate ( _{Na2SO4 )} ), magnesium sulfate (MgSO ₄ ), potassium sulfate (K ₂ SO ₄ ), calcium sulfate (CaSO ₄ ), and chromium sulfate (Cr ₂ (SO ₄ ) ₃ ). In some embodiments, the one or more salts may be potassium chloride, magnesium chloride, disodium phosphate, monopotassium phosphate, magnesium sulfate, and combinations thereof.

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

In any embodiment, each of the one or more salts may be present in the electroporation medium at a concentration of about 0.1 mM to about 200 mM, about 0.1 mM to about 150 mM, about 0.1 mM to about 100 mM, about 0.1 mM to about 80 mM, about 0.1 mM to about 60 mM, about 0.1 mM to about 40 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 1 _mM to about 20 mM, or about ₁ mM to about 10 mM. For example, salts such as _KH2PO4 , _Na2HPO4 , and _MgSO4 may be present in the electroporation medium at a concentration of about 0.1 mM to about 20 mM. The sugar may be present in the electroporation medium at a concentration of about 20 mM to about 300 mM, about 20 mM to about 200 mM, or about 40 mM to about 200 mM. The buffering agent may be present in the electroporation medium at a concentration of about 1 mM to about 100 mM, about 10 mM to about 50 mM, or about 15 mM to about 30 mM.

Other suitable media include, but are not limited to, Cw100 (0.11 S/m, 0.12 osm/l) or Cw240, as shown in Table 1. Commercially available electroporation media may be used but have been shown to be suboptimal. Such commercially available media include RPMI (1.37 S/m, 0.28 osm/l), BTX (8 mS/m, 0.27 osm/l), DMEM (Dulbecco's Modified Eagle's Medium), diluted PBS, or PBS, with or without Hepes. The media is generally conductive and may be sterilized.

In any embodiment, the cells for electroporation are at a concentration of about 1× ¹⁰ cells/ml or more, about 10× ¹⁰ cells/ml or more, about 50× ¹⁰ cells/ml or more, about 100× ¹⁰ cells/ml or more, about 150× ¹⁰ cells/ml or more, about 200× ¹⁰ cells/ml or more, about 250× ¹⁰ cells/ml or more, about 300× ¹⁰ cells/ml or more, about 400× ¹⁰ cells/ml or more, or about 500× ¹⁰ cells/ml; or from about 1× ¹⁰ cells/ml to about 300× ¹⁰ cells/ml, from about 1× ¹⁰ cells/ml to about 250× ¹⁰ cells/ml, from about 1× ¹⁰ cells/ml to about 200× ¹⁰ cells/ml, about 1×10 ⁶ cells/ml to about 150× ¹⁰ cells/ml, about 1×10 cells/ml to about 100× ¹⁰ cells/ml, about 1× ¹⁰ cells/ml to about 50× ¹⁰ cells/ml, about 1× ¹⁰ cells/ml to about 300× ¹⁰ cells/ml, about 1× ¹⁰ cells/ml to about 250× ¹⁰ cells/ml, about 10× ¹⁰ cells/ml to about 200× ¹⁰ cells/ml, about 10× ¹⁰ cells/ml to about 150× ¹⁰ cells/ml, about 10× ¹⁰ cells/ml to about 100× ¹⁰ cells/ml, about 10× ¹⁰ cells/ml to about 50× ¹⁰ cells/ml, about 100× ^{10 The} cells may be present in the electroporation medium at a cell density of from about ⁶ cells/ml to about 300× ¹⁰ cells/ml, from about 100× ¹⁰ cells/ml to about 250× ¹⁰ cells/ml, from about 100× ¹⁰ cells/ml to about 200× ¹⁰ cells/ml, or from about 100× ^{10 cells/ml to about 150×10} cells/ml.

With regard to suitable capacitances, it is believed that the capacitance may range from about 1 μF to about 150 μF. In some embodiments, the capacitance may range from about 1 to about 100 μF, about 5 to about 75 μF, about 5 to about 50 μF, about 10 to about 40 μF, or about 10 to about 30 μF, or about 10 to about 25 μF. In other embodiments, the capacitance is about 10 μF.

In some embodiments, multiple electrical pulses may be utilized with correspondingly short time constants. In some aspects, time constants of less than 30 milliseconds, less than 20 milliseconds, less than 10 milliseconds, or less than 5 milliseconds may be used. In other aspects, the time constant may range from about 0.5 to 30 milliseconds, about 1 to 20 milliseconds, and about 5 to 15 milliseconds; or about 10 milliseconds.

In some embodiments, the electric field strength for electroporation is between about 0.3 and 3 kV/cm. Lower electric field strengths (e.g., about 0.5 to 1 kV/cm) have been found to be suitable for EC-7 cells, while higher electric field strengths (e.g., about 1 to 3 kV/cm) have been found to be suitable for hank cells. Generally, electric field strengths for electroporation ranging from about 0.1 to about 5 kV/cm are contemplated herein. The voltage may be selected to generate a suitable electric field strength.

In general, the impedance (Rp) may range from about 200 Ω to infinity, or otherwise from about 200 Ω up to about 1 kΩ.

The concentration of cargo added to the electroporation reaction, e.g., material to be delivered into cells, is about 50 μg/ml or more, about 100 μg/ml or more, about 200 μg/ml or more, about 300 μg/ml or more, about 400 μg/ml or more, or about 500 μg/ml or more; about 50 μg/ml to about 500 μg/ml, about 50 μg/ml to about 400 μg/ml, about 50 μg/ml to about 300 μg/ml, about 50 μg/ml to about 200 μg/ml, about 50 μg/ml to about 100 μg/ml, about 100 μg/ml to about 500 μg/ml, about 100 μg/ml to about 400 μg/ml, about 100 μg/ml to about 300 μg/ml, or about 100 μg/ml to about 200 μg/ml. In some aspects, GFP-mRNA or GFP-DNA was added to the electroporation medium at a concentration of approximately 50 μg/ml, 60 μg/ml, 100 μg/ml, 200 μg/ml, 100-300 μg/ml, or 50-100 μg/ml, while in other embodiments, dextran 500k was added to the electroporation reaction at a concentration of approximately 50 μg/ml.

In some embodiments, a maximum of 7.33 uL/s at 4 x ¹⁰ cells/ml (13.2 mL ( ^{5.28 x 10} cells)) is achievable within 30 minutes in a single chamber apparatus. To achieve 20 x ¹⁰ cells within 30 minutes, the cell density may be increased four-fold and/or parallel processing may be performed using multiple electroporation chambers.

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

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

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

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

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

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

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

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

The methods described herein can be applied to transient or stable transfection. The term "transient transfection" refers to the introduction or transfection of a nucleic acid that is present in a cell for a limited period of time and does not integrate into the genome. The term "stable transfection" refers to transfection that results in permanent expression of a gene of interest through integration of the transfected nucleic acid into the nuclear genome or maintenance of the transfected plasmid as an extrachromosomal replicating episome within the cell.

In any embodiment, the methods described herein may further include a first cell sorting step and/or a second cell sorting step, e.g., using a microfluidic device as described herein. The first cell sorting step may involve applying pressure to cause a first solution containing cells to flow through a microfluidic chamber as described herein that includes a plurality of post arrays as described herein, deflecting the cells to the side of the chamber by the post arrays, depleting the cells from the solution exiting a first output mechanism as described herein, and concentrating the cells in the solution exiting a second output mechanism as described herein, thereby sorting the cells prior to introduction into the electroporation chamber. The cells exiting the second output mechanism may be introduced into the electroporation chamber along with the cargo. The second cell sorting step can sort the electroporated cells after they exit the electroporation chamber by applying pressure to cause a fourth solution containing the electroporated cells to flow through a microfluidic chamber as described herein that includes a plurality of post arrays as described herein, deflecting the electroporated cells to the side of the chamber by the post arrays, depleting the electroporated cells from the solution exiting a third output mechanism as described herein, and concentrating the electroporated cells in the solution exiting a fourth output 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 apparatus as described herein, e.g., in cartridge form, can be incorporated into an automated device, where electroporation can be performed in conjunction with one or more other automated processes on cells. For example, automated electroporation can be performed in conjunction with automated cell culture and harvesting, such as described in U.S. Patent Application Publication No. 2017/0037357, which is incorporated by reference in its entirety.

In another aspect, a method of in vivo transfection is provided, wherein the method is performed and the transfected cells, e.g., mixed with an isotonic buffer, may be administered to a patient to treat a disease or disorder. Examples of diseases include, but are not limited to, mitochondrial disorders, cardiac dysfunction, heart failure, autism, diabetes, and hearing loss. It is also contemplated herein that the electroporation devices described herein may be adapted for application to a target tissue of a subject to contribute to the delivery of a drug or pharmaceutical agent, e.g., insulin or a drug to treat diabetes. For example, an electrode for electroporation may be applied to the skin surface, or a needle-shaped electrode pair may be administered subcutaneously. It is also contemplated herein that the electroporation devices and methods may be used for adenovirus production, protein generation, cellular immunotherapy, or regenerative medicine.

Kits are also provided herein. For example, the kits may include an electroporation apparatus as described herein, a first container for containing cells and cargo to be transfected, a second container for containing electroporated cells, tubing for fluidly connecting the first and second containers to the electroporation apparatus, and, optionally, suitable reagents. In some embodiments, the kits may include a first package containing, for example, a first module (e.g., first module 805) containing the first container and a first portion of the tubing. The kits may further include a separate second package containing, for example, a second module (e.g., second module 815) containing the electroporation apparatus and a second portion of the tubing. The kits may also include a separate third package containing, for example, a third module (e.g., third module 825) containing the second container and a third portion of the tubing. Optionally, reagents may be present in a separate fourth package. The first, second, third, and fourth packages may be sterilized. Suitable reagents include, but are not limited to, cells to be transfected as described herein, electroporation media as described herein, and combinations thereof. Additionally or alternatively, the kit may further include electroporation tips, connecting tubing, devices, electroporation tools, electroporation buffers, additives, reagents, and combinations thereof.

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

Figures 7A-E show experimental results of electroporation reactions. Figure 7A shows various experimental conditions along with various efficiencies and viability rates. In particular, using an IOCO electroporation device, conditions of 1.5 kv/cm, Rp1k, and 10 uF resulted in greater than 80% efficiency with greater than 70% viability for electroporation of GFP-mRNA into haNK cells. The designation "reg" refers to the original micromesh, while "offset" refers to the offset micromesh. Figure 7B shows a microscopic image of GFP-mRNA being electroporated into haNK cells. Figures 7C and 7D show cell sorting results, where Figure 7C shows live cells and Figure 7D shows electroporated cells. Figure 7E shows a histogram corresponding to the cell sorting results, showing control cells and cells electroporated with GFP.

Figures 8A-8D show another set of experimental results under various electroporation conditions, showing that electroporation of GFP-mRNA into haNK resulted in an efficiency of over 50% and a viability of over 70%. Figures 8B and 8C show the results of cell sorting, where Figure 8B shows live cells and Figure 8C shows electroporated cells. Figure 8D shows a histogram corresponding to the cell sorting results, showing control cells and cells electroporated with GFP. GFP expression was counted in live cells (propidium iodide negative, PI-). CTL represents the control experiment.

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

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

Figures 10A-10E show an additional set of experimental results for EC-7 cells under various electroporation experimental conditions. Figures 10B, 10C, and 10D show cell sorting results, with Figure 10B showing live cells and Figures 10C and 10D showing electroporated live cells. Figure 10E shows control cells and GFP-electroporated cells along with histograms corresponding to the cell sorting results. GFP expression was counted in live cells (propidium iodide negative, PI-). CTL represents the control experiment.

Figures 11A-11C show images of GFP-DNA successfully electroporated into EC-7 cells using the electroporation apparatus and offset chamber of Figures 3A-3C.

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

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

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

Figures 12A and 12B show further results from transfecting EC7 cells using the methods and devices described herein. Figure 12A shows a table of electroporation parameters when the amount of cargo was varied between electroporation runs. Figure 12B shows images 6 days after electroporation. By 14 days after electroporation, approximately 70-80% efficiency was observed. Multiple "comet"-like clusters were observed after transfection. Approximately 17-18 million cells/minute may be transfected into EC7 cells at a cell concentration of approximately 40 mcg/ml in electroporation buffer cw100, which has a conductance of approximately 240 mS/m.

Example 3. Electroporation of Various Cell Types In other aspects, the methods and systems provided herein may be used to transfect a variety of different cell types. For example, up to ¹⁰ or more cells can be transfected using the methods and systems provided herein. The methods and systems provided herein may be used to transfect EC7 cells, haNK cells, CHO cells, and T cells. For example, the methods and techniques provided herein may be used to transfect EC7 cells using a flow rate of 0.6 ml/min and a cell concentration of 4 x ¹⁰ cells/ml. In another example, the methods and techniques provided herein may be used to transfect haNK cells using a flow rate of 0.36 ml/min and a cell concentration of 3 x ¹⁰ cells/ml. In yet another example, the methods and techniques provided herein may be used to transfect CHO cells using a flow rate of 0.45 ml/min and a cell concentration of 2.5 x ¹⁰ cells/ml. For T cells, the methods and techniques presented herein may be used to transfect T cells at a flow rate of 0.44 ml/min and a cell concentration of approximately 2 x ¹⁰ cells/ml. These conditions may be further varied to achieve optimal conditions.

Figures 13A-13D show various transfection efficiencies in these different cell lines using the methods and devices described herein. Figure 13A shows a transfection efficiency of greater than 95% with greater than 90% cell viability in haNK cells (transfected with GFP mRNA). Up to 127 million cells/minute were transfected. Figure 13B shows a transfection efficiency of greater than 90% with greater than 50% cell viability in CHO cells (transfected with GFP pmax DNA). Up to 15 million cells/minute were transfected. Figure 13C shows a transfection efficiency of greater than 95% with greater than 50% cell viability in EC7 cells (transfected with GFP adenovirus DNA). Up to 20 million or more cells/minute were transfected. Figure 13D shows that T cells (transfected with GFP mRNA) showed a transfection efficiency of over 90% with a cell viability of over 80%. Up to 8.8 million cells/min were transfected.

Example 4. Transfection of Primary T Cells In another aspect, the methods and systems presented herein are used to electroporate mRNA attached to poly(β-amino ester) into primary T cells. Certain poly(β-amino ester) polymers have been shown to act as effective RNA transfection agents.

Nanoparticles comprising RNA and poly(β-amino ester) polymers have been used to deliver mRNA to T cells (see, e.g., Moffett, et al., Nature Communications (2017) 8:389). For example, in some embodiments, nanoparticles may be fabricated with a polyglutamic acid (PGA)-based shell. To target the nanoparticles to appropriate targets, targeting molecules (e.g., antibody binding domains) may be attached to the PGA molecules. For example, to target nanoparticles to T cells, anti-CD3/anti-CD28 binding domains may be attached to the PGA molecules. PGA or any other suitable negatively charged molecule may be used for targeting. Nanoparticle uptake may occur through specific targeting or cationic membrane association.

The interior of the nanoparticle may contain mRNA and a carrier molecule such as poly(β-amino ester). Once the nanoparticle is internalized within a cell, the mRNA is released into the cell by nanoparticle degradation, osmotic swelling, or some other suitable process, and the mRNA is transcribed into its respective protein. In some cases, synthetic mRNA may be used to reduce mRNA degradation.

The methods provided herein may be used to transfect cells with mRNA, for example, by using mRNA and a poly(β-amino ester) (PbAE) carrier or by using nanoparticles encapsulating mRNA and a poly(β-amino ester) carrier. Figures 14A-14E show T cell transfection using PbAE complexed with mRNA (not 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 14B shows the results of flow cytometry cell sorting of T cells transfected with mRNA using PbAE. Figure 14C is a graph showing the results normalized to viable cells based on various ratios of mRNA to carrier molecules. Figures 14D and 14E show cell viability results under various conditions for GFP-mRNA transfection in stimulated (Figure 14D) and unstimulated (Figure 14E) T cells. In these experiments, a PbAE:RNA ratio of 60:1 resulted in high levels of viable cells while also achieving high GFP delivery rates.

Figures 15A and 15B show GFP mRNA transfection into T cells using control cells (no electroporation) and electroporated cells. In particular, Figure 15A shows control cells (no electroporation), where primarily background fluorescence is observed. Figure 15B shows electroporated cells, where GFP-mRNA is detected in the majority of cells, as shown by the histogram.

Example 5. Transfection of Adipose-Derived Mesenchymal Stem Cells (AD-MSCs) Adipose-derived mesenchymal stem cells (AD-MSCs) were detached and collected at a density of approximately 1 x 10 ⁵ million cells/milliliter. Cells were washed with PBS and electroporation buffer and suspended in electroporation buffer at a density of 3.3 million cells/milliliter. For each condition, 30 μg of DNA was electroporated at a final cell density of 0.1 milliliter. Electroporation conditions were as follows: pulse width = 100 μs, pulse duration = 340 ms, and field strength varied from 0.9 to 1.6 kV/cm, as shown in Table 4 below.

After electroporation, cells were seeded at a density of 30 x ¹⁰ cells/ ^cm² . 24 hours after electroporation, GFP expression efficiency was verified by flow cytometry. The results are shown in Figures 16A-16D. The x-axis values of 1-8 in each of Figures 16A-16C correspond to the electric field strength as shown in Table 4, and "CTL" corresponds to the control without electroporation. Figure 16A shows cell viability 24 hours after electroporation. As the electric field strength decreased, the number of live cells increased. Figure 16B shows GFP (green fluorescent protein) expression as read by flow cytometry 24 hours after electroporation, as a measure of cell transfection efficiency. As the electric field became stronger, a higher percentage of live cells expressed GFP. Figure 16C shows the median GFP fluorescence, which indicates how bright GFP was in the flow cytometry measurement. As the electric field became stronger, brighter GFP was measured. The results suggested that a field strength between 1.1 and 1.3 kv/cm may provide the best performance in terms of viability and efficiency. Note that this viability includes both adherent cells and supernatant, reflecting true overall viability. Figure 16D shows photographs of MSC transfection results. The photograph on the left shows the morphology of MSCs, and the image on the right is a fluorescence image in the same light field, showing that the majority of cells express green fluorescent protein (green).

Example 6 Transfection of EC-7 Cells, CHO-S Cells, Primary CD4/CD8 T Cells, haNK Cells, Primary NK Cells, Activated NK Cells, Dendritic Cells, and AD-MSC Cells EC-7 cells, CHO-S cells, primary CD4/CD8 T cells, haNK cells, primary NK cells, activated NK cells, dendritic cells, and AD-MSC cells were electroporated in an apparatus similar to that depicted in FIG. 2A using the electroporation parameters shown in Table 5 below.

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

The results of electroporating the cells in Table 5 according to the given parameters are presented in Table 8 below.

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

Pump 1701 may be connected to an input cartridge of I/O cartridge 1703 via tubing and one of sterile connectors 1704. One or more pumps 1701 may be used to generate a fluid flow through chip assembly 1720. Chip assembly 1720 may be selectively coupled and uncoupled from system 1700, thereby allowing different chip assemblies to be used with system 1700, as described further below. For example, system 1700 may include a chip holder structure to removably mount different chip assemblies, with an optional chip sensor disposed adjacent to the chip holder structure.

The system 1700 may include a scanner, user interface, or the like that can be programmed to identify or verify the chip assembly 1720 placed in the system 1700, select operations to perform using the chip assembly 1720, etc. In various embodiments, the chip assembly 1720 may be gamma radiation compatible. The system 1700 may allow for the use of various consumables, buffers, etc., in conjunction with the chip assembly 1720. In one embodiment, the system 1700 is disposable, with each chip assembly 1720 being a closed system design having an input receptacle and a normally closed Luer connector to the input receptacle for easy, sterile connection and disconnection of cell samples. In various embodiments, when connected outside a hood, a sterile connector, such as a CPC AseptiQuick, may be used to provide a sterile connection.

Figure 18 shows an exemplary device for electroporating cells with cargo, including an electroporation chamber 1820 (which may be referred to as a chip or chip assembly). The electroporation chamber 1820 includes a first electrode 1840 and a second electrode 1842, with three fluid channel layers 1825, 1826, and 1827 located between the electrodes 1840 and 1842. Each fluid channel layer 1825, 1826, and 1827 may include a fluid channel 1835 (which may be referred to as a patch). For example, fluid containing cells (and optional cargo) may flow laterally through the electroporation chamber 1820 via the fluid channel 1835, while the shape of the fluid channel 1835 defines the particular path along which the fluid travels.

As shown in FIG. 18 , fluid channel layers 1825, 1826, and 1827 can be aligned with one another such that the fluid channels 1835 in each layer 1825, 1826, and 1827 are also aligned. Electrodes 1840 and 1842 can be aligned with one another and with layers 1825, 1826, and 1827, thereby forming a sandwich configuration in which the perimeter of each electrode 1840 and 1842 and the perimeter of each channel layer 1825, 1826, and 1827 are substantially the same. In various embodiments, the length and/or width of one or more of channels 1825, 1826, and 1827 can be greater than the length and/or width of electrodes 1840 and 1842, thereby preventing electrodes 1840 and 1842 from unintentionally contacting one another and creating a short circuit.

Electroporation chamber 1820 may include first support layer 1815 and second support layer 1816. As shown in FIG. 18, electrodes 1840 and 1842 and channel layers 1825, 1826, and 1827 may be located between support layers 1815 and 1816. Electroporation chamber 1820 also includes first plate 1817 and second plate 1818 (which may be referred to as housing plates or cover plates). Plates 1817 and 1818 may be disposed as the outermost layers of electroporation chamber 1820, as shown in FIG. 18.

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

Plates 1817 and 1818 may be aligned with support layers 1815 and 1816, electrodes 1840 and 1842, and channel layers 1825, 1826, and 1827 so that the perimeter of each layer has approximately the same footprint in electroporation chamber 1820. In various embodiments, plates 1817 and 1818 may have a longer length and/or width than the other layers (e.g., as shown in FIG. 18), thereby preventing electrodes 1840 and 1842 or other layers from contacting the external chamber. Other embodiments of the electroporation chamber may have more or fewer layers than those shown in FIG. 18. For example, there may be more or fewer fluid channel layers, more or fewer support layers (or no support layers), etc.

As shown in FIG. 18 , the electroporation chamber includes an input 1810 and an output 1830. The input 1810 may allow cells and cargo to enter the electroporation chamber 1820, while the output 1830 allows electroporated cells to exit the electroporation chamber 1820. The input 1810 and the output 1830 may be separated by an offset distance to promote lateral flow of cells through the electroporation chamber 1820, such as through the paths of the fluid channels 1835 in the channel layers 1825, 1826, and 1827.

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

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

Channel layers 1825, 1826, and 1827 may comprise any suitable material for enabling fluid flow in fluid channel 1835. For example, channel layers 1825, 1826, and 1827 may comprise a polyethylene terephthalate (PET) material, a pressure-sensitive adhesive (PSA) material, a silicone gasket material, or the like. In various embodiments, center channel layer 1825 may comprise a PET material, while outer channel layers 1826 and 1827 comprise a PSA material, adhering channel layers 1825, 1826, and 1827 together.

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

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

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

Support layers 1815 and 1816 and plates 1817 and 1818 may comprise any suitable material that provides structural support to electrodes 1840 and 1842, which contain cell-containing fluid in fluid channel layers 1825, 1826, and 1827. Examples may include, but are not limited to, non-porous materials such as polydimethylsiloxane (PDMS), acrylic, polyethylene, polypropylene, metal plates, pressure-sensitive adhesive (PSA), PET material, acrylic glass, injection-molded plastic, and the like. In one embodiment, support layers 1815 and 1816 each comprise a double-sided PSA material to adhere plates 1817 and 1818 to electrodes 1840 and 1842, and each plate 1817 and 1818 is a ¼-inch (approximately 6.35 mm) thick acrylic sheet.

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

The tip assembly shown in FIG. 19 may be used in an electroporation apparatus, such as system 1700 of FIG. 17. For example, multiple tip assemblies may be selectively inserted into the system to electroporate various cells. After each electroporation process, or when a new cell type is desired for electroporation, the tip assembly of FIG. 19 may be removed from the electroporation system and replaced with another tip assembly.

As described further below, different chip assemblies may have different fluid channel 1835 volumes, different fluid channel 1835 flow paths, etc. Various chip assemblies may have the same size or footprint, allowing chip assemblies of different volumes or flow paths to be interchanged in an electroporation system. In one exemplary embodiment, chip assemblies having different fluid channel volumes may be selectively inserted into an electroporation system, including large-volume chip assemblies (e.g., fluid channel 1835 volumes of approximately 500 microliters), medium-volume chip assemblies (e.g., fluid channel 1835 volumes of approximately 250 microliters), and small-volume chip assemblies (e.g., fluid channel 1835 volumes of approximately 50 microliters). In other embodiments, more or fewer chip assembly designs may be used with an electroporation system, each chip assembly having larger or smaller fluid channel volumes, etc. than those provided above.

Figure 20 shows an example channel layer 2025 that may be part of an electroporation chamber, such as electroporation chamber 1820 of Figure 18. Channel layer 2025 includes a fluid channel 2035 with multiple bends and multiple straight path sections (which may be referred to as S-shaped paths). Figure 20 shows fluid channel 2035 with eight bends and nine straight sections (seven full lengths and two half lengths at each end of fluid channel 2035). Fluid channel 2035 can be considered four complete half cycles of an S-shaped curve pattern.

When the channel layer 2025 is received in an electroporation system, a fluid containing cells can be supplied to the two inlet ends of the fluid channel 2035, travel through the S-shaped curve of the fluid channel 2035, undergo electroporation, and exit through the other outlet end of the fluid channel. The volume of the fluid channel 2035 can determine the volumetric flow rate of the fluid through the channel and can be synchronized with the electrical pulses of the electroporation process. In some embodiments, the fluid channel 2035 can have a volume of approximately 500 microliters (e.g., capable of processing 200 million or more cells within a specified time period). In other embodiments, the fluid channel 2035 can include a larger or smaller fluid volume (e.g., approximately 25 microliters, 50 microliters, 100 microliters, 200 microliters, 250 microliters, 300 microliters, 400 microliters, 750 microliters, 1 milliliter, etc.), more or fewer bends, a thicker or narrower channel width, a longer or shorter channel length, other suitable path shapes, etc.

Figure 21 shows an example channel layer 2125 that may be part of an electroporation chamber, such as electroporation chamber 1820 of Figure 18. Channel layer 2125 includes a fluid channel 2135 with multiple bends and multiple straight path sections (which may be referred to as S-shaped paths). Similar to fluid channel 2035 of Figure 20, Figure 21 shows fluid channel 2135 with eight bends and nine straight sections (seven full lengths and two half lengths at each end of fluid channel 2135).

Compared to fluid channel 2035 of FIG. 20, fluid channel 2135 of FIG. 21 has a shorter length than bends. Thus, fluid channel 2135 may have a smaller fluid volume than fluid channel 2135 of FIG. 20. For example, fluid channel 2135 may have a fluid volume of approximately 250 microliters (e.g., may be capable of processing approximately 500 million cells within a specified time period). In other embodiments, fluid channel 2135 may include a larger or smaller fluid volume, more or fewer bends, a thicker or narrower channel width, a longer or shorter channel length, other suitable path shapes, etc.

Figure 22 shows an example channel layer 2225 that may be part of an electroporation chamber, such as electroporation chamber 1820 of Figure 18. Channel layer 2225 includes fluid channel 2235 with a single straight section (as opposed to the S-shaped curves of fluid channels 2035 and 2135 of Figures 20 and 21, which have multiple curves). Thus, fluid channel 2235 of Figure 22 may have a smaller fluid volume than fluid channels 2035 and 2135 of Figures 20 and 21.

When channel layer 2225 is received in an electroporation system, a fluid containing cells may be supplied to one end of fluid channel 2235, travel through the straight line of fluid channel 2235, undergo electroporation, and exit through the other end of the fluid channel. In some embodiments, fluid channel 2235 may have a volume of approximately 50 microliters (e.g., may be capable of processing approximately 500 million cells within a specified time period). In other embodiments, fluid channel 2235 may include a larger or smaller fluid volume, more or fewer bends, a thicker or narrower channel width, a longer or shorter channel length, other suitable path shapes, etc.

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

In some embodiments, numbers expressing quantities of ingredients, concentrations, reaction conditions, and other properties, etc., used to describe and claim particular embodiments should be understood to be modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments are approximations, the numerical values set forth in the specific examples are reported to the extent practicable. The numerical values presented in some embodiments may necessarily contain certain errors resulting from the standard deviation found in their respective testing measurements.

As used herein and throughout the claims that follow, the meanings of "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Additionally, as used herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise. Unless the context dictates otherwise, all ranges set forth herein should be interpreted as inclusive of their endpoints, and open-ended ranges should be interpreted as including commercially practical values. Similarly, all value lists should be considered as inclusive of intermediate values unless the context dictates otherwise.

It should also be apparent to those skilled in the art that many further modifications beyond those already described herein are possible without departing from the inventive concepts herein. Moreover, when 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 to refer to elements, components, or steps in a non-exclusive manner, indicating that a referenced element, component, or step may be present, utilized, or combined with other elements, components, or steps not specifically referenced. When a claim herein refers to at least one selected from the group consisting of A, B, C, and N, the text should be interpreted as requiring only one element from that group, not A+N, or B+N, etc.

A typical electroporation device is shown, which includes parallel plate electrodes (as shown in FIG. 1A) and a micromesh electrode with no offset between the input and output (as shown in FIG. 1B). A typical electroporation device is shown, which includes parallel plate electrodes (as shown in FIG. 1A) and a micromesh electrode with no offset between the input and output (as shown in FIG. 1B). 1 illustrates aspects of an apparatus for electroporation according to some embodiments provided herein. 1 illustrates aspects of an apparatus for electroporation according to some embodiments provided herein. 1 illustrates aspects of a first electrode and a second electrode according to some embodiments provided herein. FIG. 1 is a block diagram of an apparatus for generating and providing electrical pulses to cells within an electroporation apparatus as described herein, according to some embodiments provided herein. FIG. 1 is a block diagram of a further apparatus for generating and providing electrical pulses to cells within an electroporation apparatus as described herein according to some embodiments provided herein. 3A shows an embodiment of an apparatus for electroporation, including an electroporation device, according to some embodiments provided herein. FIG. 3A shows a diagram of the structure of an input/output chamber offset electroporation device. 3A and 3B show aspects of an apparatus for electroporation, including an electroporation device, according to some embodiments provided herein. Figure 3B shows a diagram of the electric field of an input and output chamber offset electroporation device. 3A and 3B show aspects of an apparatus for electroporation, including an electroporation device, according to some embodiments provided herein. Figure 3C shows electric field strength as a function of cell migration distance through an input and output chamber offset electroporation device. 3A and 3B show aspects of an apparatus for electroporation, including an electroporation device, according to some embodiments provided herein. Figure 3D shows an alternative configuration of an electroporation device, according to some embodiments provided herein. 1 illustrates aspects of pathways within an electroporation chamber according to some embodiments provided herein. 1 illustrates aspects of pathways within an electroporation chamber according to some embodiments provided herein. 1 illustrates aspects of pathways within an electroporation chamber according to some embodiments provided herein. 1 illustrates an alternative configuration of an electroporation apparatus according to some embodiments provided herein. 1 illustrates an alternative configuration of an electroporation apparatus according to some embodiments provided herein. 1 illustrates an alternative configuration of an electroporation apparatus according to some embodiments provided herein. 1 illustrates an example microfluidic chamber for sorting cells, according to some embodiments provided herein. 1 illustrates an example microfluidic chamber for sorting cells, according to some embodiments provided herein. 1A-1C are diagrams illustrating various aspects of post geometry and positioning within a microfluidic chamber according to some embodiments provided herein. 1A-1C are diagrams illustrating various aspects of post geometry and positioning within a microfluidic chamber according to some embodiments provided herein. 1A-1C are diagrams illustrating various aspects of post geometry and positioning within a microfluidic chamber according to some embodiments provided herein. 1A-1C are diagrams illustrating various aspects of post geometry and positioning within a microfluidic chamber according to some embodiments provided herein. 1A-1C are diagrams illustrating various aspects of post geometry and positioning within a microfluidic chamber according to some embodiments provided herein. 1A-1C are diagrams illustrating various aspects of post geometry and positioning within a microfluidic chamber according to some embodiments provided herein. 1A-1C are diagrams illustrating various aspects of post geometry and positioning within a microfluidic chamber according to some embodiments provided herein. 1 illustrates an example microfluidic chamber for changing buffer solutions, according to some embodiments provided herein. 1 illustrates an example microfluidic chamber having two separate chambers, one for cell sorting and another for buffer change, according to some embodiments provided herein. 1 illustrates an alternative configuration of an electroporation apparatus according to some embodiments provided herein. 1 illustrates an alternative configuration of an electroporation apparatus according to some embodiments provided herein. 1 illustrates a modular arrangement of an electroporation system including an electroporation apparatus according to some embodiments provided herein. 1 shows a table of parameters associated with a chamber offset electroporation apparatus according to some embodiments provided herein. 1A-1C show examples of fluid flow waveforms (pumps) and electrical waveforms (stimuli) that may be applied to cells flowing through a chamber offset electroporation device, according to some embodiments provided herein. 1A-1C illustrate various examples of micromeshes formed of different materials suitable for use in a chamber offset electroporation apparatus, according to some embodiments provided herein. 1 shows the results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein. 1 shows the results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein. 1 shows the results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein. 1 shows the results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein. 1 shows the results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein. 10 shows further results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein. 10 shows further results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein. 10 shows further results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein. 10 shows further results of electroporation experiments on haNK cells and corresponding parameters using the methods and devices described herein. 1 shows the results of electroporation experiments on EC7 cells and corresponding parameters using the methods and devices described herein. 1 shows the results of electroporation experiments on EC7 cells and corresponding parameters using the methods and devices described herein. 1 shows the results of electroporation experiments on EC7 cells and corresponding parameters using the methods and devices described herein. 1 shows the results of electroporation experiments on EC7 cells and corresponding parameters using the methods and devices described herein. 10 shows further results of electroporation experiments on EC7 cells and corresponding parameters using the methods and devices described herein. 10 shows further results of electroporation experiments on EC7 cells and corresponding parameters using the methods and devices described herein. 10 shows further results of electroporation experiments on EC7 cells and corresponding parameters using the methods and devices described herein. 10 shows further results of electroporation experiments on EC7 cells and corresponding parameters using the methods and devices described herein. 10 shows further results of electroporation experiments on EC7 cells and corresponding parameters using the methods and devices described herein. Microscopic images of electroporated EC7 cells are shown. Microscopic images of electroporated EC7 cells are shown. Microscopic images of electroporated EC7 cells are shown. Further results are presented for transfected EC7 cells using the methods and devices described herein. Further results are presented for transfected EC7 cells using the methods and devices described herein. 1 shows the various transfection efficiencies in different cell lines using the methods and devices described herein. 1 shows the various transfection efficiencies in different cell lines using the methods and devices described herein. 1 shows the various transfection efficiencies in different cell lines using the methods and devices described herein. 1 shows the various transfection efficiencies in different cell lines using the methods and devices described herein. 1 shows the results of a transfection experiment using PbAE as described herein to introduce mRNA into T cells. 1 shows the results of a transfection experiment using PbAE as described herein to introduce mRNA into T cells. 1 shows the results of a transfection experiment using PbAE as described herein to introduce mRNA into T cells. 1 shows the results of a transfection experiment using PbAE as described herein to introduce mRNA into T cells. 1 shows the results of a transfection experiment using PbAE as described herein to introduce mRNA into T cells. 1 shows the results of transfection experiments to introduce mRNA into T cells in the presence and absence of electroporation, as described herein. 1 shows the results of transfection experiments to introduce mRNA into T cells in the presence and absence of electroporation, as described herein. 1 shows the results of transfection experiments in adipose-derived mesenchymal stem cells (AD-MSCs) and corresponding parameters using the methods and devices described herein. 1 shows the results of transfection experiments in adipose-derived mesenchymal stem cells (AD-MSCs) and corresponding parameters using the methods and devices described herein. 1 shows the results of transfection experiments in adipose-derived mesenchymal stem cells (AD-MSCs) and corresponding parameters using the methods and devices described herein. 1 shows the results of transfection experiments in adipose-derived mesenchymal stem cells (AD-MSCs) and corresponding parameters using the methods and devices described herein. FIG. 1 is a block diagram of an electroporation apparatus according to another example embodiment of the present disclosure. FIG. 18 is an exploded view of an electroporation channel that may be used in the electroporation device of FIG. 17, according to another example embodiment of the present disclosure. FIG. 19 is an isometric view of the electroporation chamber of FIG. 18 with the layers assembled together. FIG. 19 is a front view of the fluid channel layer of the electroporation chamber of FIG. 18. 21 is a front view of a fluid channel layer having a different fluid volume than the fluid channel layer of FIG. 20 according to another example embodiment of the present disclosure. FIG. 10 is a front view of a fluid channel layer having straight fluid channels according to another example embodiment of the present disclosure. FIG. 21 is a front view of the fluid channel layer of FIG. 20 with example dimensions of the fluid channels.

Claims (18)

1. A chip assembly for electroporating cells with cargo, comprising:
A first electrode;
A second electrode;
a first fluid channel layer positioned between the first electrode and the second electrode, the first fluid channel layer defining a first fluid channel through which a fluid containing the cells and the cargo flows during an electroporation process;
a second fluid channel layer positioned between the first fluid channel layer and the first electrode, the second fluid channel layer defining a second fluid channel having the same shape as the first fluid channel in the first fluid channel layer;
a third fluid channel layer located between the first fluid channel layer and the second electrode, the third fluid channel layer defining a third fluid channel having the same shape as the first fluid channel of the first fluid channel layer, the fluid channels of the first fluid channel layer, the second fluid channel layer, and the third fluid channel layer being aligned with one another;
a first housing plate defining an input opening for receiving the fluid containing the cells and the cargo and supplying the received fluid to the first fluid channel of the first fluid channel layer, and defining an output opening for facilitating flow of electroporated cells out of the first fluid channel of the chip assembly;
a second housing plate;
a first support layer positioned between the first housing plate and the first electrode, the first support layer and the first electrode each defining a first opening corresponding to the input opening of the first housing plate and a second opening corresponding to the output opening of the first housing plate;
a second support layer positioned between the second electrode and the second housing plate ;
A chip assembly comprising: the second fluid channel layer and the third fluid channel layer each comprise a pressure-sensitive adhesive;
The chip assembly of claim 1 , wherein the first fluid channel layer comprises a polyethylene terephthalate (PET) material. 2. The chip assembly of claim 1, wherein the height of the aligned fluid channels of the first fluid channel layer, the second fluid channel layer, and the third fluid channel layer is uniform from the inlet of the aligned fluid channel to the outlet of the aligned fluid channel, and a uniform electric field is applied to the fluid in the aligned fluid channel during the electroporation process . The chip assembly of claim 3 , wherein the height of the aligned fluid channels of the first fluid channel layer, the second fluid channel layer, and the third fluid channel layer is 300 micrometers. 2. The chip assembly of claim 1, wherein the width of the first fluid channel is uniform from the inlet of the first fluid channel to the outlet of the first fluid channel, promoting a constant rate of cell flow through the first fluid channel. The chip assembly of claim 5 , wherein the width of the first fluid channel is 3 millimeters. 2. The chip assembly of claim 1 , wherein the first fluid channel has an S-curve configuration between the inlet of the first fluid channel and the outlet of the first fluid channel, the S-curve configuration including eight bends. The chip assembly of claim 1 , wherein the aligned fluid channels have a volume of 500 microliters. The chip assembly of claim 1 , wherein the aligned fluid channels have a volume of 250 microliters. The chip assembly of claim 1 , wherein the first fluid channel is a straight path from the inlet of the first fluid channel to the outlet of the first fluid channel. The chip assembly of claim 1 , wherein the aligned fluid channels have a volume of 50 microliters. the first electrode and the second electrode each comprise stainless steel;
the first support layer and the second support layer each comprise a pressure sensitive adhesive (PSA) material;
The chip assembly of claim 1 , wherein the first housing plate and the second housing plate each comprise an acrylic material. 2. The chip assembly of claim 1, wherein the input and output openings of the first housing plate are configured to receive Luer connectors to connect tubing and facilitate fluid flow to and from the first fluid channel. 1. A method of electroporating cells with a cargo, comprising:
flowing the cells together with the cargo into an electroporation chamber, the electroporation chamber comprising:
a first electrode;
a second electrode,
a first fluid channel layer positioned between the first electrode and the second electrode, the first fluid channel layer defining a first fluid channel through which an electroporation medium containing the cells and the cargo flows during an electroporation process;
a second fluid channel layer located between the first fluid channel layer and the first electrode, the second fluid channel layer defining a second fluid channel having the same shape as the first fluid channel in the first fluid channel layer;
a third fluid channel layer located between the first fluid channel layer and the second electrode, the third fluid channel layer defining a third fluid channel having the same shape as the first fluid channel of the first fluid channel layer, the fluid channels of the first fluid channel layer, the second fluid channel layer, and the third fluid channel layer being aligned with one another;
a first housing plate defining an input opening for receiving the electroporation medium containing the cells and the cargo and for supplying the received electroporation medium to the first fluid channel of the first fluid channel layer, and defining an output opening for facilitating flow of electroporated cells out of the first fluid channel of the electroporation chamber;
a second housing plate;
a first support layer positioned between the first housing plate and the first electrode, the first support layer and the first electrode each defining a first opening corresponding to the input opening of the first housing plate and a second opening corresponding to the output opening of the first housing plate;
a second support layer positioned between the second electrode and the second housing plate , the cells being suspended in an electroporation medium and having a cell density of about 1 x ¹⁰ cells/ml to about 500 x ¹⁰ cells/ml;
applying a plurality of electrical pulses to the cells in the electroporation chamber;
(i) each electrical pulse has the form of an exponential ejection waveform or a square waveform;
(ii) the plurality of electrical pulses are applied at a field strength of about 0.3 kV/cm to about 3 kV/cm;
(iii) the electric field strength is applied at a voltage of about 15 V to about 100 V;
(iv) the duration between each electrical pulse is about 0.1 seconds to about 10 seconds;
(v) applying each electrical pulse having a pulse width of about 10 μs to about 10,000 μs;
A method comprising: 15. The method of claim 14, wherein the flowing is performed in a stepwise manner, wherein a flow rate of about half or more of the total volume of the electroporation chamber is pumped into the electroporation chamber at a specified time interval, and the flow rate of the cells and cargo is about 0.1 ml/min to about 15 ml/min . 15. The method of claim 14 , wherein the cells are exposed to a uniform or substantially uniform electric field within the electroporation chamber. The method of claim 14 , wherein the plurality of electrical pulses is between 2 and 10 pulses. The method of claim 14 , wherein the cargo is a nucleic acid.