Systems, methods, and devices for electroporation of cell-containing fluids

Abstract

The present disclosure provides electroporation cartridges for single-use electroporation and electroporation cartridges for automated batch processing, electroporation instruments and systems, and methods of electroporation using these devices and systems. [Solution] In some embodiments, the electroporation cartridge includes an electroporation chamber defined by an elongate body, a first electrode at a proximal end, and a second electrode at a distal end of the chamber. The electroporation system of the present disclosure includes one or more components, including a pulse generator, a compartment for placing either a flow-through or single-use electroporation cartridge, components for storing cells, a cooling and pre-cooling mechanism, a removable and insertable modular casing having compartments for holding and placing the electroporation system and reagent components, one or more pumps for moving the sample through the system, and a processor and controller.

Description

[Technical Field]

[0001] This disclosure relates generally to systems, devices, and methods for treating cells with transient electric fields. More particularly, this disclosure relates to systems, devices, and methods for automated electroporation of cell-containing fluids, including single and automated batch electroporation systems, devices, and methods. [Background technology]

[0002] Since at least the 1970s, scientists have used electroporation as a technique to insert molecules into animal and plant cells. Exposing cells to transient electric fields, particularly brief high-voltage fields, makes the cell membrane permeable to molecules from the surrounding medium, allowing target macromolecules (usually proteins or nucleic acids) to enter the cell. With proper control of the voltage and duration of exposure to the electric field, electroporated cells can regain membrane permeability and normal function. However, excessive exposure to the electric field, whether for long periods or at too high a voltage, can permanently disrupt the cell's electrical potential and / or membrane integrity, potentially leading to cell death.

[0003] Traditionally, cell electroporation has been implemented using specialized cuvettes containing electrodes positioned relative to one another to create a uniform electric field between them. For example, electroporation cuvettes known in the art contain two flat electrodes attached to opposite walls of a rectangular cuvette or chamber. A suspension of cells to be electroporated is placed in the cuvette, where it is combined with an electroporation target and a high voltage and brief electric field pulse is applied via the electrodes. Most such commercially available electroporation cuvettes have limited capacity and can process only small volumes of cell suspension at a time, typically less than 1 milliliter.

[0004] However, this configuration has long been preferred and maintained because it is highly efficient and generates a uniform electric field, maintaining a more standard distribution of current through the affected medium. A uniform electric field helps normalize electroporation efficiency by reducing current hot spots that can damage or destroy cells and current cold spots that lead to reduced electroporation efficiency. However, the strength of the uniform electric field depends on the potential difference or voltage between the electrodes and the distance between the electrodes. Increasing the distance between the electrodes weakens the electric field. While this can be compensated for by increasing the voltage, the delicate nature of living cells has traditionally limited how much the voltage and / or distance between the electrodes can be increased while still maintaining a uniform electric field strong enough to efficiently electroporate cells.

[0005] For example, maintaining a uniform electric field between two electrodes as the physical distance between them increases requires an increased voltage. Increasing the voltage causes the electrodes to generate more heat, which is transferred to the cell-containing fluid. This is particularly problematic in continuous flow protocols, where the combination of voltage, pulse duration, and pulse number, coupled with the starting temperature of the sample, can quickly cause the sample temperature to exceed 60°C or even locally vaporize portions of the cell-containing sample. As expected, cell viability is adversely affected by elevated temperatures, especially if the temperature is prolonged or within a range incompatible with viable cells. Heating and discharge through aqueous solutions (i.e., almost all cell-containing fluids and / or electroporation media) can also form gas bubbles by localized vaporization of water molecules and / or electrolysis of water molecules to form oxygen and hydrogen gases. The presence of gas bubbles can reduce electrical conductivity and lead to arcing, which, among other things, can reduce electroporation performance and damage or destroy the sample. As a result, the amount of cell-containing fluid that can be electroporated has traditionally been limited.

[0006] Due to the limited cuvette volume, scaling this type of electroporation model has previously been impractical due to the manually intensive process of loading and unloading the cuvette. Furthermore, while maintaining sterility is essential for nearly all applications of large-scale cell electroporation, repeatedly loading cuvettes or pooling electroporated cells is particularly impractical and prone to contamination. While this electroporation method is convenient and simple and meets the needs of many researchers performing small-scale cell electroporation, additional methods are needed, particularly those that can conveniently facilitate large-scale cell electroporation while maintaining a sterile environment.

[0007] Electroporation of large quantities of cells in a closed, sterile system would enable the use of electroporation for human cell-based therapies. To address this, several continuous-flow electroporation systems have been developed, which generally consist of parallel electrodes between which a cell-containing fluid flows continuously and steadily until the entire cell mass has passed through a high-voltage electric field. However, a problem is that repeated application of high-voltage pulses to the electrodes of continuous-flow systems generates excessive heat. Some systems compensate for this by using cooling means to prevent the electrodes and cell suspension from reaching excessively high temperatures.

[0008] Nevertheless, these continuous flow systems suffer from inefficiencies and reliability issues. For example, the hydrodynamic flow of cell-containing fluid through the electroporation chamber of a continuous flow device does not result in all cells moving through the chamber between the electrodes at the same speed. The flow rate is faster away from the chamber walls compared to near the chamber walls. Cells flowing between the electrodes toward the center of the fluid flow may pass between the electrodes in a shorter time and inefficiently receive fewer pulses, while cells flowing near the walls may take longer to pass between the electrodes and therefore receive too many pulses. Thus, most continuous flow systems are inherently flawed and inefficient due to the existence of subpopulations of cells that either pass the electrodes too quickly and are therefore insufficiently electroporated, or pass the electrodes too slowly and are damaged or destroyed by excessive exposure to the high-voltage electric field.

[0009] Between imprecise and non-uniform electroporation of cells passing through the electrodes and the continuous application of an electric field through the cell-containing fluid, which creates bubbles and generates excessive heat, continuous-flow electroporation systems have failed to achieve sufficient throughput and efficiency to meet the needs of both research and development and commercial cell therapy for rapidly and efficiently transfecting cells. Indeed, electroporation has not been able to commercially compete with viral transfection, the most efficient and widely used technique. However, there are inherent risks associated with using viruses to transfect cells that may ultimately be returned to the patient.

[0010] Thus, there are many drawbacks and problems that can be addressed in automating cell electroporation, and there is a great need for systems, devices, and methods that can automate the electroporation process with high efficiency, particularly in a manner that reduces the likelihood of contamination and / or arcing caused by bubble formation within the processed cell-containing fluid. Summary of the Invention

[0011] Implementations of the present disclosure solve one or more of the above-mentioned problems or other problems in the art with systems, devices, and methods for electroporating cell-containing fluids. The terms "cell-containing fluid" and "sample" are used interchangeably herein.

[0012] In particular, one exemplary embodiment includes an electroporation cartridge having an electroporation chamber defined by an elongate body, a first electrode disposed at a proximal end of the electroporation chamber, and a second electrode disposed at an opposite distal end of the electroporation chamber. In some embodiments, at least one of the first electrode or the second electrode is movable from a capped position for electroporation to an uncapped position for loading a sample for single-use or automated batch processing, and / or the electroporation cartridge is configurable from a sealed state to an unsealed state.

[0013] In one aspect, the elongate body of the present disclosure can comprise, be made of, or include one or more of a non-conductive plastic, glass, and / or ceramic and is configured to receive a cell-containing liquid to be electroporated within an electroporation chamber defined by the elongate body. For example, the electroporation chamber of the present disclosure can comprise, be made of, or include glass and / or ceramic. As an additional example, the electroporation chamber can comprise, be made of, or include polycarbonate and / or other non-conductive, radiation-resistant plastic.

[0014] In one aspect, at least a portion of the electroporation chamber of the present disclosure is tapered between the first and second electrodes, and if present, the tapered portion of the electroporation chamber does not substantially interfere with the generation of a uniform electric field between the first and second electrodes.

[0015] Additionally or alternatively, the electroporation chamber of the present disclosure has a uniform cross-section along the length of the reaction chamber. This uniform cross-section can optionally extend the entire length of the electroporation chamber between the first and second electrodes such that the electroporation cartridge is configured to generate a uniform electric field within the electroporation chamber. For example, the electroporation chamber of the present disclosure can be a cylindrical cavity defined by an elongated body such that the uniform cross-section is circular.

[0016] In one aspect, an electroporation cartridge of the present disclosure includes a proximal sidewall defined between a proximal opening of the elongate body and an inflection point on the sidewall defining the electroporation chamber, the proximal sidewall narrowing from a first diameter defined by the proximal opening to a second, smaller diameter defined at a location distal to the inflection point.

[0017] In one aspect, the first electrode includes a bulbous extension. The bulbous extension, in one aspect, can have a substantially flat distal surface. Preferably, however, the bulbous extension has a distal surface with a convex or angled contour, which functions to expel one or more air bubbles associated with the cell-containing liquid being electroporated within the electroporation chamber when securing the first electrode within the electroporation chamber or otherwise sealing the electroporation chamber. In either embodiment, the bulbous extension can be separated from the base portion of the first electrode by a narrow stem.

[0018] In one aspect, the electroporation cartridge of the present disclosure includes a sealing member disposed between the first electrode and the proximal surface of the elongate body, the sealing member functioning to form a fluid-tight joint between the first electrode and the elongate body. The first electrode can further include a first electrode flange, and the elongate body can include a proximal body flange. The proximal body flange can lie in a plane substantially parallel to the first electrode flange, with the sealing member disposed between the first electrode flange and the proximal body flange to form a fluid-tight joint therebetween.

[0019] In one embodiment, the first electrode functions to set the electroporation cartridge between a sealed and unsealed state, and can do so without the use of an additional removable cap piece.

[0020] In one embodiment, the first electrode is itself a cap. In one embodiment, the first electrode is itself a removable cap.

[0021] In one aspect, the electroporation cartridge of the present disclosure includes a removable cap secured to the first electrode. The removable cap can include a coupling member for selectively securing the first electrode to the elongate body.

[0022] In one aspect, the diameter of the proximal end of the second electrode is substantially equal to the cross-section of the electroporation chamber. Additionally or alternatively, the second electrode can include a protruding portion extending from the distal end of the elongate body into the electroporation chamber, e.g., having a shape complementary to the inner surface of the elongate body defining the electroporation chamber. Additionally or alternatively, the second electrode can include a first sealing member disposed between the second electrode and the distal surface of the elongate body, the first sealing member functioning to form a fluid-tight joint between the second electrode and the distal surface of the elongate body. For example, the second electrode can include an electrode flange, the elongate body can include a distal body flange lying in a plane substantially parallel to the electrode flange, and the sealing member can be disposed between the electrode flange and the distal body flange to form a fluid-tight joint therebetween. Additionally or alternatively, the second electrode may include or be coupled to a second sealing member disposed around the protruding portion of the second electrode and distal to the proximal face of the second electrode, the second sealing member functioning to form a fluid-tight joint between the protruding portion and the inner surface of the elongate body defining the electroporation chamber.

[0023] In some embodiments, the proximal surface of the second electrode comprises a flat, uniform surface and / or the proximal surface of the second electrode can be perpendicular to the longitudinal axis of the electroporation chamber.

[0024] In one aspect, the electroporation cartridge of the present disclosure includes a fixation pin coupled to the second electrode and configured to secure the second electrode to the elongate body. For example, the second electrode can define a channel sized and shaped to receive the fixation pin, which can mate with a pair of openings defined by the sidewalls of the elongate body to receive the fixation pin and thereby secure the second electrode in a fixed position relative to the elongate body. The channel can be formed by a central region of a protruding portion of the second electrode distal to the first sealing member and / or second sealing member.

[0025] In one embodiment, the volume of the electroporation chamber is less than about 5 mL, preferably less than about 3 mL, more preferably less than about 1 mL, or between about 100 μL and 1 mL.

[0026] In one aspect, an electroporation cartridge of the present disclosure includes a volume reduction sleeve configured to fit within an electroporation chamber, the volume reduction sleeve defining a secondary electroporation chamber having a smaller volume than the electroporation chamber and a distal opening configured to interface with a second electrode when secured within the electroporation chamber.

[0027] In one aspect, the volume reduction sleeve includes an air vent located adjacent the proximal end of the volume reduction sleeve configured to allow air to pass during insertion or removal of the volume reduction sleeve from the electroporation chamber of the present disclosure, preventing a vacuum from forming between the secondary electroporation chamber and the electroporation chamber, thereby allowing electroporated cell-containing fluid to fill the secondary electroporation chamber during insertion of the volume reduction sleeve and to exit the secondary electroporation chamber during insertion or removal of the volume reduction sleeve.

[0028] The volume reduction sleeve of the present disclosure can further include a radial sealing member configured to secure the volume reduction sleeve within the electroporation chamber of the present disclosure, the radial sealing member forming a fluid-tight seal with the sidewall defining the electroporation chamber to prevent leakage of cell-containing fluid within the secondary electroporation chamber through the distal opening of the volume reduction sleeve.

[0029] The first electrode can be configured to selectively mate with the volume reduction sleeve to form a fluid-tight seal.

[0030] In some embodiments, the electroporation cartridge of the present disclosure can have a space defined between the outer surface of the volume reduction sleeve and the inner sidewall of the elongate body to form a fluid overfill space configured to receive an overfill volume displaced by the first electrode when the electroporation chamber is sealed.

[0031] In one aspect, the electroporation cartridge includes a fluid overfill space coupled to a proximal region of the electroporation chamber and configured to receive an overfill volume displaced by the first electrode upon sealing the electroporation chamber.

[0032] In one aspect, the electroporation cartridge of the present disclosure is a flow-through electroporation cartridge. The flow-through electroporation cartridge may include a port coupled to a first electrode defining a lumen therein, such that the lumen is fluidly connected to the electroporation chamber. Alternatively, the flow-through electroporation cartridge of the present disclosure may include a port coupled to a proximal portion of the elongate body configured to exhaust air displaced from the electroporation chamber when the electroporation chamber is filled and / or to introduce filtered or purified air into the electroporation chamber when the electroporation chamber is drained. In some cases, the flow-through electroporation cartridge includes a chamber inlet and a chamber outlet, each fluidly connected to the electroporation chamber. One or more of the chamber inlet or chamber outlet may be positioned above the proximal surface of the second electrode, and / or the lumen of the chamber inlet and / or chamber outlet may be substantially parallel to the proximal surface of the second electrode. Additionally or alternatively, one or more of the chamber inlets or chamber outlets can be coupled with plugs and / or valves to control the inward flow of cell-containing fluid to be electroporated within the electroporation chamber and / or to control the outward flow of electroporated cell-containing fluid from the electroporation chamber.

[0033] The electroporation cartridges disclosed herein can include a fluid overfill space coupled to the first electrode and / or the elongate body configured to receive an overfill volume displaced from the electroporation chamber when filling the electroporation chamber and / or sealing the electroporation chamber with a sealing cap.

[0034] An exemplary electroporation system configured to provide flow-through electroporation of a sample includes a modular casing having multiple compartments for holding and positioning multiple electroporation system components. The electroporation system of the present disclosure can include one or more pumps configured to move the sample through the system and an electroporation compartment configured to accept a flow-through electroporation cartridge configured to hold a subvolume of the sample within the electroporation chamber for subvolume electroporation. The electroporation system of the present disclosure can further include tubing routed through the casing to fluidly connect the multiple electroporation system components between inlets and outlets.

[0035] In one aspect, an electroporation system of the present disclosure includes a bag compartment configured to receive and support an input bag and / or an output bag. The bag compartment can include an insert slidably connected to the bag compartment so that the bag compartment can be selectively withdrawn from or inserted into the casing, and can include one or more magnetic latches to hold the bag compartment in a sealed position within the casing. In one aspect, the electroporation system includes one or more hooks for hanging one or more bags on the outside of the casing.

[0036] In one aspect, the electroporation system of the present disclosure includes one or more mechanisms for regulating the temperature of the sample by cooling and / or heating (e.g., according to a predetermined target temperature). For example, the electroporation system can include a cooling module in thermal contact with the electroporation chamber and configured to regulate the temperature of the electroporation chamber. In some embodiments, the cooling module can include a ceramic block cooled by thermoelectric cooling. Other embodiments of cooling modules used in the systems and devices of the present disclosure can additionally or alternatively utilize air cooling, liquid cooling, or other temperature regulation mechanisms known in the art. As explained below, such components are described herein as "cooling modules" based on their typical functions, but they can also be configured to provide such heat in applications where heating is desired.

[0037] In one aspect, the electroporation system of the present disclosure includes a mixer reservoir disposed downstream of the inlet and upstream of the electroporation cartridge, the mixer reservoir including a mixing element configured to mix portions of the sample contained within the mixing reservoir. The mixing element can be formed as a mixing blade or other mixing device that avoids contact between the magnetic-containing component and the sample fluid.

[0038] In one aspect, the mixer reservoir includes a mixer magnet assembly mechanically coupled to the mixing element, the mixer magnet assembly positioned so as not to contact the portion of the sample contained within the mixer reservoir. For example, the mixer reservoir can have a cover, and the mixer magnet assembly can be positioned on or near the cover. The electroporation system of the present disclosure can further include a mixer driver having a magnet magnetically coupled to the mixer magnet assembly and configured to indirectly drive rotation of the mixer magnet assembly via a magnetic connection to the mixer magnetic assembly.

[0039] In one aspect, the electroporation system of the present disclosure may comprise or include a sample input assembly configured to facilitate transfer of a sample between the input and the mixer reservoir. The sample input assembly, in some embodiments, includes a main section of tubing disposed between the input and the mixer reservoir, and an intermediate section of tubing coupled to the main section of tubing in a manner that allows air to pass from the intermediate section to the main section of tubing, the intermediate section of tubing extending from the main section to a distal end of the tubing. The distal end of the intermediate section of tubing has access to air (e.g., an air reservoir or a filter open to the atmosphere), allowing the intermediate section to pass air to the main section of tubing when a sufficient pressure drop occurs in the main section of tubing. For example, if an input container coupled to the inlet is empty or nearly empty of its contents, continued pumping will reduce the pressure in the main section of tubing, drawing air from the intermediate section of tubing into the main section of tubing.

[0040] In one aspect, the electroporation system of the present disclosure includes a chamber sealing assembly operably coupled to an electroporation chamber and configured to regulate pressure within the chamber during electroporation, thereby limiting bubble formation. The chamber sealing assembly includes one or more linear actuators configured to advance a plunger toward or retract the plunger from the chamber, thereby opening and closing corresponding ports to regulate fluid flow through and pressure within the chamber.

[0041] In one aspect, the electroporation system includes a pre-cooling assembly disposed upstream of the electroporation chamber and configured to regulate the temperature of a sub-volume of sample prior to electroporation of the sub-volume. In some embodiments, the pre-cooling assembly can comprise or include a cooling block and a section of tubing disposed within or adjacent to the cooling block. In some embodiments, the cooling block can be cooled, for example, by thermoelectric cooling. In other embodiments, the cooling block can additionally or alternatively utilize air cooling, liquid cooling, or other temperature regulation mechanisms known in the art. As explained below, such components are described herein as "pre-cooling modules / assemblies" based on their typical functions, but they can also be configured to provide heat in applications where such heating is desired. The electroporation system of the present disclosure can also include a flexible biasing element that biases a section of tubing disposed within or adjacent to the cooling block against the cooling block.

[0042] In one aspect, the electroporation system of the present disclosure can include one or more flow sensors configured to detect flow (or the absence thereof) through a particular section of the tubing. The flow sensors can be positioned between the mixer reservoir and the electroporation chamber. In some non-limiting embodiments, the flow sensors can be, for example, ultrasonic sensors.

[0043] In one aspect, the casing of the electroporation system of the present disclosure is configured to route one or more sections of tubing within a channel, such as along the exterior of the casing, which provides a visual indication of flow through the section of tubing.

[0044] In one aspect, the casing of the electroporation system of the present disclosure includes one or more handles. In a non-limiting embodiment, the one or more handles can include a handle having a catch configured to engage with the instrument panel to attach the instrument panel to the casing.

[0045] In one aspect, the electroporation system of the present disclosure includes an electroporation cartridge attachment feature coupled to the electroporation cartridge and configured to allow selective attachment and detachment of the electroporation cartridge to the casing. The attachment feature can include a flexible biasing element that biases the electroporation cartridge toward the cooling module.

[0046] In one aspect, the electroporation system of the present disclosure comprises or includes a capping mechanism configured to engage with an inserted electroporation cartridge and actuate one of the electrodes of the electroporation cartridge to move it from a capped position for electroporation to an uncapped position for venting. In some embodiments, the electroporation cartridge of the present disclosure can comprise or include a spring mechanism that allows overtravel of the capping mechanism relative to the displacement of the electrode that is moved as a result of actuation of the capping mechanism.

[0047] In one aspect, the electroporation cartridge of the present disclosure comprises one or more bellows structures each configured to accommodate a moving component of an electroporation chamber.

[0048] In one aspect, the electroporation system of the present disclosure includes an electroporation assembly electrically coupled to an electroporation chamber of an electroporation cartridge. In some embodiments, the electroporation assembly includes a conductivity sensor for measuring conductivity across the electroporation chamber. In some embodiments, the electroporation assembly is communicatively coupled to a controller having one or more processors and one or more hardware storage devices.

[0049] In one aspect, the controller is configured to implement a method for determining the conductivity of a sub-volume within an electroporation chamber of the present disclosure and charge a capacitor accordingly to increase the reproducibility and / or accuracy of electroporation pulses delivered throughout the electroporation of a sample volume (e.g., in a single-use electroporation cartridge of the present disclosure) or through successive sub-volumes (e.g., in a flow-through electroporation cartridge of the present disclosure).

[0050] In one aspect, a controller is configured to implement a method for predicting the risk of arcing during electroporation by determining a predicted temperature rise of a subvolume within an electroporation chamber of the present disclosure. In some embodiments, the controller can determine the conductivity of a subvolume within an electroporation chamber of the present disclosure via a conductivity sensor. In some embodiments, the controller can then (or later) determine a predicted temperature rise of the subvolume based on the determined conductivity, a set pulse voltage, and a set pulse duration. Next, if the predicted temperature rise reaches a temperature of the subvolume that is higher than a predetermined threshold temperature, e.g., about 60°C or about 70°C, the controller can send an arc risk alert. In some embodiments, the controller can further cause the electroporation system to eject the subvolume from the electroporation chamber to protect the sample.

[0051] In one aspect, a controller is configured to implement a method for predicting the risk of arcing during electroporation by determining the presence of bubbles in an electroporation chamber of the present disclosure. In this aspect, the controller can determine the conductivity of a subvolume in the electroporation chamber using a conductivity sensor. The controller can then send an arc risk alert if the determined conductivity is below a predetermined threshold, indicating the presence of one or more bubbles in the electroporation chamber. The controller can further cause the system to evacuate the subvolume from the electroporation chamber to protect the sample.

[0052] In one aspect, the controller is configured to implement a method for determining a calibrated step volume moved by the system between each electroporation event, corresponding to the fill volume of the electroporation chamber. In this aspect, the controller can determine the number of rotations, N, of the drive pump required to move a sample volume sufficient to fill the tubing located between the flow sensor and the electroporation chamber and completely fill the electroporation chamber. This number, N, therefore, corresponds to the volume between the flow sensor and the outlet of the electroporation chamber. Determining that the electroporation chamber is full can be accomplished by a conductivity sensor.

[0053] The controller then causes the drive pump to move the sample backward to a location upstream of the flow sensor using a fixed number of revolutions k, where k corresponds to the volume between the upstream location of the flow sensor and the inlet of the electroporation chamber. The controller can then determine the number of revolutions x of the drive pump required to move the sample from the upstream location of the flow sensor to the flow sensor, where x corresponds to the volume between the upstream location of the flow sensor and the flow sensor. Thus, the number (kx) corresponds to the volume between the flow sensor and the inlet of the electroporation chamber, and the number N-(kx) corresponds to the fill volume of the electroporation chamber.

[0054] In another aspect, the controller is configured to implement a method for determining a calibrated step volume corresponding to the fill volume of the flow-through electroporation chamber, which is moved by the system to the flow-through electroporation chamber between each electroporation event. In one embodiment, such a method includes a first fill of the flow-through electroporation chamber until the sample contacts (touches) the upper electrode (the first electrode described in some embodiments) of the electroporation chamber. At this time (i.e., during the first fill), the electroporation system monitors the electrical resistance within the electroporation chamber for a decrease from several thousand ohms to a stable value in the range of approximately 600-800 ohms. Once this stable electrical resistance value is reached, the first fill is stopped.

[0055] In some embodiments, for the second fill (and subsequent fills), the total fill sample volume is derived from a combination of empirical data and theoretical calculations. Thus, for the second fill (and subsequent fills), the electroporation system (e.g., a controller therein) determines the number of rotations "N rev ”, i.e., determine the number of rotations of the drive pump required to move a sufficient sample volume to completely fill the electroporation chamber (i.e., count the number of rotations from when the sample fluid enters the electroporation chamber from a fixed inlet position until the sample fluid contacts the upper electrode (reaching the sample volume). Additionally, determine the inner tube diameter “d” of the pump tube. i " and the number of rollers "n" of the pump are empirically determined. In a non-limiting example, the pump may be a peristaltic pump, and in some embodiments, may have six rollers (e.g., n=6).

[0056] N revTo determine this value, we consider the number of revolutions of the peristaltic pump required to fill the electroporation chamber from the inlet position to the upper electrode (corresponding to the drop in the resistance of the sample fluid to 600-800 ohms), the inner diameter of the tubing in the pump, d i ", the number of pump rollers "n", the volume of fluid per complete revolution of the pump ("a" μL), the volume of fluid per one roller movement "b" μL, the minimum diameter of the electroporation chamber area where the electroporation sample will reside, and / or the electrical resistance or conductivity of the fluid in the electroporation chamber (measured using a voltmeter, conductivity sensor, etc.), followed by N rev This includes theoretical calculations leading to the

[0057] This is followed by capping the top electrode after the second fill (and any subsequent fills) and measuring the electrical resistance. If the electrical resistance is within the stable range determined from the first fill (i.e., within approximately 600-800 ohms), the fill is complete and you can proceed to electroporate the sample. However, if the electrical resistance is not within the stable range determined from the first fill (i.e., not within approximately 600-800 ohms) after the second fill, you can uncap the top electrode and perform additional electroporation. rev Amount (n of pump rev A step of micro-filling the electroporation chamber with a fluid of N is performed. This is followed by measuring the electrical resistance after the micro-fill by attaching a cap to the upper electrode. If the electrical resistance is within the stable value determined from the first fill (i.e., within approximately 600-800 ohms), the second fill is complete and you can proceed to electroporate the sample. If not, repeat the above file-fill and electrical resistance steps until the electrical resistance is within the stable value determined from the first fill (i.e., within approximately 600-800 ohms). Subsequent fills (third fill, fourth fill, etc.) are performed with N. rev +n revThis is done by x (number of microfill attempts) pump revolutions. In some embodiments, the stable value of electrical resistance is about 700 ohms, for example, in the range of 650-750 ohms and any value therebetween.

[0058] In one embodiment, N rev is calculated as follows: In one embodiment of the system of the present disclosure, with a peristaltic pump with six rollers and tubing with an inner diameter of 2.4 mm, empirical data showed that 172 μL of fluid was dispensed for each full and complete revolution of the pump. From empirically determined data, 28 μL of fluid was dispensed per rotational movement of the pump per roller distance (60 degrees in this case). Following this, theoretical calculations were performed to determine the amount of fluid that could be placed into the electroporation chamber. With a nominal diameter of 6.4 mm (2r, where r = radius of the electroporation chamber), a lower limit of 6.3 mm, and an upper limit of 6.5 mm, πr 2 Using an electroporation chamber height (h) of 30 mm according to the h equation, the nominal sample volume was determined to be 965 μL, with a lower limit of 935 μL and an upper limit of 995 μL. A design tolerance of 0.2 mm for the height (h) is considered insignificant because it results in a maximum variation of only 7 μL. A lower limit of 6.3 mm for the chamber diameter was used in the calculations to reduce sample loss from the total fill due to overfilling the chamber. Based on dividing the calculated volume of 935 μL by the empirical data of 172 μL, the number of revolutions of the peristaltic pump was determined to be 5.4, but rounding to 5.5 was recommended to introduce a convex meniscus. Therefore, in this case, "N rev " is equal to 5.5 revolutions. When the chamber diameter changes from 6.3 mm to 6.5 mm, there is a difference in sample volume of approximately 30 μL per 0.1 mm change in diameter. This is very close to the empirical data of 28 μL of fluid dispensed per rotational movement of the pump per roller distance (60 degrees in this case), which we refer to as the fine filling "n" in the equation. rev " 5.5 rotations (N rev After a total fill of ), the instrument will read the conductivity and if the fill is incomplete, it will read the fine fill "n revDepending on the number of attempts "x", in the case of fine filling, the second filling (and subsequent fillings) are started with "N rev +n rev x".)

[0059] Accordingly, systems, methods, and devices for automated electroporation of cell-containing fluids are disclosed.

[0060] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an indication of the scope of the claimed subject matter.

[0061] Additional objects and advantages of the present disclosure will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the present disclosure. The features and advantages of the present disclosure may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features of the present disclosure will become more fully apparent from the following description and appended claims, or may be learned by the practice of the present disclosure as set forth hereinafter. [Brief explanation of the drawings]

[0062] To explain how the above-enumerated and other advantages and features of the present disclosure can be obtained, a more particular description of the present disclosure, briefly described above, will be rendered by reference to specific embodiments thereof, which are illustrated in the accompanying drawings. It is understood that these drawings illustrate only typical embodiments of the disclosure and therefore should not be considered as limiting its scope. The present disclosure will be described and explained with additional specificity and detail through the use of the accompanying drawings. [Figure 1A] FIG. 1 is an exploded view of an exemplary single-use electroporation cartridge, according to some embodiments of the present disclosure. [Figure 1B]FIG. 1B is a bottom perspective view of the exemplary single-use electroporation cartridge of FIG. 1A shown in a partially assembled and uncapped position according to one embodiment of the present disclosure. [Figure 1C] FIG. 1C is a longitudinal cross-sectional front view of the partially assembled, uncapped electroporation cartridge of FIG. 1B. [Figure 1D] FIG. 1B is a front view of the exemplary single-use electroporation cartridge of FIG. 1A shown in the assembled and capped position. [Figure 1E] FIG. 1E is a longitudinal cross-sectional front view of the assembled and capped electroporation cartridge of FIG. 1D. [Figure 2A] FIG. 1 is an exploded front view of an exemplary single-use electroporation cartridge according to some embodiments of the present disclosure having a volume reduction sleeve. [Figure 2B] 2B is an exploded top perspective view of the exemplary single-use electroporation cartridge and volume reduction sleeve of FIG. 2A. [Figure 2C] FIG. 2B is a front view of the exemplary single-use electroporation cartridge of FIG. 2A, showing the electroporation cartridge in a partially assembled, uncapped position and with the volume reduction sleeve attached to the removable cap. [Figure 2D] FIG. 2D is a longitudinal cross-sectional front view of the partially assembled and uncapped electroporation cartridge and volume reduction sleeve of FIG. 2C. [Figure 2E] FIG. 2B is a front view of the exemplary single-use electroporation cartridge of FIG. 2A shown assembled and capped. [Figure 2F] FIG. 2C is a longitudinal cross-sectional front view of the assembled and capped electroporation cartridge of FIG. 2E, showing a volume reduction sleeve positioned within a chamber defined by the elongated body of the cartridge to form a reduced-volume electroporation compartment between opposing electrodes. [Figure 3] FIG. 10 is a longitudinal cross-sectional front view of another single-use electroporation cartridge according to some embodiments of the present disclosure. [Figure 4] FIG. 1 is a perspective view of another exemplary single-use electroporation cartridge having an electrode cap, according to some embodiments of the present disclosure. [Figure 5] FIG. 10 is a cross-sectional view of another exemplary single-use electroporation cartridge having an authentication chip coupled to an electrode cap, according to some embodiments of the present disclosure. [Figure 6A] FIG. 10 is a perspective view of another exemplary single-use electroporation cartridge having an authentication chip coupled to an electrode cap and a gripping member coupled to a chamber body, according to some embodiments of the present disclosure. [Figure 6B] FIG. 6B is a top view of the single-use electroporation cartridge of FIG. 6A. [Figure 6C] FIG. 6B is a front view of the single-use electroporation cartridge of FIG. 6A. [Figure 6D] 6D is a cross-sectional view (taken along plane 6D shown in FIG. 6C) of the single-use electroporation cartridge of FIG. 6C. [Figure 7A] 1 shows an exploded front view of an exemplary flow-through electroporation cartridge according to some embodiments of the present disclosure, with the cartridge body shown as transparent for ease of viewing and description herein. [Figure 7B] FIG. 7B is a top perspective view of the disassembled flow-through electroporation cartridge of FIG. 7A. [Figure 7C] FIG. 7B is a front view of the exemplary flow-through electroporation cartridge of FIG. 7A assembled and shown with the cartridge body surface shaded rather than transparent. [Figure 7D] FIG. 7D is a front view of a longitudinal section of the assembled flow-through electroporation cartridge of FIG. 7C, the section bisecting the first electrode and the ports associated with the chamber inlet and outlet. [Figure 8A] FIG. 1 is a top perspective view of another embodiment of a flow-through electroporation cartridge, the illustrated flow-through electroporation cartridge having a reservoir integrated with the chamber body and shown partially assembled with the upper sleeve cap disassembled away from the chamber body. [Figure 8B] 8B is a top perspective view of the flow-through electroporation cartridge of FIG. 8A in a partially assembled state, with the upper sleeve cap mated to the proximal opening of the chamber body. FIG. [Figure 8C] FIG. 8C is a longitudinal cross-sectional view (taken along plane 8C shown in FIG. 8B) of the flow-through electroporation cartridge of FIG. 8B, where the illustrated flow-through electroporation cartridge is shown with an upper electrode attached thereto, and the arrows shown in the drawing indicate overflow fluid being discharged from the electroporation chamber in response to being collected in an associated reservoir. [Figure 9] 1 is another embodiment of a flow-through electroporation cartridge according to some embodiments of the present disclosure. [Figure 10A] 1 illustrates a schematic diagram of one embodiment of an electroporation process flow through an exemplary electroporation system of the present disclosure. [Figure 10B] 1 illustrates one embodiment of a control system communicatively coupled to one or more components of an electroporation system of the present disclosure and configured to provide one or more computer-implemented process control methods to the system. [Figure 11A] 1 illustrates an exemplary embodiment of an electroporation system or electroporation device, showing one embodiment of an electroporation device. [Figure 11B] 1 illustrates another exemplary embodiment of an electroporation system or electroporation device with the lid in an open position. [Figure 11C]1 illustrates an exemplary embodiment of an electroporation system or electroporation device, including an electroporation device and a removably attachable modular casing that can contain various electroporation components, such as cells and reagents. [Figure 11D] 1 illustrates yet another exemplary embodiment of an electroporation system or electroporation device, the electroporation device having a lid. [Figure 12A] 1 illustrates an example embodiment of a modular casing for placing various electroporation components that can be selectively attached and detached from an electroporation device. [Figure 12B] 1 illustrates another exemplary embodiment of a modular casing for positioning various electroporation components that can be selectively attached to and detached from an electroporation device. [Figure 12C] 1 illustrates another exemplary embodiment of a modular casing for positioning various electroporation components that can be selectively attached to and detached from an electroporation device. [Figure 12D] 1 illustrates another exemplary embodiment of a modular casing for positioning various electroporation components that can be selectively attached to and detached from an electroporation device. [Figure 12E] 1 illustrates another exemplary embodiment of a modular casing for positioning various electroporation components that can be selectively attached to and detached from an electroporation device. [Figure 12F] 12B-12E show different views of the empty modular casing, illustrating the attachment features that allow for removable attachment of the casing to the electroporation instrument. [Figure 12G]12B-12E show different views of the empty modular casing, illustrating the attachment features that allow for removable attachment of the casing to the electroporation instrument. [Figure 13A] 10 illustrates yet another embodiment of a modular casing for placing various electroporation components that can be selectively attached and detached from an electroporation system / instrument. [Figure 13B] 13B shows a rear view of the modular casing of FIG. 13A. [Figure 13C] 1 illustrates one embodiment of additional electroporation components that can be attached to the modular casing of the present disclosure to allow for manual dispensing of solutions / payloads / reagents into the electroporation cell mixer chamber as needed. [Figure 14A] 1 shows an example of a bag compartment that can be incorporated into the electroporation systems / devices described herein. [Figure 14B] 1 shows an example of a bag compartment that can be incorporated into the electroporation systems / devices described herein. [Figure 15A] 1 illustrates an example of a sample transfer assembly configured to provide efficient transfer of sample fluid from an input container to a mixer reservoir and an electroporation chamber, according to one embodiment of the present disclosure. [Figure 15B] 1 illustrates an example of a sample transfer assembly configured to provide efficient transfer of sample fluid from an input container to a mixer reservoir and an electroporation chamber, according to one embodiment of the present disclosure. [Figure 15C] 1 illustrates an example of a sample transfer assembly configured to provide efficient transfer of sample fluid from an input container to a mixer reservoir and an electroporation chamber, according to one embodiment of the present disclosure. [Figure 15D]1 illustrates an example sample transfer assembly showing the flow path of sample fluid from the input reservoir through various tubes and components of an electroporation system, to the input tubing, to the cell mixer reservoir, and to the electroporation chamber, according to one embodiment of the present disclosure. [Figure 15E] 1 illustrates an example sample transfer assembly showing the path of sample fluid flow from the input container through various tubes and components of the electroporation system, to the input tubing, to the cell mixer reservoir and electroporation chamber, according to one embodiment of the present disclosure. [Figure 15F] 1 illustrates an example of a sample transfer assembly showing the path of air flow while flowing sample fluid from the input tube to the electroporation chamber, according to one embodiment of the present disclosure. [Figure 15G] 1 illustrates an example of a sample transfer assembly showing the path of air flow while flowing sample fluid from the input tube to the electroporation chamber, according to one embodiment of the present disclosure. [Figure 15H] 1 illustrates an example of an airflow control assembly including an air filter, tubing, and stopcock for an electroporation system / casing module, according to one embodiment of the present disclosure. [Figure 15I] 10 illustrates a step of disassembling an electroporation casing module from an electroporation device according to one embodiment of the present disclosure. [Figure 16A] 1 shows a close-up view of an exemplary mixer reservoir configured to hold the transferred sample and maintain the sample in a homogenous suspension while successive sub-volumes of the sample are passed to the electroporation cartridge for electroporation, according to some embodiments. [Figure 16B] 1 shows a close-up view of an exemplary mixer reservoir configured to hold the transferred sample and maintain the sample in a homogenous suspension while successive sub-volumes of the sample are passed to the electroporation cartridge for electroporation, according to some embodiments. [Figure 17A] 1 illustrates an exemplary embodiment of a pre-cooling module positioned upstream of the electroporation cartridge and configured to cool samples passing therethrough prior to electroporation. [Figure 17B] 1 illustrates an exemplary embodiment of a pre-cooling module positioned upstream of the electroporation cartridge and configured to cool samples passing therethrough prior to electroporation. [Figure 17C] 1 illustrates an exemplary embodiment of a pre-cooling module positioned upstream of the electroporation cartridge and configured to cool samples passing therethrough prior to electroporation. [Figure 17D] 1 shows an example of a cooling module configured to cool the electroporation chamber itself. [Figure 18A] 1 shows an example of a high voltage touch pin of an electroporation instrument of the present disclosure not engaged with an exemplary electroporation chamber. [Figure 18B] 1 shows an example of a high voltage touch pin of an electroporation instrument of the present disclosure not engaged with an exemplary electroporation chamber. [Figure 18C] 1 shows an example of a high voltage touch pin of an electroporation instrument of the present disclosure engaging with an electroporation chamber. [Figure 18D] 1 shows an example of a high voltage touch pin of an electroporation instrument of the present disclosure engaging with an electroporation chamber. [Figure 19A] 10 illustrates an embodiment of an electroporation cartridge mounting feature configured to allow the cartridge to be mounted in the appropriate location within the cooling module. [Figure 19B] 10 illustrates an embodiment of an electroporation cartridge mounting feature configured to allow the cartridge to be mounted in the appropriate location within the cooling module. [Figure 19C] 10 illustrates an embodiment of an electroporation cartridge mounting feature configured to allow the cartridge to be mounted in the appropriate location within the cooling module. [Figure 20A] 10A-10C illustrate operation of a capping mechanism to transfer an electroporation cartridge from a capped state in preparation for electroporation to an uncapped state in preparation for filling or draining a corresponding electroporation chamber, according to some embodiments. [Figure 20B] 10A-10C illustrate operation of a capping mechanism to transfer an electroporation cartridge from a capped state in preparation for electroporation to an uncapped state in preparation for filling or draining a corresponding electroporation chamber, according to some embodiments. [Figure 20C] 10A-10C illustrate operation of a capping mechanism to transfer an electroporation cartridge from a capped state in preparation for electroporation to an uncapped state in preparation for filling or draining a corresponding electroporation chamber, according to some embodiments. [Figure 21] 10A-10C illustrate the capping mechanism and its operation in conjunction with the cap of an electroporation cartridge, and illustrate the function of a spring-based mechanism according to some embodiments that allows overtravel of the capping mechanism to ensure that the electrodes are fully moved to the desired position despite different dimensional tolerances of the cartridge. [Figure 22A] 1 illustrates in greater detail an example of a sealing mechanism and associated components of an electroporation cartridge of the present disclosure, according to some embodiments. [Figure 22B] 1 illustrates in greater detail an example of a sealing mechanism and associated components of an electroporation cartridge of the present disclosure, according to some embodiments. [Figure 22C] 1 illustrates in greater detail an example of a sealing mechanism and associated components of an electroporation cartridge of the present disclosure, according to some embodiments. [Figure 22D] 1 illustrates in greater detail an example of a sealing mechanism and associated components of an electroporation cartridge of the present disclosure, according to some embodiments. [Figure 22E]1 illustrates in greater detail an example of a sealing mechanism and associated components of an electroporation cartridge of the present disclosure, according to some embodiments. [Figure 22F] 22E show an exemplary flow-through electroporation cartridge including the sealing mechanism shown in FIG. 22E coupled to the inlet and outlet ports in various open and closed configurations, according to some embodiments. [Figure 22G] 22E show an exemplary flow-through electroporation cartridge including the sealing mechanism shown in FIG. 22E coupled to the inlet and outlet ports in various open and closed configurations, according to some embodiments. [Figure 22H] 22E shows an exemplary flow-through electroporation cartridge including the sealing mechanism shown in FIG. 22E in an open and closed configuration coupled to an outlet port, according to one embodiment. [Figure 22I] 22E shows an exemplary flow-through electroporation cartridge including the sealing mechanism shown in FIG. 22E in an open and closed configuration coupled to an outlet port, according to one embodiment. [Figure 22J] 1A and 1B show an umbrella valve in an open and closed configuration, respectively, for use as a sealing mechanism, according to one embodiment of the present disclosure. [Figure 22K] 1A and 1B show an umbrella valve in an open and closed configuration, respectively, for use as a sealing mechanism, according to one embodiment of the present disclosure. [Figure 22L] 22J and 22K are top views of the inlet ports with the corresponding umbrella valves removed from the view. [Figure 22M] 1 shows an example of a setup for pressurizing and sealing an outlet port fitted with a check valve (such as a mini-valve) of an exemplary electroporation cartridge, according to one embodiment. [Figure 22N] 1 shows an example of a setup for pressurizing and sealing an outlet port fitted with a check valve (such as a mini-valve) of an exemplary electroporation cartridge, according to one embodiment. [Figure 22O]1 shows an example of a setup for pressurizing and sealing an outlet port fitted with a check valve (such as a mini-valve) of an exemplary electroporation cartridge, according to one embodiment. [Figure 22P] 1 shows an example of a setup for pressurizing and sealing an outlet port fitted with a check valve (such as a mini-valve) of an exemplary electroporation cartridge, according to one embodiment. [Figure 22Q] 1 shows an example of a setup for pressurizing and sealing an outlet port fitted with a check valve (such as a mini-valve) of an exemplary electroporation cartridge, according to one embodiment. [Figure 22R] 1 shows an example of a setup for pressurizing and sealing an outlet port fitted with a check valve (such as a mini-valve) of an exemplary electroporation cartridge, according to one embodiment. [Figure 22S] 1 shows an example of a setup for pressurizing and sealing an outlet port fitted with a check valve (such as a mini-valve) of an exemplary electroporation cartridge, according to one embodiment. [Figure 23] 1 illustrates a method for predicting the risk of arcing during an electroporation operation based on predicted temperature changes of a sample sub-volume, according to one embodiment. [Figure 24] 1 illustrates a method for preventing arcing in an electroporation chamber based on an initial conductivity measurement of a sub-volume, according to one embodiment. [Figure 25] 1 shows a schematic diagram of an electroporation circuit, according to one embodiment. [Figure 26] 1 illustrates a method for generating reproducible and consistent electrical pulses across an electroporation chamber, according to one embodiment. [Figure 27A] 1 illustrates a method for calibrating the fill volume of an electroporation chamber, according to one embodiment. [Figure 27B] 1 illustrates a method for calibrating the fill volume of an electroporation chamber, according to one embodiment. [Figure 27C] 1 illustrates a method for calibrating the fill volume of an electroporation chamber, according to one embodiment. [Figure 27D] 1 illustrates a method for calibrating the fill volume of an electroporation chamber, according to one embodiment. [Figure 27E] 1 illustrates a method for calibrating the fill volume of an electroporation chamber, according to one embodiment. [Figure 28] 1 shows an exemplary method flow for preparing cells for transformation by one or more electroporation systems disclosed herein, according to one or more embodiments of the present disclosure. [Figure 29A] FIG. 29 shows an exemplary method flow for batch processing and transformation of cells prepared, for example, by the exemplary method outlined in FIG. 28, according to one or more embodiments of the present disclosure. [Figure 29B] 29A and 29B are graphs showing exemplary viability and transformation efficiency of primary cells prepared according to the protocols outlined in FIGS. 28 and 29A. [Figure 30A] 29 shows an exemplary method for flow-through processing and transformation of cells prepared, for example, by the exemplary method outlined in FIG. 28, according to one or more embodiments of the present disclosure. [Figure 30B] 30A and 30B are graphs showing exemplary viability and transformation efficiency of primary cells prepared according to the protocols outlined in FIG. 28 and FIG. 30A. [Figure 31] 1 is a graph showing the results of batch and flow-through processing and electroporation of primary cells according to one or more embodiments of the present disclosure. [Figure 32] 1 is a graph showing the viability and transformation efficiency of an exemplary flow-through system and method for transforming immortalized cell culture cells via electroporation, according to one or more embodiments of the present disclosure. [Figure 33]1 is a series of graphs showing an exemplary comparison of transformation efficiency and cell viability of immortalized cell cultures electroporated using the systems and methods disclosed herein versus conventional electroporation systems. [Figure 34] 1 is a graph showing the viability and transformation efficiency of cells transformed with a knock-in gene using an exemplary single-use consumable and electroporation system and method according to one or more embodiments of the present disclosure. [Figure 35] 1 is a graph showing transduction efficiency and cell viability in the generation of CAR-T cells using the flow-through and single-use cartridges and electroporation systems and methods disclosed herein, in accordance with one or more embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0063] Before describing various embodiments of the present disclosure in detail, it is understood that the present disclosure is not limited to the parameters of the particularly illustrated systems, methods, apparatus, products, processes, and / or kits, which may, of course, vary. Thus, while particular embodiments of the present disclosure will be described in detail with reference to particular configurations, parameters, components, elements, etc., the description is illustrative and should not be construed as limiting the scope of the claimed invention. Moreover, the terminology used herein is for the purpose of describing embodiments and is not necessarily intended to limit the scope of the claimed invention.

[0064] Furthermore, unless otherwise understood or stated, implicitly or explicitly, it will be understood that for any given component or embodiment described herein, any of the possible candidates or alternatives listed for that component may generally be used individually or in combination with one another. Furthermore, unless otherwise understood or stated, implicitly or explicitly, it will be understood that any listing of such candidates or alternatives is merely exemplary and not limiting.

[0065] Additionally, numerical values ​​expressing quantities, components, distances, or other measurements used in the specification and claims, unless otherwise indicated, should be understood to be modified by the term "about," as defined herein. Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the subject matter presented herein. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least 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 the subject matter presented herein are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0066] The headings and sub-headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims.

[0067] Overview and Advantages of an Exemplary Electroporation Cartridge Embodiments of the present disclosure relate to electroporation systems and their various components, including several types of electroporation chambers, electroporation devices, electroporation cartridges, electroporation modules, module casings, electroporation instruments, as well as electroporation systems including systems having computer processors, user interfaces, and computer-implemented methods.

[0068] Embodiments of the present disclosure relate to electroporation systems and related instruments and devices, such as electroporation cartridges and / or electroporation chambers useful for single electroporation experiments.Embodiments of the present disclosure relate to electroporation systems and instruments for non-automated electroporation.

[0069] Embodiments of the present disclosure relate to high-throughput electroporation systems and related instruments and devices, such as electroporation cartridges, and / or chambers and / or electroporation casings / modules.Embodiments of the present disclosure relate to electroporation systems and instruments for automated high-throughput electroporation.

[0070] As previously discussed, there are many disadvantages and problems that can be addressed in the field of electroporation. Embodiments of the present disclosure address many of the disadvantages and problems in the field of automated, high-throughput electroporation as well as in single-shot electroporation applications. For example, embodiments of the present disclosure provide devices, apparatus, systems, and methods that can perform electroporation in a functionally closed, sterile pathway, and provide one or more advantages, including minimizing or substantially reducing arcing, minimizing or substantially reducing bubble formation and associated arcing in cell-containing fluids, significantly improving or maximizing transfection efficiency and transfected cell recovery efficiency, providing a uniform electric field for efficient electroporation, etc.

[0071] In some embodiments, the systems, devices, instruments, and methods of the present disclosure combine the advantages of continuous flow electroporation with the advantages of batch or static volume electroporation, specifically the ability to automatically electroporate large numbers of cells in a sterile, closed system while minimizing arcing due to bubble formation in the cell-containing fluid and maximizing recovery efficiency.

[0072] Embodiments of the present disclosure address one or more of the aforementioned problems in the field of automated electroporation. For example, Figures 1A-6D, 10A and 10B, and 11A-11D and their corresponding descriptions illustrate various electroporation devices, such as single-use electroporation cartridges, as well as electroporation instruments and systems that enable highly efficient electroporation. In other examples, the embodiments illustrated in Figures 7A-9, 10A-22S, and their corresponding descriptions illustrate various electroporation devices, such as flow-through electroporation cartridges, electroporation modules, electroporation instruments, and systems that enable highly efficient electroporation of large volumes of cell-containing fluids using automated batch processes. Exemplary methods utilizing one or more electroporation cartridges are presented in Figures 23-33 and their corresponding descriptions.

[0073] Advantageously, the disclosed systems, devices (such as single-use and flow-through electroporation cartridges), and methods reduce bubble formation in several ways, including, for example, pressurizing the electroporation chamber prior to electroporation, which can make vaporizing aqueous cell-containing fluids and electrolyzing water molecules into oxygen and hydrogen gases more energy-intensive (and thereby more difficult or less likely). Furthermore, the disclosed single-use and flow-through electroporation cartridges additionally direct any bubbles formed during processing away from the electrode surface, thereby further reducing the likelihood of arcing. Because fewer bubbles form or collect on or near the electrode surface, embodiments of the present disclosure experience less arcing and maximize the recovery efficiency of each sample.

[0074] The disclosed flow-through devices, modules, and systems further offer the advantage of automating the manual process of loading and unloading samples into and from electroporation cuvettes, a process that many have attempted and failed to achieve. Instead of the continuous processing performed in prior art systems, which tends to reduce electroporation efficiency, the disclosed systems, cartridges, and methods enable automated batch electroporation, combining the advantages of continuous flow electroporation with the advantages of batch or static volume electroporation while minimizing the inherent drawbacks of each. For example, the devices and systems disclosed herein provide a sterile, closed system that eliminates (or significantly reduces) the risk of sample contamination previously associated with batch or static processing techniques. Preferably, the devices and systems disclosed herein comply with ISO (International Organization for Standardization) guidelines and are fabricated from materials suitable for use in cell and gene therapy applications.

[0075] Exemplary Electroporation Cartridge As discussed above, electroporation systems known in the art have many disadvantages. In particular, arcing during electroporation is undesirable because it affects transfection efficiency and cell viability. There is a need for systems and devices that minimize or prevent arcing in electroporation cartridges and electroporation chambers designed for single-use use. There is a need for systems and devices that minimize or prevent arcing in continuous-flow or continuous-batch electroporation processes.

[0076] One of the main causes of arcing is the presence of gas bubbles at or near the electrode surface and / or in the path of the current generated by applying a high voltage to the cell-containing medium during electroporation. When a high voltage is applied, as in electroporation, almost all bubbles of significant size will generate arcing. Gas bubbles are known to form as a result of heat generated from an electrical discharge passing through the cell-containing aqueous solution. This concentrated, frequently repeated heating can also cause bubble formation by localized vaporization of water molecules. The electrical discharge passing through the cell-containing solution during electroporation can cause electrolysis of water molecules, forming oxygen and hydrogen gases, which are another source of gas bubbles during electroporation.

[0077] Embodiments of the present disclosure include electroporation cartridges, associated systems, and methods for using the same that enable reduced bubble formation, even during electroporation of large volumes (e.g., 1 mL or greater) that would traditionally result in arcing. An exemplary single-use electroporation cartridge is shown in FIGS. 1A-1E, where FIG. 1A shows an exploded view of the components of the exemplary electroporation cartridge, FIGS. 1B and 1C show the electroporation cartridge of FIG. 1A in a partially assembled, uncapped position, and FIGS. 1D and 1E show a cross-sectional, surface-shaded view of the assembled cartridge in the capped position. The illustrated single-use electroporation cartridge, along with other single-use electroporation cartridges described herein, may include one or more security features to ensure that they are used only once. For example, single-use electroporation cartridges may include locking features, separation features, and / or other mechanical features that prevent reuse of the electroporation cartridge after insertion and removal from an electroporation system. The single use of the single-use electroporation cartridges may additionally or alternatively be assured electronically, for example, via an electronic tag or code associated with each single-use electroporation cartridge and scanned / recorded by the electroporation system.

[0078] 1A-1E, an electroporation cartridge 100 includes an electroporation chamber 102 defined by an elongated body 104, a first electrode 106 disposed at a proximal end 110 of the electroporation chamber 102, and a second electrode 108 disposed at an opposite distal end 114 of the electroporation chamber 102, wherein at least one of the first electrode 106 or the second electrode 108 is movable from a capped position for electroporation to an uncapped position for sample loading (e.g., as shown between FIGS. 1B, 1C, 1D, and 1E). As particularly shown in FIG. 1A, the elongated body has two open ends (proximal end 110 defining a proximal opening 112 and distal end 114 defining a distal opening 116) at which the first electrode 106 and the second electrode 108 are disposed, respectively. According to one embodiment, the first electrode 106 is coupled with a removable cap 107 that allows for selective coupling of the first electrode 106 to the elongate body 104. In some embodiments, the second electrode 108 of the cartridge 100 is inserted into a distal opening 116 of the elongate body 104, where it is secured in place by a distal cap 118. In some embodiments, the second electrode 108 is secured within and / or attached to the distal end 114 of the elongate body 104 by the distal cap 118 via a locking feature to prevent the bottom cap from being removed. This may advantageously reduce the likelihood of a user becoming confused about which end to use to add and / or remove cell-containing fluid for electroporation, and also provides the benefit of a modular construction that can be partially assembled to create a structurally sound cartridge.

[0079] 1C , the second electrode 108 further includes a first sealing member 120 disposed between the second electrode 108 and a distal surface 122 of the elongate body 104, the first sealing member 120 operative to form a fluid-tight joint between the second electrode 108 and the distal surface 122 of the elongate body 104. In some cases, the fluid-tight joint is provided by compressing a sealing member (e.g., an O-ring or other gasket) between the electrode flange 124 and a distal body flange 126 that lies in a plane substantially parallel to the electrode flange 124. In some embodiments, the distal cap 118 engages and locks with the elongate body using the sealing member 120 disposed between the electrode flange 124 and the distal body flange 126 to form a fluid-tight joint therebetween.

[0080] 1C , in some embodiments, second electrode 108 may include a protruding portion 128 that extends from distal end 114 of elongate body 104 into electroporation chamber 102 to define a bottom of electroporation chamber 102. In some embodiments, second electrode 108 may further include a second sealing member 130 disposed around protruding portion 128 and distal to a proximal face of the second electrode that forms a fluid-tight joint with an inner wall of electroporation chamber 102. The first sealing member 120 forms a fluid-tight seal between the second electrode 108 and the elongate body 104 by compressing the distal cap 118, sealing the distal end of the electroporation chamber 102 from the external environment, while the second sealing member 130 forms a fluid-tight joint between the protruding portion 128 of the second electrode 108 and the inner sidewall of the electroporation chamber 102 by creating a seal near the wetted proximal surface of the second electrode 108 to prevent fluid from permeating around the second electrode 108, thereby minimizing dead volume.

[0081] 1C, the circumference of the protruding portion 128 can have a shape complementary to the contour of the inner surface of the elongated body 104 that defines the electroporation chamber 102, such that the diameter of the proximal end of the second electrode 108 is substantially equal to the cross-section of the electroporation chamber 102. Additionally, the proximal surface of the second electrode can be a flat, uniform surface disposed perpendicular to the longitudinal axis of the electroporation chamber.

[0082] The inclusion of one or more of the aforementioned structural features in the shape and / or location of the second electrode relative to the electroporation chamber can provide certain advantages. For example, generating a uniform electric field is one factor for successful and efficient electroporation. To have a uniform electric field, it is advantageous for the opposing first and second electrodes to be substantially parallel and have essentially the same cross-sectional shape as the electroporation chamber. A uniform electric field is most effectively generated in an electroporation chamber having a uniform cross-section (e.g., a constant diameter). Thus, in some embodiments, the electroporation cartridge disclosed herein can include an electroporation chamber having a uniform cross-section along the length of the reaction chamber. The uniform cross-section can extend the entire length of the electroporation chamber between the first and second electrodes such that the electroporation cartridge is configured to generate a uniform electric field within the electroporation chamber disposed between the first and second electrodes. As a non-limiting example of the foregoing, the electroporation chamber can be defined as a cylindrical cavity having a circular cross-section extending along the entire length of the cylindrical cavity between the first and second electrodes.

[0083] Alternatively, the uniform cross-section can extend along less than the entire length of the electroporation chamber. In such embodiments, at least a portion of the electroporation chamber can be tapered between the first and second electrodes. Preferably, the tapered portion of the electroporation chamber does not substantially interfere with the generation of a uniform electric field between the first and second electrodes. For purposes of this disclosure, the tapered portion of the electroporation chamber does not substantially interfere with the generation of a uniform electric field when the electric field generated between opposing first and second electrodes is defined by field lines that are substantially parallel and equally spaced within a 10% tolerance. For clarity, the taper in the electroporation chamber can include a constriction in the proximal sidewall defined between the proximal opening of the elongate body and an inflection point on the sidewall defining the electroporation chamber, where the proximal sidewall narrows from a first diameter defined by the proximal opening to a second, smaller diameter defined distal to the inflection point.

[0084] It should be understood that the presence of a taper or uniform cross-section along the entire length of the electroporation chamber may affect the methods available for efficiently and / or cost-effectively manufacturing the electroporation chamber. Preferably, the elongate body and / or electroporation chamber are made of or include a non-conductive, radiation-resistant plastic, ceramic, and / or glass. For example, the elongate body and / or electroporation chamber may be made of polycarbonate or another non-conductive, gamma-ray-resistant plastic. Alternatively, both glass and ceramic are more electrically insulating and more thermally conductive than polycarbonate, and advantageously, either material can be mass-produced with zero draft sidewalls and can provide a constant cross-section.

[0085] In some embodiments, the chamber is made of a material that can be sterilized without impairing the functionality of the chamber (e.g., without reducing electroporation efficiency or affecting cell viability) by one or more of steam sterilization, flash sterilization, hydrogen peroxide sterilization, vaporized hydrogen peroxide sterilization, gamma sterilization, peracetic acid sterilization, ethylene oxide sterilization, chlorine dioxide gas sterilization, electron beam sterilization, etc.

[0086] As previously mentioned, a uniform electric field can be generated between two opposing electrodes with a cross-section that approximates the uniform cross-sectional shape and size of the electroporation chamber. However, the inventors discovered that in manufacturing various cartridges with these characteristics and shapes, air tends to become trapped more easily when the chamber is sealed. Trapped air can reduce electrical conductivity within the electroporation chamber, resulting in arcing and potentially adversely affecting the electroporation process and performance.

[0087] To overcome the problem of air entrapment, the electroporation cartridge disclosed herein takes advantage of surface tension at the liquid-air interface, where the mutual attraction of liquid molecules is greater than that of molecules in air. This creates a convex meniscus, which, when capped with the upper electrode, forces the liquid to be expelled around the electrode, preventing air from being trapped between the sample and the distal surface of the first electrode. To facilitate this, the distal portion of the first electrode 106 can have a bell-shaped or bulbous protrusion 132 separated from the base region 134 by a narrow stem 136, as shown in Figures 1A-1E. The bulbous protrusion 132 can have a diameter smaller than the cross-section of the electroporation chamber 102, forming a gap between it and the electroporation chamber 102. This gap between the bulbous protrusion and the chamber body allows small air bubbles to escape from the electroporation volume. The narrow stem 136 has the effect of creating a larger volume of air in the proximal portion of the chamber, which is the volume that is compressed during electroporation, minimizing the pressure buildup in the chamber caused by partial vaporization of the sample.

[0088] The bell-shaped or bulbous shape of the distal end of the first electrode can provide additional benefits during electroporation. Gas bubbles can form during electroporation due to electrolysis and / or water vaporization. The bulbous extension can function to expel one or more gas bubbles generated during electroporation, removing them from the electrode surface before they coalesce into bubbles large enough to cause arcing. For example, the bulbous extension can have an arc-shaped or convex surface that encourages gas bubbles rising from the electroporation volume to pass along the surface of the bulbous extension and rise to the sample-air interface proximate the stem.

[0089] Furthermore, it has been found that, advantageously, higher pressures can reduce the intensity of vaporization, resulting in smaller and / or fewer bubbles forming during electroporation. By sealing the electroporation chamber prior to electroporation, the chamber behaves like a pressure chamber due to the release of energy and electrolysis that occurs during electroporation. Increasing the pressure within the chamber delays the formation of bubbles from reaching a significant size that could result in arcing.

[0090] 1A-1E, the first electrode 106 is coupled with a sealing member 138 that functions to form a fluid-tight joint between the first electrode and the elongated body 104. In some embodiments, axial compression to maintain the fluid-tight seal can be provided by a removable cap 107 that couples to the first electrode 106. In some embodiments, the removable cap 107 is threadedly sealed to the elongated body 104, although it should be understood that other forms of coupling are contemplated herein (e.g., friction fitting, snap fitting, etc.). As shown in FIGS. 1A-1E, each of the caps 107, 118 features flanges that extend beyond the outer surface of the electrode. These flanges can provide additional balance and stability to the device, such as when placed on a flat surface. Without the illustrated flanges, the cartridge's narrow footprint and midline center of gravity could potentially cause instability.

[0091] As the first electrode 106 is removed by a user during normal operation, the sealing member may tend to become lost or detached from the electrode when it is detached from the elongate body. To prevent this, the electrode may include a retention feature 140 proximal to the stem 136 configured to allow the sealing member 138 to extend over but be retained thereon, thereby abutting a sealing surface of the first electrode 106. This sealing surface creates a functionally closed system at the upper end of the chamber when the cap 107 is coupled thereto.

[0092] In some embodiments, the first electrode 106 can further include a cap retention feature 142 to prevent the end cap from becoming dislodged from the electrode after it has been attached. The cap retention feature 142 can function as a snap-fit ​​barb, can be threadedly secured to the cap, or can be retained by any other means known in the art. In particular, in some embodiments, it can be beneficial to secure the first electrode to the cap such that the cap rotates independently of the first electrode, so that the attached sealing member is only subjected to axial compression.

[0093] In some embodiments, the electrodes 106, 108 are made from or plated with a conductive material that does not adversely affect cells by introducing harmful or toxic elements, either passively or during electroporation. For example, plating the electrodes with pure gold can impart beneficial electrical conductivity to the electrodes that is unlikely to introduce harmful or toxic elements into the electroporation medium. Furthermore, it should be understood that the electrodes 106, 108 can be connected to a high-voltage circuit and can function as anodes or cathodes, or alternate between the two, depending on the electroporation protocol. Alternatively, other non-toxic and / or non-reactive metals or materials can be used, as known in the art.

[0094] In some embodiments, the volume within the electroporation chamber of the present disclosure is larger than that of a conventional electroporation cuvette. In some embodiments, the electroporation chamber has an internal volume of about 10 mL to about 1 mL. In some embodiments, the electroporation chamber has an internal volume of about 1 mL to about 100 μl.

[0095] In some embodiments, exemplary electroporation chambers of the present disclosure can have a volume of less than about 5 mL, preferably less than about 3 mL, or in some embodiments, less than about 4 mL, less than about 2 mL, or less than about 1 mL. In some embodiments, when the internal volume of the electroporation chamber is about 10 mL to about 1 mL, the distance between the first and second electrodes is between about 20 mm and 100 mm, e.g., between about 30 mm and 50 mm, between about 40 mm and 70 mm, and / or between about 60 mm and about 100 mm, in preferred voltage ranges for electroporating most cell samples at these volumes.

[0096] In another exemplary embodiment, the volume of the electroporation chamber is about 1 mL, or about 100 μL to 1 mL. In some embodiments, at this volume and at a preferred range of voltages for electroporating most cell samples, the distance between the first and second electrodes is between about 20 mm and 40 mm, e.g., between about 22 mm and 38 mm, between about 25 mm and 35 mm, and preferably about 30 mm. Having an electroporation chamber configured to this size is advantageous because a standard 1 mL pipette can easily fit into the body of the 1 mL electroporation chamber, thus aspirating nearly 100% of the electroporated sample, without requiring any specialized equipment beyond that typically used to transfer such volumes of liquid in a laboratory or clinical setting.

[0097] Volume reduction sleeve In some cases, the sample volume is less than 1 mL. If the distance between the two electrodes remains the same, the diameter of the chamber must be significantly reduced to accommodate the smaller volume. The problem is that existing standard pipette tips are unlikely to fill or eject small-volume chambers.

[0098] 1A-1E 。 Essentially, the elongate body is fitted with a volume-reducing sleeve that forms a fluid-tight junction with the second (lower) electrode and defines an inner, secondary electroporation chamber having a smaller volume than that defined by the original elongate body 104. In this way, the electroporation cartridge described above can be easily adapted for small sample volumes while maintaining the benefits and advantages of the original design.

[0099] 2A-2F illustrate an exemplary embodiment of the electroporation cartridge of FIGS. 1A-1E configured with a volume reduction sleeve. As shown in these figures, the volume reduction sleeve defines a secondary electroporation chamber with a volume of approximately 200 μL. However, it should be understood that this volume is exemplary in nature, and other volumes are contemplated herein and can be achieved by adjusting the cross-sectional circumference of the volume reduction sleeve. For example, a 1 mL elongated body can support volumes from 100 μL to 1 mL using sleeves of various sizes. Specifically, smaller volume sleeves, such as 100 μL sleeves, can be created by reducing the cross-sectional circumference of the sleeve. Alternatively, larger volume sleeves, such as 200 μL, 250 μL, 400 μL, 450 μL, or 500 μL sleeves, can be created by increasing the cross-sectional circumference of the volume reduction sleeve.

[0100] 2A-2F, Figures 2A and 2B are exploded views of an exemplary single-use electroporation cartridge and volume reduction sleeve. Figures 2C and 2D show the cartridge and sleeve of Figures 2A and 2B in an assembled, uncapped position, and Figures 2E and 2F show the cartridge and sleeve of Figures 2A and 2B in an assembled, capped position.

[0101] In the embodiment shown in Figures 2A-2F, sleeve assembly 200 replaces the standard upper electrode assembly 150 (including removable cap 107, first electrode 106, and sealing member 138) of the 1 mL single-use cartridge shown and described in Figures 1A-1E. Lower electrode assembly 155 (including distal cap 118, second electrode 108, and sealing members 120, 130 of Figures 1A-1E) remains unchanged. Essentially, the interior volume of secondary electroporation chamber 202 defined by sleeve 204 is 200 μL (or other defined volume less than 1 mL as disclosed herein), preferably mimicking the shape of a 1 mL chamber.

[0102] 2C and 2D, the lower electrode assembly 155 is shown coupled to the elongate body 104 to seal the distal portion of the elongate body from the environment, as described above. The sleeve assembly 200 is assembled in FIGS. 2C and 2D, and in particular, the sleeve electrode 206 can be coupled to the removable cap 207 in the same or similar manner as described above. Additionally, the proximal sealing member 238 of the sleeve assembly 200 can also be held in place by a retaining member and can be configured to form a fluid-tight joint with the respective electrode and elongate body flange, as described above.

[0103] In some embodiments, the sleeve 204 can further include a radial sealing member 210 that seals against the inner surface of the elongate body 104. The radial sealing member 210 forms a fluid-tight seal with the sidewall defining the electroporation chamber 102 to prevent cell-containing fluid from leaking from the secondary electroporation chamber 202 through the distal opening of the volume reduction sleeve and into the electroporation chamber 102. The distal end of the sleeve 204 can include a groove 216 to receive and / or mate with the radial sealing member 210. In one embodiment, the bottom surface of the sleeve is flat, minimizing dead volume by accounting for any areas where fluid may become trapped.

[0104] In operation, a user places a desired amount of sample into the elongated body 104, as they normally would with the cartridge 100 of FIGS. 1A-1E, and then places the sleeve 204 into the elongated body 104. When inserted into the elongated body 104, the sleeve 204 expels additional fluid into its hollow center. The sleeve is designed to completely wet the underside of the sleeve electrode 206 with a 200 μL volume. The sleeve assembly 200 includes a vent 212 to allow air to escape from the secondary electroporation chamber 202 during insertion. The vent 212 also allows the electroporated sample to remain inside the elongated body 104 when the sleeve 204 is removed after electroporation. The vacuum created within the chamber while removing the sleeve 204 draws the electroporated sample from the secondary electroporation chamber 202 into the elongated body 104. This design allows users to aspirate and dispense smaller volumes (e.g., 200 μL) into the elongated body 104 using a standard pipette without special technique. It also allows for high-voltage electroporation of small volumes while maintaining a "capillary-style" shape. In some embodiments, a slot may be present near the vent to ensure the vent is unobstructed. Such a slot is useful when removing the sleeve if the vent is very close to the inner surface of the elongated body.

[0105] The volume reduction sleeve 200 may be made of or include a non-conductive plastic, glass, or ceramic and may include any of the other structural features described above with respect to the elongate body defined thereby and / or electroporation chamber 102. Additionally, the sleeve body may be injection molded and may have external ribs for rigidity and to guide the sleeve during insertion / removal.

[0106] In some embodiments, the sleeve 204 is secured to the sleeve electrode 206 by adhesive, welding, or geometric (physical) locking features to form the integrated sleeve assembly 200. In such embodiments, the sleeve 204 is introduced into the elongate body 104 when the proximal cap 214 is secured to the elongate body 104. Like the first electrode 106, the sleeve electrode 206 can be made from or plated with a conductive material that does not adversely affect cells by introducing harmful or toxic elements, either passively or during electroporation. For example, plating the electrode with pure gold can impart beneficial conductive properties to the electrode that make it less likely to introduce harmful or toxic elements into the electroporation volume. Furthermore, it should be understood that the sleeve electrode 206 can be connected to a high-voltage circuit and can function as an anode or a cathode, or alternate between the two, depending on the electroporation protocol. It should be further appreciated that the sleeve electrode 206 may include the same or similar structural features (appropriately scaled to fit the reduced cross-section of the sleeve) as described above with respect to the bulbous protrusion 132, stem 136, and base region 134 of the first electrode 106 to mitigate air bubbles and beneficially reduce the likelihood of arcing during electroporation.

[0107] 3, a longitudinal cross-section of yet another embodiment of a single-use electroporation cartridge 100' is shown having additional features. For example, the electroporation chamber 102 of the chamber body 104 is sealed at the bottom by two sealing members: a first proximal sealing member 120 and a secondary sealing member 130. The first proximal sealing member 120 forms a fluid-tight seal with the inner surface of the chamber body 104, while the secondary sealing member 130, which also forms a fluid-tight seal with the inner surface of the chamber body 104, serves as a backup seal to prevent leakage past the first proximal sealing member 120.

[0108] The cartridge 100′ further includes a locking pin 119 for securing the second electrode to the elongated body 104, which can beneficially prevent the second electrode from shifting when pressure is applied to the chamber 102. To accommodate the locking pin 119, in some embodiments, the second electrode 108 defines a channel configured to receive the locking pin and mate with a pair of openings defined by the sidewalls of the elongated chamber body 104 that are also configured to receive and / or retain the locking pin 119, thereby securing the second electrode in a fixed position relative to the chamber body 104.

[0109] 3 may further include a fluid overfill space 125 that functions to accommodate an overfill volume that is expelled from the electroporation chamber when the electroporation chamber is sealed with the sealing cap and / or first electrode. A sealing member 138 similar to that described above may also be present to form a fluid-tight joint between the first electrode 106 and the elongate body / chamber body 104.

[0110] 4, another embodiment of the exemplary single-use cartridge 100" can include a fluid overfill reservoir 125' that can be attached to the proximal outlet of an associated electroporation chamber 104'. The fluid overfill reservoir 125' can include a port 127 that is adapted to a filtered air source and / or tubing associated therewith to assist in forming and breaking a vacuum seal within the electroporation chamber 104' during repeated rounds of filling, electroporating, and draining cells within the chamber 104'.

[0111] As further illustrated by the cartridge 100″ in FIG. 4 , the upper electrode 106 can function as a selectively movable cap that functions to position the cartridge 100″ in a capped or uncapped position. When in the uncapped position, the electrode 106 can be moved away from the proximal end of the electroporation chamber 104′, allowing the distal sealing member 138 associated therewith to disengage from the proximal end of the electroporation chamber 104′, thereby breaking the fluid-tight seal formed therebetween. The upper electrode 106 can then move back toward the proximal end of the electroporation chamber 104′, where it presses the distal sealing member 138 against the proximal end of the electroporation chamber 104′ to reform the fluid-tight seal therebetween. In this exemplary embodiment, the upper electrode 106 can be used without an associated removable cap piece to form, break, and reform a fluid-tight seal with the electroporation chamber and repeatedly contact the electrode with the cell-containing fluid to be electroporated.

[0112] Although not shown in FIG. 4, it should be understood that a single-use or flow-through device may also include an upper electrode that functions to seal and unseal the electroporation chamber without the use of an associated removable cap piece.

[0113] 5, there is shown a cross section of an exemplary single-use electroporation cartridge 450. The components and functionality of cartridge 450 may be similar to cartridge 100' of FIG. 3 above, with the addition of an authentication chip 452 associated with an electrode cap 454. Authentication chip 452 may be any type of authentication or use restriction device known in the art and may provide any of several desired functions to the disclosed cartridge and system.

[0114] For example, the authentication chip can be or include non-volatile memory that can be used to embed manufacturing characteristics and operating parameters, data storage, security, and / or manage the limited use and reuse of the associated cartridge. This can prevent the use of unauthorized aftermarket consumables and / or ensure the authenticity and use of cartridges manufactured by the original equipment manufacturer. The non-volatile memory can also provide the additional functionality of enabling factory calibration of the cartridge such that the cartridge can transmit a predetermined run protocol to an associated electroporation system during use. In this manner, the cartridge can specify one or more run-time parameters of the electroporation system and / or be optimized for use with various cell types or electroporation targets.

[0115] The authentication chip can take a variety of forms, and in some cases may depend on the type of sterilization protocol (if any) used in the manufacture and / or packaging of the cartridge. In general, gamma radiation (a common sterilant in the manufacture of medical or laboratory-grade equipment) is not directly compatible with semiconductor devices such as erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), and flash memory, which traditionally incorporate floating gate memory technology used in many non-volatile memories. In embodiments where sterilization by gamma radiation is desired, alternative non-floating gate technologies may be used, including those user-programmable non-volatile memory devices known in the art.

[0116] Other forms of authentication chips can be used and are within the scope of this disclosure. For example, simple electronics can be used as a reliable method for limiting a cartridge to single use or a predetermined number of uses. In an exemplary use, a cartridge utilizing circuitry with a fuse built into the authentication chip can be associated with an electroporation system. After performing a specific electroporation protocol with the associated cartridge, the electroporation system can pass a large current through the authentication chip to blow the fuse. If the cartridge is removed and later reconnected, the lack of electrical continuity through the fuse can be a signal to keep the associated electroporation system inactive, preventing the system from completing circuits, etc., necessary for function.

[0117] An alternative authentication chip is a radio frequency identification (RFID) tag. An RFID tag can be associated with each cartridge, and when the cartridge is loaded into a compatible electroporation system equipped with an RFID reader, the RFID tag can transmit information to the electroporation system related to the particular cartridge. In this manner, the RFID tag can beneficially provide both usage restriction and anti-counterfeiting features. For example, the RFID tag can include the type of cartridge associated with it, the number of times the cartridge can be used / used, a key or manufacturer-specific authentication, etc.

[0118] 5, authentication chip 452 can be housed within a space 456 formed by electrode cap 454. Access to the chip (e.g., to communicate with an electroporation system) can be achieved via a contactless communication protocol or through one or more pin ports formed in electrode cap 454.

[0119] 6A-6D show various views of another exemplary single-use electroporation cartridge 460 having an authentication chip 465 housed within an electrode cap 462. As shown in the cross-sectional view of FIG. 6D, the authentication chip 465 rests within a recess formed by a lower portion 478 of the electrode cap 462 and is retained therein by an upper portion 476 of the electrode cap 462. The upper portion 476, in some embodiments, can be secured to the lower portion 478 in a manner that prevents tampering with or otherwise removing the authentication chip 465 from the electrode cap 462 after manufacture.

[0120] The cartridge 460 in FIGS. 6A-6D is shown as having multiple pin ports 464 formed in the top 476 of the electrode cap 462. The number and orientation of the pin ports 464 can vary, and in some embodiments, they can correspond to the number and / or orientation of pins associated with the connector / bus of the electroporation system that contacts and communicates with the authentication chip. For example, the authentication chip shown in FIGS. 6A-6D includes six pin ports 464 that correspond to and mate with six pins of the connector / bus of the electroporation system (not shown). In some embodiments, the number of pin ports can be fewer or nonexistent (e.g., when the authentication chip is an RFID tag). Thus, it should be understood that the electrode cap can be configured in any manner to accommodate a desired authentication chip and communicator / bus associated with a mating electroporation system.

[0121] Continuing with reference to Figures 6A-6D, the body 466 of the cartridge 460, in some embodiments, can include one or more stabilizers 468. The illustrated embodiment shows two stabilizers 468. In some embodiments, the stabilizer 468 itself can be sized and shaped to more easily manipulate the cartridge 460 and can include one or more gripping members 470 to assist a user in securely gripping and manipulating the cartridge 460. For example, when loading the cartridge 460 into an electroporation system, a user can grasp the stabilizer 468 (e.g., the gripping member 470) and easily guide the cartridge 460 into position. Similarly, the stabilizer 468 can be used to remove the cartridge 460 from the electroporation system after use. The gripping member 470 can include one or more protrusions extending from the surface of the stabilizer 468, as shown, or can include other contours on the surface of the stabilizer 468. In some embodiments, the gripping members may additionally or alternatively include a different material (e.g., rubber or silicone) designed to increase friction between the user's grip and the stabilizer.

[0122] The stabilizer may further include, in some embodiments, legs 472 positioned flush with the base (or second electrode) of cartridge 460, thereby providing an effectively broad base for supporting cartridge 460. Legs 472 act to stabilize the cartridge when placed on, for example, a benchtop or other flat surface, preventing cartridge 460 from tipping over if it is brushed against it, and otherwise increasing the stability of the cartridge in an upright position.

[0123] As shown in Figure 6B, the stabilizer may further include a signal or indicia 474 for passively communicating the orientation or direction of the cartridge 460 when inserted into or associated with an electroporation system. Figure 6B shows the indicia 474 as an arrow, but it should be understood that other signals, indicia, or designations may be placed thereon as known in the art.

[0124] In some embodiments, electroporation cartridges, such as those depicted and described in FIGS. 1-5, 6A-6D (and FIGS. 9 and 22A-22S, described later in the description), are designed to form a convex meniscus when fluid is dispensed into these cartridges. In some embodiments, the formation of a convex meniscus in the electroporation cartridges of the present disclosure (as opposed to the concave meniscus formed in some prior art electroporation cartridges) prevents air bubbles from being trapped during capping or sealing of the chamber. In some embodiments, the formation of a convex meniscus in the electroporation cartridges of the present disclosure (as opposed to the concave meniscus formed in some prior art electroporation cartridges) substantially reduces the trapping of air bubbles during capping or sealing of the chamber.

[0125] Some embodiments of the electroporation cartridge described herein can be implemented within a sequential batch processing system, which is described in more detail below. One such embodiment is disclosed in FIGS. 7A-7D. FIGS. 7A and 7B show an exploded view of an exemplary flow-through electroporation cartridge 300, while FIGS. 7C and 7D show the flow-through electroporation cartridge 300 in an assembled configuration. As shown in FIGS. 7A-7D, the flow-through electroporation cartridge 300 includes the same or similar components as described above with respect to the electroporation cartridge of FIGS. 1A-1E. However, instead of being a single-use (or limited batch use) electroporation cartridge requiring manual manipulation between individual electroporation events, the flow-through electroporation cartridge 300 shown in FIGS. 7A-7D can be used to repeatedly electroporate successive samples without user intervention. Such a flow-through electroporation cartridge can be used to process tens to hundreds of milliliters of sample in a single or multiple separate runs.

[0126] As shown in FIGS. 7A-7D, the chamber body 304 of the continuous flow cartridge 300 includes a flow-through electrode 306 inserted into the upper open end of the chamber body 304. Like the other electrodes disclosed herein, the flow-through electrode 306 is made from or plated with a conductive material, such as gold, that does not adversely affect cells by introducing harmful or toxic elements. The flow-through electrode 306 is operable to connect to a high-voltage circuit and can function as an anode, a cathode, or alternate between the two. In some embodiments, the flow-through electrode 306 further includes a bell-shaped or bulbous protrusion with a slight convex curvature, which aids in the exit of bubbles from the electroporation volume by moving them radially outward and away from the distal electrode surface, thereby reducing the possibility of arcing during electroporation.

[0127] In some embodiments, the flow-through electrode 306 can have additional physical features to accommodate continuous flow functionality. For example, one or more apertures or through-holes 308 can be formed near the flow-through electrode 306 and axially perpendicular to the flow-through electrode 306. The through-holes 308 can intersect with blind holes or lumens 310 that are on the axis of the flow-through electrode 306 (see FIG. 7D ). The through-holes 308 and lumens 310 can function as vents that allow filtered air to enter and exit the electroporation chamber 302 when filling and removing fluid therefrom, respectively. When the chamber 302 is filled with fluid to be electroporated, air present within the chamber 302 exits the chamber 302 through these through-holes 308 and lumens 310. When the electroporated fluid is pumped out of the chamber 302, filtered air enters the chamber 302 through these through-holes 308 and 310, preventing a vacuum from forming within the chamber 302. In some embodiments, such as those shown in Figures 7A-7D, the top of the flow-through electrode 306 is shaped as a barb or port 312 to help maintain sterility. This port can be fitted with tubing and a micron filter to open the chamber to the atmosphere. Port 321 can be connected to a sterile air source or air filter. During electroporation, pressure buildup can be dissipated through through-hole 308 and lumen 310. As with other electrodes disclosed herein, in some embodiments, the flow-through electrode 306 is coupled to a cap such that it can rotate independently and only (or primarily) the sealing member 314 is subjected to axial compression.

[0128] The flow-through electroporation cartridge 300 may further include a chamber inlet 316 and a chamber outlet 318 fluidly connected to the electroporation chamber 302. The chamber inlet 316 may be fluidly connected to a source of cell-containing fluid to be electroporated, and the chamber inlet 316 may be used to move the fluid into the electroporation chamber 302 through the action of one or more pumps in the system. Conversely, the chamber outlet 318 may be fluidly connected to an output reservoir of electroporated cells, and through the action of one or more pumps associated with the system, the chamber outlet 318 may be used as an exit point for the electroporated cells from the electroporation chamber 302. The chamber inlet 316 and the chamber outlet 318 may each be associated with a plug and / or a valve to control the inward flow of the cell-containing fluid to be electroporated within the electroporation chamber and / or the outward flow of the electroporated cell-containing fluid from the electroporation chamber. In some embodiments, the chamber inlet / outlet can be positioned above the proximal surface of the second electrode and / or the lumen of the chamber inlet / outlet can be substantially parallel to the proximal surface of the second electrode.

[0129] 8A-8C, another embodiment of a flow-through electroporation cartridge 400 is shown, having a reservoir 402 with an inlet and an outlet, integrated with an electroporation chamber body 404. In exemplary operation, the fluid to be electroporated is loaded into the chamber body 404 using a standard pipette, and an electrode adapter 406 can be attached to the opening. An electrode 106 can be attached to the adapter to seal the chamber 408 (see, e.g., FIG. 8C). The fluid can overfill the chamber, forming a convex meniscus at the top of the chamber 404. Insertion of the electrode can then cause the overfill of fluid to flow out of the adjacent opening into the reservoir 402, ensuring properly wetted electrodes (e.g., as shown in FIG. 8C, which is a cross-section taken along plane A of the flow-through electroporation cartridge of FIG. 8B). The arrows in FIG. 8C indicate overflow fluid being expelled from the electroporation chamber in response to collection in the associated reservoir.

[0130] Referring now to FIG. 9, an exemplary flow-through electroporation cartridge 400′ having an inlet and an outlet is shown. The components and functionality of cartridge 400′ are similar to cartridge 400 of FIGS. 8A-8C above, with the addition of an authentication chip 452 associated with an electrode cap 454. Authentication chip 452 may be any type of authentication or use restriction device known in the art and may provide any of several desired functions to the disclosed cartridge and system. Some examples of authentication chips described in the sections above can also be used here. Some aspects and additional embodiments of the electroporation cartridge and how it functions to refill and electroporate a batch of cells are described below in FIGS. 22A-22S.

[0131] Overview of the electroporation system Below, we first provide an overview of an exemplary electroporation system and the corresponding process for electroporating a sample. As will be appreciated, the embodiments described herein can provide effective electroporation of relatively large sample volumes, offering, among other advantages, high electroporation efficiency, high cell viability, and a safe and relatively easy user experience. As will be seen in more detail below, the system is functionally closed such that all contacting parts are shielded from the ambient environment, thereby limiting potential contamination and enhancing safe operation of the device. Furthermore, as described in more detail below, the disclosed system can retrieve the sample in the event of a system error, an excessively high temperature reading, an arcing risk reading, and / or other warning event. In the event of such an event, for example, the relevant subvolume of the sample may be pumped out of the electroporation chamber and returned to the mixing reservoir or even the sample input bag from which it originated.

[0132] Figure 10A schematically illustrates the process of an exemplary flow-through electroporation system 500. Figure 10A is intended to provide an overview of an exemplary process flow that the systems described herein may be utilized to perform. Various electroporation system components that may be included within system 500 are described in more detail below. It will be understood that various alternative embodiments of different electroporation system components may be combined together in various ways to create an electroporation system such as that generally shown in Figure 10A.

[0133] In the illustrated system 500, an input bag 502 containing a sample may be fluidly attached to a first pump 504. The first pump 504 is configured to drive fluid flow from the input bag 502 to a mixer reservoir 508. A first flow sensor 506 may be disposed between the pump 504 and the mixer reservoir 508 to enable the system to determine when fluid has flowed through a corresponding section of tubing. The mixer reservoir 508 is fluidly attached to a second pump 510. The second pump 510 is configured to drive fluid flow from the mixer reservoir 508 to an electroporation cartridge 516. In some embodiments, the electroporation cartridge may be a flow-through electroporation cartridge, such as any of the flow-through electroporation cartridges described above (see, e.g., FIGS. 7A-9 and 22A-Q, above).

[0134] As shown, in some embodiments, a second flow sensor 512 and / or a pre-cooling module 514 (also referred to as a pre-cooling assembly) can be disposed between the second pump 510 and the electroporation cartridge 516. As explained in more detail below, the pre-cooling module is beneficially provided upstream of the electroporation cartridge 516 to help reduce the temperature at which the sample is passed (or adjust the temperature at which the sample is passed, if necessary, by heating) to a lower temperature (e.g., toward a target temperature) that is more suitable for electroporation and less prone to forming bubbles.

[0135] 10A, the electroporation cartridge 516 is fluidly connected to a third pump 518 configured to drive the sample fluid from the electroporation cartridge 516 toward an output, such as an output bag 526. A third flow sensor 520 may be disposed between the third pump 518 and the output bag 526.

[0136] During operation, electroporation cartridge 516 operates to deliver electroporation pulses to a portion of the sample contained therein. The terms "subvolume" and "sample subvolume" are used herein to refer to a discrete portion of the sample contained within electroporation cartridge 516 at any given time, to distinguish it from the larger overall volume intended to be processed by system 500. System 500 advantageously operates to transfer a series of consecutive subvolumes via electroporation cartridge 516, from electroporation of one subvolume to the next, without requiring removal of electroporation cartridge 516.

[0137] This system 500 advantageously enables the effective electroporation of relatively large sample volumes. For example, a single "run" of electroporating several successive subvolumes until the entire sample volume is electroporated can process total sample volumes from about 5 mL up to about 25 mL, or even up to about 50 mL. Sample volumes greater than 50 mL could conceivably be processed with minor additional setup, limited only by the fact that most standard sample bags are not made that large. Thus, the system can process even larger volumes, as long as the input and output containers are configured to accommodate such volumes.

[0138] As shown, system 500 may also include one or more air filters or other air venting devices to provide air venting and aid in the movement of sample fluid through the system. For example, a vent line and / or air filter 522 may be coupled to input bag 502, and a vent line and / or air filter 523 may be coupled to electroporation cartridge 516. Vent line and / or air filter 521 may also be connected to an intermediate section of tubing 524 extending from the main section of tubing between first pump 504 and mixer reservoir 508. As described in more detail below, this functionality may be utilized to identify and / or control when sample transfer from input bag 502 to mixer reservoir 508 occurs.

[0139] Specific examples of the aforementioned components are described in more detail below, although it will be understood that alternative embodiments of electroporation components may additionally or alternatively be included. For example, while the pump may be described as a peristaltic pump, other embodiments may include one or more gear pumps, diaphragm pumps, rotary displacement pumps, pneumatic pumps, in-line pumps, other types of pumps, or other suitable fluid displacement mechanisms known in the art. Furthermore, while three pumps are shown in this particular example, other embodiments may include fewer or more pumps.

[0140] As a further example, while the flow sensors may be described as ultrasonic sensors, in other embodiments they may include one or more tachometers, spring and piston flow meters, turbine / paddle sensors or other positive displacement flow meters, vortex flow meters, Pitot tubes, Hall effect sensors, or other suitable flow sensors known in the art. Furthermore, while three flow sensors are shown in this particular example, other embodiments may include fewer or more flow sensors. Furthermore, while the following examples describe the input and output containers as bags, other suitable sample containers may be utilized as long as their contents can be effectively transferred from the input container to the electroporation system and then discharged into the output container.

[0141] Additionally, while the cooling module and pre-cooling module components described herein are described in the context of cooling, it is understood that, at least in some embodiments, they can also be operated to heat a sample and / or sample subvolume. For convenience, these components are referred to herein as "cooling" or "pre-cooling" modules, as typical operations benefit more from cooling than heating. However, this should not be considered limiting of their ability to provide heating in addition to, or as an alternative to, their ability to provide cooling.

[0142] Additionally, while the specific examples described herein show a single system with a single input bag, other implementations may utilize multiple systems and / or multiple input bags per system to process greater volumes through parallel operations.

[0143] 10B illustrates a control system 600 (used interchangeably herein with the terms "computer system" and "computing system") that may be included as part of an electroporation system. For example, control system 600 may form part of and / or be communicatively coupled to one or more components of electroporation system 500 shown in FIG. 10A, or to any of the other electroporation systems described herein.

[0144] The control system 600 includes a controller 601 having one or more processors 602 and one or more hardware storage devices (for holding memory 603). The controller 601 is communicatively coupled to one or more of the various electroporation components of the electroporation system to receive data and / or send instructions to one or more of the electroporation components. As shown, the controller 601 can be communicatively coupled to a pump 604, a flow sensor 606, a mixer driver 608 (for controlling mixing in the mixer reservoir), a pre-cooling module 614, a cooling module 615, and an electroporation assembly 617.

[0145] Controller 601 operates to provide control over various coupled electroporation components. For example, controller 601 may be configured to control the operation, direction, and / or speed of pump 604, to receive flow data from flow sensor 606, to control the operation, direction, and / or speed of mixer driver 608, thereby controlling mixing in the mixer reservoir accordingly, and to control the temperature of pre-cooling module 614 and / or cooling module 615.

[0146] The electroporation assembly 617 includes various components configured to interact with the electroporation cartridge. As shown, the electroporation assembly 617 can include a conductivity sensor 630 configured to measure the conductivity across an electroporation chamber of the electroporation cartridge. As described in more detail below, a controller can use information from the conductivity sensor to control one or more electroporation parameters, such as whether to deliver a pulse and a target voltage for delivering the pulse.

[0147] The electroporation assembly 617 also includes components that make up an electroporation circuit configured to generate and send an electrical pulse across the electroporation chamber. These components include a charger 632 configured to act as a voltage source for charging a capacitor 634, and one or more discharge resistors 636 and / or other safety components configured to allow the capacitor to safely discharge when not immediately discharged for normal operation. As known in the art, additional or alternative circuit components may be included to generate and send the electrical pulse across the electroporation chamber.

[0148] The electroporation assembly 617 may also include a capping mechanism 638 configured to mechanically engage the electroporation cartridge when the electroporation cartridge is properly inserted into the electroporation assembly 617. As described in more detail below, in some embodiments, the capping mechanism 638 operates to transition the electroporation cartridge between a capped state, which allows electroporation, and an uncapped state, which allows evacuation from and fluid transfer to the electroporation chamber. A sealing mechanism 640 operates in conjunction with the capping mechanism 638 to seal and unseal the electroporation chamber as the system moves from electroporation of one subvolume to the next.

[0149] Various exemplary electroporation components are now described in more detail. Any of the following embodiments can be incorporated into electroporation system 500 and / or control system 600.

[0150] 11A-11D illustrate an exemplary embodiment of an electroporation system of the present disclosure. Figures 11A and 11B illustrate an exemplary electroporation system / apparatus 605' (components of systems 500 and 600 are shown but not numbered) that includes one or more components of electroporation system 500 and one or more components of electroporation system 600, according to one embodiment. The embodiment shown in Figure 11A includes a door that can be closed while electroporation is in progress, and a user interface panel 606' that may allow a user to select electroporation parameters, run a protocol, monitor the progress of a run, receive any error messages to correct potential problems, and the like, via a graphical user interface and computer-implemented methods.

[0151] In one embodiment, the electroporation system / apparatus 605′ of FIGS. 11A and 11B includes at least one or more pumps, at least one or more sensors for sensing liquid (e.g., but not limited to, ultrasonic sensors), an area for removably inserting at least one cell mixer mechanism and / or cell mixer, an area for removably inserting an electroporation chamber (e.g., a single-use electroporation chamber for a single electroporation run or a flow-through chamber for batch / continuous electroporation), corresponding electropins (e.g., but not limited to, high-voltage electropins that make electrical contact with electrodes located on the inserted electroporation chamber), an authentication chip reader (configured to read the chip of the electroporation chamber), one or more air bubble sensors, one or more pre-cooling chambers or modules, a shell or plate for inserting modular casings having various electroporation device components. a lace holder (e.g., an instrument panel or modular casing such as, but not limited to, 700, 701, 703, 800, or 803 shown in Figures 12A-12G and 13A-13C), a pressure sensor, a locking mechanism for clamping to the modular casing and / or electroporation chamber, a handle for easy insertion and removal of the modular casing and / or electroporation chamber, a stopcock and positioning mechanism, an air filter, tubing for sample flow from one component to another, tubing for air flow, one or more stopcocks for controlling air flow and / or liquid flow, a sterile air source and / or air filter, vents, valves, a rotating mechanism for opening and closing the chamber or cell inlet and outlet, a pressure sensing mechanism, a conductivity sensor, hooks or drawers for attaching a sample input bag and a sample output bag (bags containing cells in fluid before and after electroporation, respectively), a processor system (e.g.,11A and 11B ), some of the components listed above may reside within the housing of 605′, while some of the components may reside on an interior surface, such as 607, of the system / apparatus 605′.

[0152] In some embodiments, the one or more pumps of system 605' can comprise a first pump (e.g., a peristaltic pump) for transferring a sample, e.g., a sample containing cells to be electroporated from an input bag / reservoir, to a cell mixer, and a second pump (e.g., a peristaltic pump) for transferring a processed sample, e.g., a sample containing electroporated cells from the electroporation chamber to an output bag / reservoir, to the cell mixer.

[0153] In some embodiments, the system 605' can include a pre-cooling module located just before the cells enter the electroporation chamber so that the cells are cooled to an optimal temperature. In some embodiments, an additional pre-cooling module can be located in the area where the electroporation cartridge is removably inserted into the instrument. Pre-cooling modules are described in the section above.

[0154] Figure 11C shows an exemplary electroporation system comprising the electroporation apparatus of Figure 11A and a removably attachable modular casing (which may be similar to 700, 701, 703, 800, 801, 803, etc.) for positioning various electroporation components (additional details of the modular casing are shown in Figures 12A-12G, 13A-13C, and 15A-15I below). The modular casing is suitable for processing large volumes of samples for electroporation by electroporating small batches of samples at a time using flow-through electroporation chambers.

[0155] Electroporation instruments and systems 605' and / or 600' typically have a lid to allow a user to close the system while electroporation is being performed. FIG. 11B shows an embodiment in an open position with a sliding lid. FIG. 11D shows another embodiment of an electroporation instrument with a different lid that can be opened upward. Various other types of lids can be used. The lid can provide a sterile environment as well as safety from the high voltages used by the user.

[0156] In some embodiments of electroporation system 605′ or 600′, for example, when using single-use electroporation chambers, a modular casing with multiple components is not required. In such systems configured for a single electroporation event (as opposed to batch processing / continuous flow), 605′ or 600′ can include components such as an electrical pulse generator, a bubble sensor, a conductivity sensor, a fluid sensor, a pressure sensor, a pre-cooling module for directly cooling the electroporation chamber, a slot for inserting an electroporation chamber with appropriate electrical contacts (e.g., high-voltage pins) available for delivering electrode pulses to electrodes in the electroporation chamber, a locking mechanism for clamping the electroporation chamber, an air filter, a computer controller and processor, and optionally, a user interface. The single-use electroporation chamber can be placed into a corresponding slot to receive the electroporation chamber and connected to the electrode contacts and other components in the electroporation instrument to perform electroporation.

[0157] Electroporation System and Modular Casing FIG. 12A illustrates an example embodiment of an electroporation system 700, including a modular casing 703 and an instrument panel 701. In some embodiments, the casing 703 is attached to and detachable from the instrument panel 701 (see FIG. 12A). Many conventional electroporation systems require the use of multiple consumable components and suffer from easily tangled tubes and bags that get in the way of other components and complicate setup and operation. The modular casing 703 is intended to beneficially locate the various electroporation components and tubes in one location in an organized, easily managed layout. This, along with other design features of the systems described herein, helps reduce errors and maximize electroporation efficiency. For example, the electroporation cartridge design described herein beneficially reduces user touchpoints where mistakes or problems can occur.

[0158] 12A, casing 703 is selectively attachable / detachable from instrument panel 701. Casing 703 may include one or more attachment features that engage with instrument panel 701 and allow for selective attachment. Casing 703 may be attached to one side of instrument panel 701 as shown. Additionally or alternatively, instrument panel 701 may be configured to surround all or a portion of casing 703.

[0159] FIG. 12A (and FIG. 11C (including an instrument panel / modular casing similar to 701 / 703) shows an exemplary layout of the various electroporation components. An input bag 702 is fluidly coupled via tubing to a first pump 704. The first pump 704 pumps sample fluid from the input bag 702 through a first flow sensor 706 and into a mixer reservoir 708. A second pump 710 then pumps the sample fluid from the mixer reservoir 708 through a second flow sensor 712, through a pre-cooling module 714, and into the electroporation chamber of an electroporation cartridge 716. Following electroporation of the sample subvolume, a third pump pumps the fluid from the electroporation chamber through a third flow sensor (not shown) and towards an output chamber (e.g., an output bag) (not shown).

[0160] As shown, casing 703 and instrument panel 701 can each separately include one or more hooks or other attachment features for supporting input bag 702 and optionally also output bag. In the illustrated embodiment, input bag 702 can be attached to casing hook 707 during transport and while mounting casing 703 to instrument panel 701. After attachment, input bag 702 can be moved to instrument hook 709 to move it out of the field of view of other electroporation components and / or to a more suitable location for transport and electroporation processing.

[0161] The modular casing of the present disclosure, along with the components of system 700, are encased in an outer shell or lid 720 that can be removably placed into electroporation instrument 600' or system 605' (e.g., as depicted in FIGS. 11A-11D) and manually opened and closed (e.g., upward from the bottom of the system, or slid upward or sideways / opened) to access the instrument panels to expose and provide access to those components, as shown in FIG. 11A. In some embodiments, lid 720 is rotatable about a hinge to expose and provide access to the internal components of system 700.

[0162] 12B-12E further illustrate a slightly different embodiment of electroporation system 800 having a casing 803 configured to be attached to a corresponding instrument panel 801. FIG. 12B shows the back side of casing 803, FIG. 12C shows the front side of casing 803, FIG. 12D shows the attachment of input and output bags to the tubes of casing 803, and FIG. 12E shows the attachment of casing 803 to instrument panel 801.

[0163] Similar to system 700, system 800 includes a casing 803 and tubing routed through the casing 803 from an inlet 805 (configured to attach to input bag 802) to an outlet 825 (configured to attach to output bag 826). The tubing connects to a mixer reservoir 808, a pre-cooling module 814, one or more flow sensors 812, and an electroporation cartridge 816. The tubing also passes through one or more cut-out sections of the casing (flow indicators 844 and 846) that allow for flow visualization. The casing 803 may also include one or more side handles 852 and one or more top handles 850 to aid in moving and handling the casing 803 (e.g., from a lab bench to an instrument panel 801).

[0164] Casing 803 includes an arrangement of compartments 842 that correspond to the layout of pumps (e.g., pumps 804 and 810, other pumps not shown) on instrument panel 801 (shown in FIG. 12E) such that when casing 803 is attached to instrument panel 801, the pumps are received in compartments 842. As best shown in FIG. 12C, casing 803 also includes mixer compartment 827 that is attached to instrument panel 801 and configured to provide space adjacent mixer reservoir 808 for receiving a corresponding mixer driver.

[0165] 12C, system 800 may also include an air reservoir 848 pneumatically coupled to mid-section 824 of the tubing disposed between inlet 805 and mixer reservoir 808. As described in more detail below, air reservoir 848 is configured to supply air to the mid-section of the tubing after sample is drawn into the mixer reservoir.

[0166] FIGS. 12F and 12G show different views of casing 803 to illustrate one embodiment of attachment features that allow casing 803 to be attached to instrument panel 801. In this embodiment, side handle 852 is flexible and includes catch 856 that can engage with corresponding structure on instrument panel 801. Handle 852 can be configured to be biased toward a neutral, straight position and to bend when grasped and pulled outward. As shown in FIG. 12G, a user can cause handle 852 to bend outward by placing their fingers on grip surface 854 and pulling on grip surface 854. Releasing handle 852 returns it to its neutral position, allowing catch 856 to move inward and engage with the instrument panel. Other embodiments may additionally or alternatively include other attachment features known in the art, including latches, alignment pins, hooks, clamps, etc.

[0167] 13A-13C illustrate another exemplary modular casing embodiment that can be attached to an electroporation device / system such as 605' or 600'. FIG. 13A illustrates a front view of modular casing 700' for positioning various electroporation components that can be selectively attached and detached from the electroporation device. FIG. 13B illustrates a rear view of modular casing 700' (of FIG. 13A) showing additional components.

[0168] Similar to system 700, system 700′ includes a casing 703′ and tubing routed through casing 703′ from inlet tube 739 configured to attach to an input bag (not shown) that hangs on input bag hook 707. Input tube 739 connects to cell mixer reservoir 708, then through a first pump (not shown as it is located on the surface of the device / system such as 605′ / 600′), to tubing 737 (which is the tubing exiting mixer reservoir 708) shown as mixer tube 737, which is visible through a slot in modular casing 708′ for the mixer pump (mixer pump not shown), and then through a pre-cooling module as pre-cooling tube 736 (the pre-cooling module is connected to 605′). 17C above), however, a slot in the casing for pre-cooling module 714′ is shown, and further tubing continues to an electroporation cartridge such as 300, 400, or 400′ (located / housed in electroporation cartridge retainer 878) and then to outlet tubing 735 (configured to attach to an output bag (not shown)). The output bag attaches to output bag hook 707′. In some cases, tubing passes through one or more cutout sections of the casing to allow flow visualization. Casing 703′ may also include one or more side handles 752′, which may have grippers such as 709′, and one or more top handles 750′ to aid in movement and handling of casing 703′ (e.g., from a lab bench to an instrument panel of instrument / system 605′ or 600′ or an instrument / system such as 701 or 801).

[0169] As shown in the embodiment of Figures 13A and 13B, the cell mixer reservoir 708 is positioned at an angle within the modular casing. The inventors have discovered that the angled cell mixer 708 results in an unexpected and surprising reduction in sample loss by reducing or preventing sample from becoming trapped in the cell mixer 708. In some embodiments, the cell mixer 708 is angled at an angle of about 5 degrees to about 20 degrees, and any value therebetween. In some embodiments, the cell mixer 708 is angled at an angle of about 10 degrees, e.g., about 8 degrees, about 8.5 degrees, about 9 degrees, about 9.5 degrees, about 10 degrees, about 10.5 degrees, about 11 degrees, about 12 degrees, etc.

[0170] 13A, 13B, and 13C, the system 700′ can include an air reservoir (not explicitly shown) pneumatically coupled to a mid-section of the tubing, the air inlet tubing 738, and positioned between the sample inlet tubing 739 and the mixer reservoir 708. The air reservoir, configured to supply air to the mid-section of the tubing after sample is drawn into the mixer reservoir, is described in detail elsewhere in this application.

[0171] 13B shows the arrangement of air inlet tubes 738 on the mixer reservoir 708, passing through several air filters 722 to stopcocks (not explicitly shown) with stopcock adapters 730 that hold the stopcocks on the modular casing 700'. The operation of the stopcocks and stopcock adapters 730 is described in the section above. Icons 731 can be used as visual indicators to the user of airflow direction. The air inlet tubes connecting to the air filters 722 allow air exchange to different zones to allow fluid flow and / or avoid pressure buildup in the mixer chamber or electroporation chamber.

[0172] FIG. 13C illustrates an embodiment of a modular casing 700′ that is provided with an additional input chamber 740 (e.g., a payload chamber) with its own additional input tube 741 for adding one or more additional components to the sample. The input tube 741 can have an inlet to the mixer reservoir 708 in addition to the inlet to the sample inlet tube 739. In one example, the additional substance can include a payload (a material to be electroporated into cells (e.g., nucleic acid, DNA, RNA, protein, drug, or any other molecule or substance desired to be electroporated into cells) that needs to be added later and that needs to be prevented from being exposed to the cells or other contents of the cell bag during setup and assembly of the electroporation system prior to performing electroporation). In some non-limiting embodiments, the additional input chamber 740 can be a syringe containing additional substance that can be manually injected into the cell mixer when and as needed (as shown in FIG. 13C ). However, other chambers, such as input bags, containers, etc., can be used as chamber 740.

[0173] The electroporation cartridge 300, 400, or 400' is inserted into a slot in the instrument via the cartridge casing 878. The casing 878 and its function are described hereafter in Figures 19A-19C.

[0174] bag compartment 14A and 14B show an example of a bag compartment 858 that can be incorporated into an electroporation system as described herein. During use, the sample container bag should be supported in an upright position with the port facing downwards to minimize residual liquid (in the case of an input bag) and minimize air bubble accumulation (in the case of an output bag). Preferably, the bag also fits within a small footprint to keep the size of the device small and minimize obstruction to the view of other components of the system. Additionally, although not required, it is beneficial to seal the bag during use of the system to prevent the user from handling it during high-voltage electroporation operations.

[0175] In the illustrated bag compartment 858 (with the remainder of the casing removed for clarity), an insert 860 is slidably connected to the bag compartment 858 such that it can be selectively extended from or inserted into the casing. The insert 860 may be connected to the bag compartment 858 via, for example, a track and rail system 862. The insert 860 may include a hook 864 or other suitable mounting feature for supporting an input bag and a support 866 for supporting an output bag. The bag compartment 858 may include one or more latches (e.g., magnetic latches), clamps, stops, etc. for holding the insert 860 in a desired position.

[0176] Transfer of input samples 15A-15C illustrate one example of an assembly configured to provide effective transfer of sample fluid from an input bag to a mixer reservoir 808. Sample input can vary from process to process based on sample type, sample volume, sample fluid properties (e.g., viscosity), and bag type. Therefore, it is beneficial to be able to accurately determine when sample fluid has been completely transferred from the input bag to the mixer reservoir. Additionally, it is desirable to minimize sample loss due to residual volume remaining in the bag and / or in the tubing between the bag and mixer reservoir 808.

[0177] The illustrated transfer assembly is configured to indicate when the input bag is substantially emptied of sample fluid so that the controller can configure the process control accordingly. The transfer assembly is also configured to minimize sample waste by removing the tubing between the sample inlet 805 and the mixer reservoir 808 at the end of the sample transfer process.

[0178] Figure 15A shows the sample transfer assembly before the end of sample transfer, and Figures 15B and 15C show the sample transfer assembly at the end of sample transfer. In Figure 15A, air has not yet passed from air reservoir 848 through mid-section 824 of the tubing to main section 829 of the tubing. That is, air reservoir 848 remains full while sample fluid is being transferred from inlet 805 to mixer reservoir 808 via main section 829 of the tubing.

[0179] 15B and 15C, once the input bag is nearly empty, continued operation of pump 804 further reduces the pressure in the tubing, drawing air from mid-section 824 of the tubing into main section 829 of the tubing. This additional bolus of air helps flush / clean main section 829 of the tubing of any remaining sample, helping to maximize the amount of sample delivered to mixer reservoir 808.

[0180] The air reservoir 848 may also be associated with a sensor (e.g., a proximity sensor) that senses a decrease in the volume of the air reservoir, thereby signaling the progress or completion of the sample transfer process. In the illustrated embodiment, the plunger 849 moves when the air reservoir 848 is depleted, such that movement of the plunger 849 indicates pressure within the main section 829 of the tubing, and thereby the progress of the sample transfer process.

[0181] Other embodiments may additionally or alternatively utilize other means for determining the progress or completion of the sample transfer process. For example, electroporation system 600′ or 605′ shown in FIGS. 11A-11B omits the syringe and plunger layout of system 800. Instead, system 700 includes a mid-section of tubing extending from the main section of tubing and terminating in an air filter open to the atmosphere. Air flow within the mid-section of tubing can be controlled by adjusting the degree to which the mid-section of tubing is open, for example, by using a pinch mechanism to open and close the mid-section of tubing. Similar to the systems of FIGS. 15A and 15B, if the pressure in the main section of tubing becomes sufficiently low near the end of the sample transfer process, air can be drawn through the mid-section of tubing into the main section of tubing. As previously indicated, system 700 also includes a first flow sensor 706 positioned between the input and mixer reservoir 708 to help determine when sample transfer is complete.

[0182] Figures 15D-15E show an example embodiment of sample flow through the modular casing shown in system 700' of Figures 13A-13C. Like part numbers have like functions as in Figures 13A-13C. Arrows on different sections of tubing in Figures 15D and 15E indicate the flow of a sample (e.g., a sample containing cells in a fluid containing a substance to be electroporated) through modular casing system 700' shown in the front view (Figure 15D) and back view (Figure 15E) of modular casing 700'. For example, as indicated by the arrows, sample enters inlet tubing 739, enters mixer reservoir 708 through the inlet (see arrows to 708 in FIGS. 15D and 15E), exits through the outlet of 708 (see FIG. 15E), continues through the mixer pump (pump not shown), through mixer tubing 737, through a pre-cooling chamber (not shown) via tubing 736, and into the inlet to electroporation chamber 300, 400, or 400′. Additional payload can be loaded using input device 740 (such as a syringe) to manually dispense the payload into the sample in mixer reservoir 708. After electroporation of the sample, the electroporated sample exits electroporation chamber 300, 400, or 400′ through the outlet (see arrows to outlet tubing 735 in FIG. 15E). The electroporated sample continues, in the embodiment of FIG. 15E, through outlet tube 735 to the top of the electroporation chamber and casing 700' and into an outlet bag (not shown) which can be hung on a hook 707'.

[0183] Figures 15F-15G show an example embodiment of airflow through modular casing system 700' (of Figures 13A-13C). Arrows on different sections of tubing indicate the airflow through modular casing system 700', showing air entering air inlet tube 738 (see Figure 15F). Figure 15G shows the airflow arrows through tube 738 and tube 734 (the tubing exiting electroporation chamber 300, 400, or 400' during filling and subsequent filling of the chamber via air filter 722 and stopcock 731 located behind stopcock retainer 730).

[0184] Figure 15H shows additional details of the stopcock mechanism for air flow, with the shaded arrow indicating rotation of the stopcock. The instrument operator rotates stopcock 731 90 degrees clockwise to allow air exchange and returns it to its original position (0 degrees) to stop air exchange. This allows the input pump (or first pump) to function as air pushes sample / liquid out of tubing 737 into the subsequent stage, mixer reservoir 708, and from there through mixer tubing 737 to the mixer pump. The stopcock adapter 730 acts as a retainer to hold stopcock 731 on casing module 700'. The stopcock adapter 730 also serves as an easier lead for the user to properly load it into the stopcock drive of an instrument such as 605' or 600'.

[0185] FIG. 15I shows three steps for removing modular casing 700′ from instrument or system 600′ or 605, consisting of first removing the electroporation chamber by removing the knob on cartridge retainer 878, then removing stopcock adapter 730, and then using the handle and grip (if present) to remove modular casing 700′ from system 605′ or instrument 600′.

[0186] Mixer Reservoir 16A and 16B show close-up views of an exemplary mixer reservoir 808. The mixer reservoir 808 beneficially functions to keep the sample fluid (e.g., containing cells) in a homogenous suspension while successive subvolumes of the sample are electroporated and transferred to the output bag. The inclusion of the mixer reservoir 808 advantageously allows for the use of a wide variety of input bag types. Because bags can vary in volume, shape, stiffness, etc., attempting to transfer directly from the bag itself to the electroporation cartridge can lead to uneven cell density between subvolumes, cell settling, clogged tubing lines, and other undesirable problems. These problems are avoided by transferring the sample volume to the mixer reservoir 808.

[0187] 16A, mixer reservoir 808 can include mixer elements 868. Mixer elements 868 are preferably configured as blades (as shown), impellers, etc., as opposed to free-moving elements such as magnetic stir bars. Centrifugal mixers are also less preferred because they tend to concentrate cells in the outer portions of the circulating fluid.

[0188] The magnetic elements preferably do not contact the sample fluid. Therefore, the mixer element 868 preferably does not include magnetic materials and is formed from a medical-grade polymer or other suitable material. The mixer element (not shown in FIG. 16B) can be coupled to a mixer magnet assembly disposed in the cover 811. When the casing is attached to the instrument panel, a mixer driver (not shown, part of the instrument panel) is received in the mixer compartment 827. The mixer driver includes one or more magnets that are magnetically coupled to the mixer magnet assembly, thereby indirectly driving rotation of the mixer magnet assembly via the magnetic connection.

[0189] The illustrated mixer reservoir 808 includes a reservoir input 869 and a reservoir output 870. As shown, the reservoir input 869 is preferably located at the top of the mixer reservoir 808 so that the incoming sample fluid collects at the bottom after flowing down the interior sides of the mixer reservoir 808. This configuration tends to limit the formation of air bubbles as they enter the mixer reservoir 808.

[0190] cooling mechanism 17A and 17B illustrate one embodiment of the pre-cooling module 814. In this embodiment, the pre-cooling section 831 of the tubing upstream of the electroporation chamber is brought into contact with a cooling block 871 by one or more mounting clamps 876. Other mounting methods can be used, and the clamps 876 are an example embodiment. The cooling block 871 can be a ceramic block or comprise any other material capable of effective heat transfer and conduction with the pre-cooling section 831 of the tubing. The cooling block 871 can be cooled according to methods known in the art, preferably by thermoelectric cooling. Other embodiments can additionally or alternatively utilize air cooling, liquid cooling, or other temperature regulation mechanisms known in the art.

[0191] In the illustrated embodiment, the pre-cooling section 831 of the tube is arranged in a looped or serpentine layout. This beneficially provides greater contact between the pre-cooling section 831 of the tube and the cooling block 871, allowing greater heat transfer to occur. However, it has been found that a looped or serpentine layout can also lead to the generation of air bubbles, which can adversely affect subsequent electroporation of the sample subvolume. Therefore, in some embodiments, the pre-cooling section 831 of the tube is maintained in a substantially straight line, such as, for example, pre-cooling module 714 in the embodiment of FIG. 12A and pre-cooling module 814 in the embodiment of FIG. 12B.

[0192] 17B, ​​the pre-cooling module 814 can further include a flexible biasing element 872. In this embodiment, the flexible biasing element 872 is disposed on a side of the cooling block 871 opposite the pre-cooling section of the tube 831. The flexible biasing element 872 includes features that bias against the cooling block 871 and tend to press the cooling block 871 against the pre-cooling section of the tube 831, thereby helping to maintain good thermal contact between the cooling block 871 and the pre-cooling section of the tube 831.

[0193] Figure 17C shows a perspective view of the embodiment of pre-cooling module 714 shown in Figure 12A. A section of substantially straight linear tubing (e.g., 831 in Figure 12A or 737 in Figures 13A-13C) having a sample travels through pre-cooling module 714 to cool the sample prior to electroporation.

[0194] 17D shows an example of a cooling module 873 configured to cool an electroporation chamber of the present disclosure. The illustrated cooling module 873 is sized and shaped to receive a corresponding electroporation chamber portion of an electroporation cartridge of the present disclosure. FIG. 17D also shows upper electrode contacts 874 and lower electrode contacts 875 positioned to contact corresponding upper and lower electrodes of an inserted electroporation cartridge. Like the pre-cooling module, the cooling module 873 can include a ceramic block or other suitable thermally conductive material and can operate by thermoelectric cooling and / or other cooling methods known in the art (e.g., air cooling and / or liquid cooling).

[0195] Figures 18A and 18B show an example of a high-voltage touch pin of an electroporation device of the present disclosure that does not engage with an exemplary electroporation cartridge. Figure 18A shows a rear cross-sectional view of an electroporation device or system showing the area where an electroporation cartridge can be removably positioned (cartridge not explicitly shown). Figure 18A shows a cam 921 of the engagement portion. Figure 18B shows the corresponding area in cross-sectional top view, showing the high-voltage pin 920 of the system where an electroporation cartridge can be removably positioned exactly where the end of the high-voltage pin terminates. Spring 922 and sensor 923 are also shown. The stroke of the linear actuator is approximately 10 mm. Figure 18B shows the high-voltage pin 920 released from the electroporation cartridge when cam 921 is engaged. Figures 18C and 18D show an example of a high-voltage touch pin of an electroporation device of the present disclosure that engages with an electroporation chamber. Figure 18C shows a rear cross-sectional view of the electroporation device or system showing the area where an electroporation cartridge can be removably placed (cartridge not explicitly shown). A cutaway portion of cam 921 is shown in Figure 18C, which corresponds to the engagement of high voltage pin 920 with the electroporation cartridge. Figure 18D shows the corresponding area in a top cross-sectional view, showing the high voltage pin 920 of the system engaging with the electroporation cartridge (cartridge not shown). The stroke of the linear actuator is approximately 2 mm.

[0196] Electroporation cartridge attachment function 19A, 19B, and 19C illustrate an embodiment of an electroporation cartridge mounting feature configured to allow the electroporation cartridge to be mounted in an appropriate position within the cooling module 815. In the illustrated embodiment, the electroporation cartridge 816 includes a cartridge body 877 and a retainer 878. The cartridge body 877 is adapted to be attached to the electroporation cartridge 816 by one or more catches 883. The retainer 878 is then attached to the cartridge body 877. The retainer 878 includes two catches 879 that extend beyond the cartridge body 877 when the retainer 878 is attached to the cartridge body 877 to engage corresponding structure on the cooling module 815, thereby allowing the cartridge body 877 and electroporation cartridge 816 to be positioned within the cooling module 815.

[0197] As shown, the retainer 878 can also include a flexible biasing element 872 configured to bias the cartridge body 877 and the electroporation cartridge 816 toward the cooling module 815. The flexible biasing element 872 beneficially accounts for differences in part tolerances and ensures that small variations are compensated for so that the electroporation cartridge 816 is in effective thermal contact with the cooling module 815, whatever the case may be.

[0198] The attachment features are also configured to allow for selective release and removal of the electroporation cartridge. Pressing the proximal section 880 of the retainer 878 inward bends the catch 879 outward, allowing removal from the cooling module 815. A tab 881 on the retainer 878 fits into a corresponding slot 882 in the cartridge body 877, allowing the user to pull the entire cartridge out of the cooling chamber upon disengaging the catch 879.

[0199] Capping mechanism for electroporation chamber 20A-20C illustrate the operation of capping mechanism 838, which moves the electroporation cartridge between a capped state in preparation for electroporation and an uncapped state in preparation for filling or draining the electroporation chamber. Capping mechanism 838 functions as a linear actuator that engages cap 885 of electroporation cartridge 816. Cap 885 is, in turn, coupled to an upper electrode 884 of the electroporation cartridge, such that movement of capping mechanism 838 controls the up and down movement of upper electrode 884.

[0200] Figures 20A-20C also show a chamber inlet plunger 886, which is controlled by a chamber inlet driver 887, and a chamber outlet plunger 888, which is controlled by a chamber outlet driver 889. Figure 20A shows the upper electrode 884 in an uncapped position, the inlet plunger 886 in a retracted, open position, and the outlet plunger 888 in an advanced, closed position. Figure 20A represents the relative positions of these components during a fill operation.

[0201] Figure 20B shows the upper electrode 884 in the closed position, the inlet plunger 886 in the advanced closed position, and the outlet plunger 888 remaining in the advanced closed position, representing the relative positions of these components when in a sealed state in preparation for an electroporation operation.

[0202] Figure 20C shows the upper electrode 884 in an uncapped position, the inlet plunger 886 remaining in an advanced, closed position, and the outlet plunger 888 moved to a retracted, open position. Figure 20C represents the relative positions of these components during an ejection operation, for example, after electroporation of a subvolume within the electroporation chamber.

[0203] FIG. 21 illustrates in further detail the capping mechanism (element 838 in FIGS. 20A-20C) and its operation in conjunction with cap 885 (shown in cross section). As shown, cap 885 includes a spring 890 disposed within a spring chamber and an elongated member 891 that is mechanically coupled to spring 890 such that linear motion of elongated member 891 can be transferred to spring 890 and such that force stored in spring 890 can be transferred to elongated member 891. For example, as shown, elongated member 891 can include a flange extending outwardly and sized to engage the coils of spring 890, while the remainder of elongated member 891 extends through the lumen of spring 890.

[0204] An elongated member 891 extends from the cap 885 and is mechanically coupled to the upper electrode 884. When the cap 885 is moved in response to actuation of the capping mechanism 838, the linear motion, absent resistance, causes the elongated member 891 and, therefore, the upper electrode 884, to move accordingly. However, once the electrode 884 tops out or bottoms out, the elongated member 891 can no longer move. At that point, the continued movement of the cap 885 deforms the spring 890. That is, the spring 890 allows overtravel of the cap 885 after the upper electrode 884 reaches the upper or lower position of the terminal. The mechanically allowed overtravel ensures that the electrode 884 is moved to the proper position despite actuation drift and / or potential tolerances from one electroporation cartridge to the next.

[0205] As shown, the upper limit of electrode 884 may be defined by hard stop 892. The top of the electroporation chamber can similarly function as a hard stop corresponding to the lower limit of electrode 884. Some embodiments may include one or more spring pins 893 located on the top of the electroporation chamber to define a "home" or position useful during initial calibration of the instrument. Spring pins 893 are configured so that the downward force during the initial downward movement of upper electrode 884, when assisted by force from spring 890 from the over-travel of cap 885, overcomes spring pin 893 and depresses it, allowing electrode 884 to reach the fully capped position.

[0206] Sealing mechanism of the electroporation chamber 22A and 22B show an example sealing mechanism 840 in more detail. By providing an effective seal of the electroporation chamber 895, bubble formation and associated arcing during electroporation is beneficially minimized. The ability to maintain a higher relative pressure within the electroporation chamber 895 may reduce vaporization of the fluid (i.e., increase the boiling point of the fluid) and may also reduce the growth rate of oxygen and hydrogen bubbles formed as a result of the electrolysis of water in the sample fluid.

[0207] For purposes of illustration, outlet plunger 888 is shown here, although inlet plunger 886 can be similarly configured. When plunger 888 is in the retracted position, as shown in Figure 22A, outlet port 894 is exposed, allowing fluid to exit electroporation chamber 895. However, when plunger 888 is in the advanced position, as shown in Figure 22B, outlet port 894 is sealed, preventing fluid from passing from the electroporation chamber.

[0208] The outer portions of the inlet plunger 886, the outlet plunger 888, and / or the upper electrode 884 may also be covered by a bellows 896. The bellows 896 beneficially functions to encase the moving parts of the device and helps to isolate the internal environment to minimize the possibility of particles entering the electroporation chamber 895.

[0209] A detailed view of outlet plunger 888 shown in Figure 22B shows that the plunger may include a rubber cap 897 for forming a watertight seal with the outlet channel. Alternate embodiments may additionally or alternatively include other sealing members, such as one or more O-rings. Movement of inlet plunger 886 and outlet plunger 888 may be controlled by respective linear actuators 887 / drivers 889 (see Figures 20A-20C).

[0210] 22C shows another view of plunger 888, showing a rubber cap 897 on a first end and an attachment 898 on a second end. The attachment is configured to engage a corresponding outlet driver 889 to mechanically transfer drive motion to plunger 888. Attachment 898 can be configured as an aperture, as shown here, or alternatively can be configured as a catch, clamp, magnetic coupling, or other suitable mechanical linkage.

[0211] 22D shows an alternative embodiment of a plunger 1088 having a threaded portion 1098 and configured to convert rotational motion to linear motion. The plunger 1088 may have an O-ring seal member 1097 as shown, or alternatively may have a rubber cap as in other plunger embodiments, or other suitable sealing member. Other means for controlling the linear motion of the plunger may additionally or alternatively be included. For example, some embodiments may include a track and rail assembly, a worm gear assembly, or a pneumatically or hydraulically powered actuator.

[0212] 22E-22G illustrate an alternative embodiment of the plunger of FIGS. 22C and 22D. As shown, the sealing mechanism can include a rotatable sealing mechanism 1000 having a semi-cylindrical head 1002 that can be rotated to selectively block or allow entry into the electroporation chamber 1020 through the inlet 1018a and / or selectively prevent exit from the electroporation chamber 1020 through the outlet 1018b. In exemplary operation, the body 1006a of the sealing mechanism 1000 can be engaged with a rotating piston 1014a operable by the electroporation system to rotate the head 1002a to an open position (e.g., as shown in FIGS. 22F and 22G). Fluid can enter the electroporation chamber 1020 via the inlet port 1016 and through the inlet 1018a. The opposing seal mechanism 1000b is engaged by a complementary rotating piston 1014b and rotated to a closed position (e.g., as shown in FIG. 22G), thereby allowing fluid to fill the electroporation chamber 1020. Once filled, the seal mechanism 1000a can be rotated so that the head 1002a blocks the inlet 1018a. Electroporation can then be performed as described herein. Electroporated cells can be removed from the electroporation chamber 1020 by rotating the head 1002b of the seal mechanism 1000b associated with the outlet 1018b to an open position. The electroporated cells can then be removed via the outlet port 1016b.

[0213] In some embodiments, the sealing mechanism 1000 includes a sealing ring 1004 configured to form a fluid-tight seal between the body of the sealing mechanism and the cartridge 1010, which can prevent sample leakage and protect the sterility of the consumable. The cartridge 1010 shown in Figure 22F can also include an authentication chip 1012, as described above with respect to Figures 5 and 6A-6D.

[0214] The electroporation system of the present disclosure can include a linear piston, a rotary piston, or a combination thereof. For example, as shown in FIG. 22H, a linear piston 1022 is coupled to the inlet 1018 and a rotary piston is coupled to the outlet 1016. In FIG. 22H, the linear piston is actuated to block the inlet, and the rotary piston is actuated to move the head 1002 to a closed position over the outlet 1016. In this configuration, cells within the chamber 1020 can be retained for electroporation. To vent the chamber 1020 and release the cells therefrom, the rotatable sealing mechanism 1000 can be rotated so that the head is oriented in an open position, thereby opening the outlet 1016, as shown in FIG. 22I.

[0215] In some embodiments, the ports can be angled to allow more efficient flow into and out of the chamber, such as those inlet and outlet ports in Figures 22F-22I shown in association with a rotatable sealing mechanism. In contrast to the right angle formed with the inlet in Figure 22I, which can form bubbles and impede or prevent flow therethrough, the outlet port 1016 in Figure 22I is angled, which can allow more efficient fluid flow therethrough and reduce bubble formation between interfaces.

[0216] In some embodiments, the inlet can be coupled to a valve, such as the umbrella valve 1026 shown in Figures 22J and 22K. Under pressure in a first direction (e.g., from fluid flow), the umbrella valve transitions to an open position 1024a by flexibly deforming a head portion 1028 away from an opening 1030 formed beside the inlet 1032, as shown by arrow C in Figure 22J. As shown in Figures 22J and 22K, the umbrella valve 1026 allows fluid to flow unidirectionally through the inlet. When pressure is applied to the head portion 1028 (as shown by arrow D in Figure 22K), the head portion pushes against the opening 1030, causing the valve to assume a closed position 1024b and maintaining a seal over the opening 1030 of the inlet 1032.

[0217] 22L shows a top view of an exemplary inlet 1032 having multiple peripheral openings 1030 formed around a central opening 1034. The central opening 1034 is sized and shaped to receive an umbrella valve whose head portion extends over the peripheral openings 1030 when in the closed position. It should be understood that the openings 1030 can be in any number or configuration, so long as they are covered (and sealed) by the head portion of the complementary umbrella valve when in the closed position.

[0218] FIG. 22M shows an example setup for pressurizing and sealing an outlet port fitted with a check valve (e.g., a mini-valve). As shown in FIG. 22M, a rotary translation piston 1014 with attached 1002 is coupled to an inlet 1018, and the rotary piston 1014 is coupled to an outlet 1016. In FIG. 22N, the rotary translation piston is actuated to block the inlet 1018, and the rotary translation piston 1014 is actuated to move the head 1002 to a closed position over the outlet 1016. In this configuration, cells in the chamber 1020 can be retained for electroporation. To vent the chamber 1020 and release the cells therefrom, the rotatable sealing mechanism 1000 of the rotary piston 1014 can be rotated so that the head is oriented in an open position, thereby opening the outlet 1016. 1022 is a rubber plug that is assembled to 1014.

[0219] The rotatable sealing mechanism 1000 of the rotary-translating piston 1014 in FIG. 22M is also referred to as a catch. The catch or rotatable sealing mechanism 1000 couples to an adapter attached to a rotary motor. A pin 1000′ (e.g., a dowel pin) can act as a cam to guide the movement of the rotary piston 1014, as shown in FIG. 22N. According to one embodiment, during operation, the catch or rotatable sealing mechanism 1000 rotates, driven by a rotary motor on the instrument. As shown in FIG. 22O, the piston 1014 latches to the catch 1000 via an “I” shape 1015. The piston 1014 is driven by the catch 1000 as it rotates.

[0220] As shown in Figures 22P and 22Q, when protrusion 1017 is aligned with pin 1000', inlet 1018 is blocked and electroporation chamber 1020 is sealed. As shown in Figures 22R and 22S, catch 1000 is rotated 180 degrees to open inlet 1018. The linear position of piston 1014 is guided by groove and pin 1000'. Piston 1014 and head 1002 move away from the chamber surface of electroporation chamber 1020.

[0221] It should be appreciated that in some embodiments, the electroporation chamber of the cartridge is pressurized during electroporation (even if only slightly above atmospheric pressure), and therefore an umbrella valve, if utilized at all, is preferably utilized on the inlet side where the increased pressure within the chamber acts to push the head portion of the valve into a closed configuration. If an umbrella valve is used on the outlet, the pressure required to open the valve should ideally be greater than the pressure applied to the valve during electroporation.

[0222] Arc Detection and Prevention Arcing has a detrimental effect on both cell viability and transfection efficiency. Because samples for electroporation are often valuable, it is desirable to minimize waste and loss of yield. A primary cause of arcing is the formation of gas bubbles. The systems and methods of the present invention advantageously include features that reduce the occurrence of arcing, or at least detect the risk of arcing, allowing for sample recovery before a portion of the sample is wasted as a result of arcing.

[0223] 23 illustrates a method 900 for predicting the risk of arcing during an electroporation operation. Method 900 may be performed as a computer-implemented method performed by controller 601 (see FIG. 10B) using data received from and / or sending instructions to one or more communicatively coupled system components.

[0224] In the first step of method 900, the controller determines the initial temperature of a subvolume within the electroporation chamber of the electroporation cartridge (step 902). Determining the temperature of the subvolume is preferably accomplished non-invasively. That is, the initial temperature determination is preferably accomplished without the use of a temperature probe. The use of a temperature probe has been found to disrupt the uniformity of the electric field within the electroporation chamber. Temperature probes and associated sensing circuitry have also been found to have a limited lifetime in the high-voltage environment of the chamber. External infrared temperature sensors are also less preferred because their measurements are affected by the sidewalls of the chamber.

[0225] In a preferred embodiment, the initial temperature of the sub-volume is determined indirectly using a predetermined correlation between conductivity and temperature. For a given known chamber geometry, measuring the conductivity and estimating the temperature by conversion using the predetermined correlation has been found to yield temperatures with an accuracy of ±2°C.

[0226] If it has not already been done, the controller can determine the conductivity of a subvolume within the electroporation chamber of the electroporation cartridge via a conductivity sensor (step 904). The controller can then determine the predicted temperature rise of the subvolume based on the determined conductivity, the set pulse voltage, and the set pulse duration (step 906). Arcing risk is related to the voltage level, temperature, bubble formation, and the total energy transferred to the subvolume. As the temperature rises, a portion of the subvolume undergoes a phase change from liquid to vapor, increasing the likelihood of bubble formation.

[0227] Using known electrical principles, the total energy transferred by a given electrical pulse can be calculated from the voltage used to deliver the pulse, the resistance of the subvolume between the electrodes (determined by a conductivity sensor), and the set pulse duration. The law of conservation of energy can then be applied by assuming that all electrical energy applied to the subvolume is converted to heat (Joule heating effect). The specific heat capacity of the subvolume can be measured or assumed to be substantially the same as that of water. Using the specific heat capacity, the expected temperature rise can be easily determined.

[0228] The controller may then determine a predicted resultant temperature of the sub-volume based on the determined initial temperature and the predicted temperature rise (step 908). The controller may then determine whether the resultant temperature exceeds a predetermined threshold (step 910). The threshold may be, for example, about 60°C or about 70°C. If the determination is yes, the controller may send an arc risk alert (step 912), which may include an alarm sent to an input / output device with which a user can interact, a shutdown command, a pause in the process that must be manually overridden, etc. Optionally, the controller may be configured to return the sub-volume to the mixer reservoir to allow further cooling or other intervention steps to occur (step 914).

[0229] Alternatively, if a determination is made that the predicted temperature will not exceed the threshold, then the controller may cause the system to continue with the electroporation protocol (step 916).

[0230] FIG. 24 illustrates a related method 940 for preventing arcing within an electroporation chamber. Similar to method 900, method 940 may be implemented by controller 601 (see FIG. 10). In the illustrated method, the controller determines the conductivity of a subvolume within an electroporation chamber of an electroporation cartridge via a conductivity sensor (step 942). The controller can then determine whether the measured conductivity is below a predetermined threshold (step 944). Large air bubbles significantly reduce conductivity in a manner that can be easily detected by measuring conductivity.

[0231] If the determination is "yes," the conductivity of the sub-volume is below the threshold, and the controller may send a bubble detection signal (step 946). Similar to step 912 of method 900, this may include an alarm sent to an input / output device with which the user can interact, a shutdown command, a pause in the process that must be manually overridden, etc. Optionally, the controller may be configured to return the sub-volume to the mixer reservoir to protect the sample (step 948).

[0232] Alternatively, if it is determined that the conductivity is not below the threshold, then the controller can cause the system to continue with the electroporation protocol (step 950).

[0233] Optimization of electroporation pulses 25 shows a schematic diagram of an electroporation circuit 930 including a charger 932 and a capacitor 934 electrically connected to an electroporation chamber 995. High voltage electroporation systems can utilize voltage pulses ranging from about 500 V to about 2500 V, and it is desirable to achieve such high voltage based electrical pulses with reproducibility and precision.

[0234] Calibration of the charger 932 and / or capacitor 934 may reduce some of the variation. However, as shown in schematic circuit 930, there will be an inherent amount of inherent circuit resistance 938. This may be due, for example, to circuit protection components, discharge resistors, or other safety features of the circuit, and / or the resistance of the high voltage switch when in the on position.

[0235] Additionally, the resistance of the electroporation chamber 995 varies depending on the subvolume characteristics and temperature. For example, if the chamber has a volume of 1 mL, its resistance can typically vary from about 500 ohms to 2,000 ohms. Resistance variations from one subvolume to another can lead to inconsistent electrical pulses, which can lead to inconsistent electroporation results.

[0236] 26 illustrates a method 960 that may be implemented by a computer system, such as controller 601, for generating repeatable and consistent electrical pulses across an electroporation chamber. In method 960, the controller causes the system to determine the conductivity of a subvolume within the electroporation chamber of an electroporation cartridge using a conductivity sensor (step 962) and use the determined conductivity to determine the voltage drop across the electroporation chamber (step 964). The controller can then determine the resistance of the electroporation circuit between the capacitor and the electroporation chamber (step 966), which represents the inherent fixed circuit resistance.

[0237] The controller can then charge the capacitor to a voltage level that exceeds the determined voltage drop across the electroporation chamber to compensate for the additional resistance between the capacitor and the electroporation chamber (step 968). That is, the controller can add the series resistance and the measured resistance across the electroporation chamber to determine the resistance of the entire circuit and then charge the capacitor accordingly. By taking into account both the fixed portion of the circuit resistance and the variable resistance of the electroporation chamber, a finer-tuned voltage charge can be determined, so that the actual delivered electrical pulse is more consistent from one sample subvolume to the next.

[0238] The method may also optionally include iteratively increasing the charger voltage based on the previous input voltage and the corresponding previously measured actual voltage applied to the electroporation chamber (step 970). Because the charger output tolerance can vary ±100V, rather than simply operating the charger based on a set voltage level, the controller can apply an iterative voltage compensation method to continuously reduce the voltage error (e.g., within about ±5V) without making sudden adjustments that could risk significantly changing the output.

[0239] The iterative voltage compensation method can begin by first selecting a target pulse voltage (e.g., 2500 V) and offsetting the input voltage by a predetermined amount (e.g., 200 V). The actual pulse voltage delivered to the electroporation chamber is then measured. In the next iteration, the input voltage is varied as follows:

[0240] Charger Input V = (Previous Input V / Previously Measured Output V) x (Target V - Offset)

[0241] The "offset" can decrease from one iteration to the next. For example, as mentioned above, if the initial offset is 200V, the offset in the next iteration could be 100V, then 5V, and eventually decrease to 0. These offsets are merely exemplary; in other implementations, the offset can decrease more quickly or more slowly depending on the needs and preferences of the application. Furthermore, the offset can be applied as a positive or negative offset, depending, for example, on whether charger errors are expected to initially result in a charge greater or less than the target.

[0242] Electroporation Chamber Fill Calibration The high-throughput electroporation systems described herein include many different components that work in conjunction with one another. Consequently, multiple mechanical tolerances are stacked against one another, which can cause variations in electroporation chamber volume between different consumable sets and / or even between different subvolumes within the same consumable set. As noted above, the adverse effects of failing to properly fill an electroporation chamber include underfilling, which can lead to arcing, and overfilling, which can lead to loss of yield.

[0243] 27A, 27B, and 27C graphically illustrate a method for calibrating the fill volume of an electroporation chamber. Shown are a mixer reservoir 708, a second pump 710, a second flow sensor 712, a pre-cooling module 714, and an electroporation cartridge 716. A flowchart of method 980 is shown in FIG. 27D. Method 980 can be implemented by a computer system, such as controller 601.

[0244] In a first step, the controller can determine the number of rotations, N, of the drive pump required to fill the tubing located between the flow sensor and the electroporation chamber and move a sufficient volume of sample to completely fill the electroporation chamber (step 982). This is shown graphically in FIG. 27A. The number, N, corresponds to the volume between the flow sensor and the outlet of the electroporation chamber. Determining that the electroporation chamber is full can be done using a conductivity sensor. That is, the electroporation chamber can be determined to be full when the conductivity sensor measures a conductivity that indicates that fluid extends all the way from the lower electrode to the upper electrode.

[0245] The controller can then cause the electroporation chamber to drain (step 984). At this point, the controller can cause the drive pump to move the sample backward to a location upstream of the flow sensor using a fixed number of revolutions, k (step 986). This is shown graphically in FIG. 27B. The fixed number, k, corresponds to the volume between the upstream location of the flow sensor and the inlet of the electroporation chamber. The controller can then determine the number of revolutions, x, of the drive pump required to move the sample from the upstream location of the flow sensor to the location where it is detected by the flow sensor (step 988). This is shown graphically in FIG. 27C. The number, x, corresponds to the volume between the upstream location of the flow sensor and the flow sensor. Therefore, the number, (kx), corresponds to the volume between the flow sensor and the inlet of the electroporation chamber. Therefore, the volume between the inlet and outlet of the electroporation chamber (i.e., the volume of the electroporation chamber) is equal to N-(kx). The controller can then set this volume as a step volume such that the associated pump operates N-(kx) times repeatedly between each subvolume electroporation (step 990).

[0246] Figure 27E illustrates another method for filling a flow-through electroporation chamber of the present disclosure. As shown, in the first step (1050), the first filling of the chamber is stopped when the sample fluid contacts the upper electrode, closing the electrical circuit. For example, this occurs when the electrical resistance reading drops from several thousand to approximately 700 ohms (or in the 600-800 ohm range).

[0247] In the second step (1060), a nominal value, e.g., N rev The total filling is done by and the top electrode is capped to read the resistance value.

[0248] At this stage, in a third step (1070), the electrical resistance (or conductivity) is read. If the electrical resistance or conductivity is within range (e.g., 1X times 700 ohms), as determined from the first or any previous fill value, then the second fill is complete and you can proceed to electroporate the sample (step 1090).

[0249] However, if it is out of range (e.g., greater than 1.X times 700 ohms), determine it from the initial or any previous fill value, then uncap the top electrode and add an additional n rev Proceed to step 1080 which involves fine filling the volume. For subsequent fills, the pump value is N rev +n rev The result is x (x = number of trials).

[0250] Thus, in some embodiments, in an electroporation system of the present disclosure that includes a controller having one or more processors and one or more hardware storage devices, the one or more hardware storage devices storing computer-executable instructions that, when executed by the one or more processors, configure the controller to determine a step volume to be moved by the system between each electroporation event, calibration is performed by performing at least the following: performing a first fill while monitoring that, during the first fill of the electroporation chamber, the electrical resistance of the electroporation chamber drops from several thousand ohms to a stable value in the range of about 600-800 ohms; and stopping the first fill when this voltage plateau is reached.

[0251] Then, for the second fill, N from the peristaltic pump rev This is followed by a total filling of the electroporation chamber based on a calculated value (e.g., the number of revolutions of the peristaltic pump to fill the chamber until a stable resistive volume is reached). In some embodiments, the second filling of the electroporation chamber is performed using a total filling of N revIn addition to the values, the values ​​are further based on calculations using one or more empirical values ​​such as the inner diameter of the tubing, the number of rollers of the pump, the diameter of the electroporation chamber, and / or the height of the electroporation chamber.

[0252] N rev After the second fill, the electroporation chamber is completely filled with the desired rotational velocity. The upper electrode is then capped after the second fill and the electrical resistance is measured. If the electrical resistance is within the stable range determined from the first fill, the second fill is complete and you can proceed to electroporate the sample. If the electrical resistance is not within the stable range determined from the first fill, you can remove the cap from the upper electrode and perform an additional fill. rev After the first fill, cap the top electrode and measure the electrical resistance. If the electrical resistance is within the stable value determined from the first fill, complete the second fill and proceed to electroporation of the sample. If not, repeat the steps of fine filling and measuring the electrical resistance as described above until the electrical resistance is within the stable value determined from the first fill. Subsequent fills (third fill, fourth fill, etc.) are performed with N. rev +n rev This is done by rotating the pump x (number of microfill attempts). In some embodiments, the stable value of the electrical resistance is about 700 ohms.

[0253] In some of these aspects, the controller is configured to implement a method for determining a calibrated step volume to be moved by the system into the flow-through electroporation chamber between each electroporation event, corresponding to the fill volume of the flow-through electroporation chamber. In one embodiment, such a method includes a first fill of the flow-through electroporation chamber until the sample contacts (touches) the upper electrode (the first electrode described in some embodiments) of the electroporation chamber. At this time (i.e., during the first fill), the electroporation system monitors the electrical resistance within the electroporation chamber for a decrease from several thousand ohms to a stable value in the range of approximately 600-800 ohms. Once this stable electrical resistance value is reached, the first fill is stopped.

[0254] For the second fill (and subsequent fills), the total fill sample volume is derived from a combination of empirical data and theoretical calculations. Therefore, for the second fill (and subsequent fills), the electroporation system (e.g., the controller therein) determines the number of rotations, N rev ”, i.e., the number of rotations of the drive pump required to move a sufficient sample volume to completely fill the electroporation chamber (i.e., count the number of rotations from when the sample fluid enters the electroporation chamber from a fixed inlet position until the sample fluid contacts the upper electrode (reaching the sample volume)). Additionally, the inner diameter of the pump tubing, d i " and the number of rollers "n" of the pump are empirically determined. In a non-limiting example, the pump may be a peristaltic pump, and in some embodiments, may have six rollers (e.g., n=6).

[0255] N revTo determine this value, we consider the number of revolutions of the peristaltic pump required to fill the electroporation chamber from the inlet position to the upper electrode (corresponding to the drop in the resistance of the sample fluid to 600-800 ohms), the inner diameter of the tubing in the pump, d i ", the number of pump rollers "n", the fluid volume per complete revolution of the pump ("a" μL), the fluid volume per one roller movement "b" μL, the minimum diameter of the electroporation chamber area where the electroporation sample resides, and / or the electrical resistance or conductivity of the fluid in the electroporation chamber (measured using a voltmeter, conductivity sensor, etc.), followed by N rev This includes theoretical calculations leading to the

[0256] This is followed by capping the top electrode after the second fill (and any subsequent fills) and measuring the electrical resistance. If the electrical resistance is within the stable range determined from the first fill (i.e., within approximately 600-800 ohms), the fill is complete and you can proceed to electroporate the sample. However, if the electrical resistance is not within the stable range determined from the first fill (i.e., not within approximately 600-800 ohms) after the second fill, you can uncap the top electrode and perform additional electroporation. rev Amount (n of pump rev A step of micro-filling the electroporation chamber with a fluid of N is performed. This is followed by measuring the electrical resistance after the micro-fill by attaching a cap to the upper electrode. If the electrical resistance is within the stable value determined from the first fill (i.e., within approximately 600-800 ohms), the second fill is complete and you can proceed to electroporate the sample. If not, repeat the above file-fill and electrical resistance steps until the electrical resistance is within the stable value determined from the first fill (i.e., within approximately 600-800 ohms). Subsequent fills (third fill, fourth fill, etc.) are performed with N. rev +n revThis is done by x (number of microfill attempts) pump revolutions. In some embodiments, the stable value of electrical resistance is about 700 ohms, for example, in the range of 650-750 ohms and any value therebetween.

[0257] In one example embodiment, N rev is calculated as follows: In one embodiment of the system of the present disclosure, with a peristaltic pump with six rollers and tubing with an inner diameter of 2.4 mm, empirical data showed that 172 μL of fluid was dispensed for each full and complete revolution of the pump. From empirically determined data, 28 μL of fluid was dispensed per rotational movement of the pump per roller distance (60 degrees in this case). Following this, theoretical calculations were performed to determine the amount of fluid that could be placed into the electroporation chamber. With a nominal diameter of 6.4 mm (2r, where r = radius of the electroporation chamber), a lower limit of 6.3 mm, and an upper limit of 6.5 mm, πr 2 Using an electroporation chamber height (h) of 30 mm according to the formula for h, the nominal sample volume was determined to be 965 μL, with a lower limit of 935 μL and an upper limit of 995 μL. A design tolerance of 0.2 mm for variation in height (h) is considered insignificant because it results in a maximum variation of only 7 μL. A lower limit of 6.3 mm for the chamber diameter was used in the calculations to reduce sample loss from the total fill due to overfilling the chamber. Based on dividing the calculated volume of 935 μL by the empirical data of 172 μL, the number of revolutions of the peristaltic pump was determined to be 5.4, but rounding to 5.5 was recommended to introduce a convex meniscus. Therefore, in this case, "N rev " is equal to 5.5 revolutions. When the chamber diameter changes from 6.3 mm to 6.5 mm, there is a difference in sample volume of approximately 30 μL per 0.1 mm change in diameter. This is very close to the empirical data of 28 μL of fluid dispensed per rotational movement of the pump per roller distance (60 degrees in this case), which we refer to as the fine filling "n" in the equation. rev " 5.5 rotations (N rev ) total fill, the instrument will read the conductivity and if the fill is incomplete, it will read the fine fill "n revDepending on the number of attempts "x", in the case of fine filling, the second filling (and subsequent fillings) are started with "N rev +n rev x".)

[0258] Computer system of the present disclosure It will be appreciated that computer systems are taking an increasingly wide variety of forms. In this description and in the claims, the terms "controller," "computer system," or "computing system" include any device or system, or combination thereof, that includes at least one physical and tangible processor and physical and tangible memory capable of holding computer-executable instructions that can be executed by the processor. As used herein, the term "computer system" or "computing system" is intended to include, by way of example and not limitation, personal computers, desktop computers, laptop computers, tablets, handheld devices (e.g., cell phones, PDAs, pagers), microcomputer-based or programmable consumer electronics, minicomputers, mainframe computers, multiprocessor systems, network PCs, distributed computing systems, data centers, message processors, routers, switches, and even devices not traditionally considered to be computing systems, such as wearables (e.g., eyeglasses).

[0259] Memory can take any form and may depend on the nature and form of the computing system. Memory may be physical system memory, including volatile memory, non-volatile memory, or a combination of the two. The term "memory" may also be used herein to refer to non-volatile mass storage devices such as physical storage media.

[0260] A computing system has multiple structures thereon, often referred to as "executable components." For example, executable components may be included in the memory of a computing system. The term "executable component" is a name well understood by those skilled in the art of computing as a structure that may be software, hardware, or a combination thereof.

[0261] For example, when implemented in software, those skilled in the art will understand that the structure of an executable component may include software objects, routines, methods, etc. that can be executed by one or more processors on a computing system, whether such executable component resides on the computing system's heap or whether the executable component resides on a computer-readable storage medium. The structure of the executable component resides on a computer-readable medium in a form that, when executed by one or more processors of the computing system, functions to cause the computing system to perform one or more functions, e.g., the functions and methods described herein. Such structure may be directly computer-readable by a processor, such as when the executable component is binary. Alternatively, the structure may be structured to be sequentially interpretable (whether in a single step or multiple steps) and / or compiled to generate a binary that is directly sequentially interpretable by a processor.

[0262] The term "executable component" is well understood by those skilled in the art to include structures that are implemented exclusively or nearly exclusively in hardware logic components, such as field programmable gate arrays (FPGAs), application specific integrated circuits (ASICs), program specific standard products (ASSPs), system-on-chip systems (SOCs), complex programmable logic devices (CPLDs), or other specialized circuitry. Thus, the term "executable component" is a term well understood by those skilled in the art of computing for structures whether implemented in software, hardware, or a combination thereof.

[0263] Terms such as "component," "service," "engine," "module," "control," "generator," and the like may also be used in this description. As used in this description, these terms, with or without modifiers, are intended to be synonymous with the term "executable component," and as such, have a structure that is well understood by those skilled in the computing arts.

[0264] While not all computing systems require a user interface, in some embodiments, a computing system includes a user interface for use in communicating information from / to a user. A user interface may include output mechanisms as well as input mechanisms. The principles described herein are not limited to the precise output or input mechanisms, which as such depend on the nature of the device. However, output mechanisms may include, for example, speakers, displays, haptic output, projections, holograms, etc. Examples of input mechanisms may include, for example, microphones, touchscreens, projections, holograms, cameras, keyboards, stylus, mouse or other pointer inputs, any type of sensor, etc.

[0265] Accordingly, the embodiments described herein may include or utilize special-purpose or general-purpose computing systems. The embodiments described herein also include other physical computer-readable media for carrying or storing computer-executable instructions and / or data structures. Such computer-readable media may be any available media that can be accessed by a general-purpose or special-purpose computing system. Computer-readable media that store computer-executable instructions are physical storage media. Computer-readable media that carry computer-executable instructions are transmission media. Thus, by way of example, and not limitation, the embodiments disclosed or contemplated herein may include at least two distinctly different kinds of computer-readable media: storage media and transmission media.

[0266] Computer-readable storage media include RAM, ROM, EEPROM, solid-state drives ("SSD"), flash memory, phase-change memory ("PCM"), CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or other physical and tangible storage media that can be used to store desired program code in the form of computer-executable instructions or data structures, and that can be accessed and executed by a general-purpose or special-purpose computing system to implement the disclosed functions of the present invention. For example, computer-executable instructions may be embodied on one or more computer-readable storage media to form a computer program product.

[0267] Transmission media may be used to carry desired program code means in the form of computer-executable instructions or data structures and may include a network and / or data links accessible by a general-purpose or special-purpose computing system. Combinations of the above are also intended to be included within the scope of computer-readable media.

[0268] Furthermore, after reaching the various computing system components, program code in the form of computer-executable instructions or data structures may be automatically transferred from a transmission medium to a storage medium (or vice versa). For example, computer-executable instructions or data structures received over a network or data link may be buffered in RAM within a network interface module (e.g., a "NIC") and eventually transferred to the computing system's RAM and / or a less volatile storage medium. Thus, it should be understood that storage media may be included in computing system components that also (or even primarily) utilize transmission media.

[0269] Those skilled in the art will further appreciate that a computing system may also include communication channels that enable it to communicate with other computing systems, for example, over a network. Accordingly, the methods described herein may be implemented in network computing environments with many types of computing systems and computing system configurations. The methods of the present disclosure may also be implemented in distributed system environments in which tasks are performed by both local and / or remote computing systems that are linked over a network (either by wired data links, wireless data links, or a combination of wired and wireless data links). In a distributed system environment, processing, memory, and / or storage functionality may also be distributed.

[0270] Those skilled in the art will also understand that the disclosed methods can be implemented in a cloud computing environment. A cloud computing environment may be distributed, but this is not required. If distributed, the cloud computing environment may be distributed internationally within a single organization and / or have components owned across multiple organizations. For purposes of this description and the claims that follow, "cloud computing" is defined as a model for enabling on-demand network access to a shared pool of configurable computing resources (e.g., networks, servers, storage, applications, and services). The definition of "cloud computing" is not limited to any of the many other benefits that can be derived from such a model when properly deployed.

[0271] Cloud computing models may consist of various characteristics, such as on-demand self-service, wide area network access, resource pooling, rapid scalability, and scalable services. Cloud computing models may be offered in the form of various service models, such as, for example, Software as a Service ("SaaS"), Platform as a Service ("PaaS"), and Infrastructure as a Service ("IaaS"). Cloud computing models may also be deployed using various deployment models, such as private cloud, community cloud, public cloud, and hybrid cloud.

[0272] List of defined term abbreviations To aid in understanding the scope of this written description and the appended claims, a selection of terms are directly defined below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0273] As used herein, the terms "approximately," "about," and "substantially" refer to an amount or condition that is close to a specific recited amount or condition that still performs a desired function or achieves a desired result. For example, the terms "approximately," "about," and "substantially" can refer to an amount or condition that deviates from a specifically recited amount or condition by less than 10%, or less than 5%, or less than 1%, or less than 0.1%, or less than 0.01%.

[0274] As used herein, the term "electroporation" is intended to include the process of exposing cells to an electric field, typically a brief, high-voltage field, to cause the uptake of an electroporation target from the surrounding electroporation medium into the electroporated cell. It should be understood that the cell can be any living cell, and the electroporation systems and methods disclosed herein can be used with prokaryotic and / or eukaryotic cells. As known to those skilled in the art, the process of electroporating a target into a prokaryotic organism, such as a bacterium, is referred to as "transformation," while the process of electroporating a target into a eukaryotic organism, such as a primary cell or cell line, is typically referred to as "transfection." In this disclosure, the terms "transformation" and "transfection" have the same meaning, unless otherwise specified, regardless of the type or variety of organism being transformed. Thus, the term "electroporation" or any form thereof is intended to include the transformation / transfection of a living cell (prokaryotic or eukaryotic) with an electroporation target.

[0275] The term "electroporation target" as used herein is intended to be understood as any molecule, compound, or substance intended to be introduced into a target cell via electroporation. By way of example and not limitation, an electroporation target may include a protein, peptide, nucleic acid, drug, or another compound. Proteins may include purified, folded, or unfolded proteins with native, mutated, or engineered sequences, and peptides are understood to include any amino acid sequence and may include portions of a protein sequence. Nucleic acids include sequences derived from biological or environmental sources and may be any one or more of genes, regulatory sequences, intergenic sequences, genomic DNA, plasmid DNA, cDNA, or various known forms of RNA. As outlined herein, electroporation targets can take any of the aforementioned forms, but in preferred embodiments, the electroporation target constitutes a nucleic acid for transfecting primary cells or cell lines.

[0276] As used herein, the term "primary cells" is intended to mean cells isolated directly from the tissues or fluids of an organism that have a finite lifespan without intervention and have limited in vitro proliferation capacity using standard cell culture techniques. Primary cells are typically not associated with homogenous genotypic and phenotypic characteristics. In contrast to primary cells, the term "cell line" is intended to include cells that have acquired homogenous genotypic and phenotypic characteristics (e.g., from continuous passage over an extended period of time). As known to those skilled in the art, cell lines include finite or continuous cell lines. Immortalized or continuous cell lines have acquired the ability to proliferate indefinitely, either through genetic mutation or artificial modification.

[0277] As used herein, the term "sealing member" is intended to include any structural element or mechanism known in the art that facilitates, is shaped to seal, or functions to seal a joint between two surfaces. The sealing members disclosed herein preferably include O-rings or other gaskets that selectively allow the disclosed and associated electroporation cartridges to function as functionally closed environments. O-rings or similar gaskets provided within the present disclosure can be made of or include any suitable material known in the art, such as, by way of example and not limitation, non-conductive materials such as rubber or silicone.

[0278] Various aspects of the present disclosure, including devices, systems, and methods, may be described with reference to one or more embodiments or implementations that are exemplary in nature. As used herein, the term "exemplary" means "serving as an example, instance, or illustration," and should not necessarily be construed as preferred or advantageous over other embodiments disclosed herein. Furthermore, references to "implementations" of the disclosure or invention include specific references to one or more embodiments thereof, and vice versa, and are intended to provide illustrative examples without limiting the scope of the invention, which is indicated by the appended claims rather than by the following description.

[0279] As used herein, unless otherwise understood or stated, implicitly or explicitly, words appearing in the singular include their plural equivalents, and words appearing in the plural include their singular equivalents. Accordingly, it should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a reference to a single referent (e.g., "a widget") includes one, two, or more referents unless otherwise understood or stated, implicitly or explicitly. Similarly, a reference to a plural referent should be construed as including a single referent and / or multiple referents unless the content and / or context clearly dictates otherwise. For example, a reference to a plural referent (e.g., "widgets") does not necessarily require that a plurality of such referents exist. Instead, it will be understood that one or more referents are contemplated herein, regardless of the number of referents that may be inferred, unless otherwise specified.

[0280] As used herein, directional terms such as "top," "bottom," "left," "right," "up," "down," "upper," "lower," "proximal," "distal," "adjacent," and the like, are used herein merely to indicate relative directions and are not intended to otherwise limit the invention of the present disclosure and / or claims. Summary of Exemplary Embodiments: An itemized list of some non-limiting exemplary embodiments of the present disclosure is provided below. 1. An electroporation cartridge comprising an electroporation chamber defined by an elongated body, a first electrode disposed at a proximal end of the electroporation chamber, and a second electrode disposed at an opposite distal end of the electroporation chamber, wherein at least one of the first electrode or the second electrode is movable from a capped position for electroporation to an uncapped position for loading a sample, and / or the electroporation cartridge is configurable from a sealed state to an unsealed state. 2. The electroporation cartridge of paragraph 1, wherein the elongated body is made of or includes one or more of non-conductive plastic, glass, or ceramic and is configured to receive a cell-containing liquid to be electroporated within an electroporation chamber defined by the elongated body. 3. The electroporation cartridge of paragraph 2, wherein the electroporation chamber is made of or includes glass and / or ceramic. 4. An electroporation cartridge according to paragraph 2 or 3, wherein the electroporation chamber is made of or includes polycarbonate or other non-conductive, radiation-resistant plastic. 5. The electroporation cartridge of any one of items 1 to 4, wherein at least a portion of the electroporation chamber is tapered between the first electrode and the second electrode. 6. The electroporation cartridge of paragraph 5, wherein the tapered portion of the electroporation chamber does not substantially impede the generation of a uniform electric field between the first electrode and the second electrode. 7. The electroporation cartridge of any one of claims 1 to 6, wherein the electroporation chamber comprises a uniform cross-section along the length of the reaction chamber. 8. The electroporation cartridge of paragraph 7, wherein the uniform cross-section extends the entire length of the electroporation chamber between the first electrode and the second electrode, such that the electroporation cartridge is configured to generate a uniform electric field within the electroporation chamber disposed between the first electrode and the second electrode. 9. The electroporation cartridge of paragraph 7 or 8, wherein the electroporation chamber comprises a cylindrical cavity and has a uniform cross section that is circular. 10. The electroporation cartridge of any one of claims 1 to 7, further comprising a proximal sidewall defined between a proximal opening of the elongate body and an inflection point in the sidewall defining the electroporation chamber, the proximal sidewall narrowing from a first diameter defined by the proximal opening to a second, smaller diameter defined at a location distal to the inflection point. 11. The electroporation cartridge of any one of claims 1 to 10, wherein the first electrode comprises a bulbous extension having a substantially flat distal surface. 12. The electroporation cartridge of any one of claims 1 to 10, wherein the first electrode comprises a bulbous extension having a distal surface with a convex or angled contour. 13. The electroporation cartridge of paragraph 11 or 12, wherein the bulbous extension is separated from the base portion of the first electrode by a narrow stem. 14. An electroporation cartridge according to any one of claims 11 to 13, wherein the bulbous extension functions to expel one or more air bubbles associated with the cell-containing liquid being electroporated within the electroporation chamber when the first electrode is secured within the electroporation chamber. 15. An electroporation cartridge according to any one of claims 1 to 14, further comprising a sealing member disposed between the first electrode and the proximal surface of the elongate body, the sealing member functioning to form a fluid-tight joint between the first electrode and the elongate body. 16. The electroporation cartridge of paragraph 15, wherein the first electrode includes a first electrode flange, the elongated body includes a proximal body flange, the proximal body flange rests in a plane substantially parallel to the first electrode flange, and a sealing member is disposed between the first electrode flange and the proximal body flange to form a fluid-tight joint therebetween. 17. The electroporation cartridge of any one of claims 1 to 16, wherein the first electrode functions to configure the electroporation cartridge in a sealed or non-sealed state. 18. The electroporation cartridge of paragraph 17, wherein the first electrode functions to configure the electroporation cartridge between a sealed state and an unsealed state without an additional removable cap piece. 19. The electroporation cartridge of paragraph 17 or 18, wherein the first electrode is a removable cap. 20. The electroporation cartridge of any one of claims 1 to 17, further comprising a removable cap secured to the first electrode, the removable cap including a coupling member for selectively securing the first electrode to the elongate body. 21. The electroporation cartridge of any one of claims 1 to 20, wherein the diameter of the proximal end of the second electrode is substantially equal to the cross-section of the electroporation chamber. 22. The electroporation cartridge of any one of claims 1 to 21, wherein the second electrode includes a protruding portion that extends from the distal end of the elongate body into the electroporation chamber. 23. The electroporation cartridge of paragraph 22, wherein the circumference of the protruding portion includes a shape complementary to the inner surface of the elongated body that defines the electroporation chamber. 24. The electroporation cartridge of paragraph 22 or 23, wherein the second electrode further includes a first sealing member disposed between the second electrode and the distal surface of the elongate body, the first sealing member functioning to form a fluid-tight joint between the second electrode and the distal surface of the elongate body. 25. The electroporation cartridge of paragraph 24, wherein the second electrode includes an electrode flange, the elongate body includes a distal body flange, the distal body flange lies in a plane substantially parallel to the electrode flange, and a sealing member is disposed between the electrode flange and the distal body flange to form a fluid-tight joint therebetween. 26. An electroporation cartridge according to any one of claims 22 to 25, wherein the second electrode further includes a second sealing member disposed around the protruding portion of the second electrode and distal to the proximal surface of the second electrode, the second sealing member functioning to form a fluid-tight joint between the protruding portion and the inner surface of the elongated body defining the electroporation chamber. 27. An electroporation cartridge according to any one of claims 22 to 26, wherein the proximal surface of the second electrode comprises a flat, uniform surface. 28. An electroporation cartridge according to any one of claims 22 to 27, wherein the proximal surface of the second electrode is perpendicular to the longitudinal axis of the electroporation chamber. 29. The electroporation cartridge of any one of claims 1 to 28, further comprising a fixation pin coupled to the second electrode and configured to fix the second electrode to the elongate body. 30. The electroporation cartridge of paragraph 29, wherein the second electrode defines a channel sized and shaped to receive a fixation pin, the channel being aligned with a pair of openings defined by the sidewalls of the elongate body and configured to receive the fixation pin, thereby securing the second electrode in a fixed position relative to the elongate body. 31. The electroporation cartridge of paragraph 30, wherein the channel is formed by a central region of a protruding portion of the second electrode distal to the first sealing member and / or the second sealing member. 32. The electroporation cartridge of any one of items 1 to 31, wherein the volume of the electroporation chamber is less than about 5 mL, preferably less than about 3 mL, more preferably less than about 1 mL, or between about 100 μL and 1 mL. 33. The electroporation cartridge of any one of claims 1 to 32, further comprising a volume reduction sleeve configured in size and shape to fit within the electroporation chamber. 34. The electroporation cartridge of paragraph 33, wherein the volume-reducing sleeve defines a secondary electroporation chamber having a smaller volume than the electroporation chamber. 35. The electroporation cartridge of paragraph 33 or paragraph 34, wherein the volume reduction sleeve includes a distal opening configured to interface with a second electrode when secured within the electroporation chamber. 36. The electroporation cartridge of any one of claims 33 to 35, wherein the volume reduction sleeve includes an air vent positioned adjacent the proximal end of the volume reduction sleeve and configured to allow air to pass through the volume reduction sleeve during introduction or withdrawal of the volume reduction sleeve from the electroporation chamber, thereby preventing a vacuum from forming between the secondary electroporation chamber and the electroporation chamber, thereby allowing electroporated cell-containing fluid to fill the secondary electroporation chamber upon insertion of the volume reduction sleeve and to exit the secondary electroporation chamber upon withdrawal of the volume reduction sleeve. 37. The electroporation cartridge of any one of claims 33 to 36, wherein the volume reduction sleeve includes a radial sealing member configured to secure the volume reduction sleeve within the electroporation chamber. 38. An electroporation cartridge according to paragraph 37, wherein the radial sealing member forms a fluid-tight seal with the sidewall defining the electroporation chamber to prevent leakage of cell-containing fluid within the secondary electroporation chamber through the distal opening of the volume reduction sleeve. 39. The electroporation cartridge of any one of claims 33 to 38, wherein the first electrode is configured to selectively couple with the volume reduction sleeve to form a fluid-tight seal. 40. An electroporation cartridge according to any one of claims 33 to 39, wherein a space is defined between the outer surface of the volume reduction sleeve and the inner sidewall of the elongate body, forming a fluid overfill space configured to receive an overfill volume displaced by the first electrode when sealing the electroporation chamber. 41. The electroporation cartridge of any one of claims 1 to 40, further comprising a fluid overfill space associated with the proximal region of the electroporation chamber and configured to receive an overfill volume displaced by the first electrode when sealing the electroporation chamber. 42. An electroporation cartridge according to any one of claims 1 to 41, further comprising one or more springs longitudinally disposed proximal to the elongate body and configured to position the first electrode in an uncapped position at a distance from the electroporation chamber. 43. The electroporation cartridge of paragraph 42, wherein the electroporation cartridge in the capped position is configured such that one or more springs are compressed and a first electrode is positioned within the electrode chamber to function to electroporate a cell-containing fluid disposed therein. 44. The electroporation cartridge of any one of claims 1 to 43, wherein the electroporation cartridge comprises a flow-through electroporation cartridge. 45. The electroporation cartridge of paragraph 44, further comprising a port coupled to the first electrode, the port defining a lumen within the first electrode that is fluidly connected to the electroporation chamber. 46. ​​The electroporation cartridge of paragraph 44, further comprising a port coupled to the proximal portion of the elongate body, the port configured to exhaust air displaced from the electroporation chamber when the electroporation chamber is filling and / or to introduce filtered or purified air into the electroporation chamber when the electroporation chamber is draining. 47. The electroporation cartridge of any one of claims 44 to 46, further comprising a chamber inlet and a chamber outlet, each of the chamber inlet and chamber outlet being fluidly connected to the electroporation chamber. 48. The electroporation cartridge of paragraph 47, wherein one or more chamber inlets or chamber outlets are located on the proximal surface of the second electrode. 49. The electroporation cartridge of paragraph 47 or paragraph 48, wherein the lumen of the chamber inlet and / or the chamber outlet is substantially parallel to the proximal surface of the second electrode. 50. An electroporation cartridge according to any one of claims 47 to 49, wherein one or more of the chamber inlets or chamber outlets are coupled to plugs and / or valves to control the inward flow of cell-containing fluid to be electroporated within the electroporation chamber and / or to control the outward flow of electroporated cell-containing fluid from the electroporation chamber. 51. An electroporation cartridge according to any one of claims 44 to 50, further comprising a fluid overfill space associated with the first electrode and / or the elongated body, the fluid overfill space configured to receive an overfill volume displaced from the electroporation chamber when filling the electroporation chamber. 52. An electroporation cartridge according to any one of claims 44 to 50, further comprising a fluid overfill space associated with the sealing cap, the fluid overfill space configured to receive an overfill volume expelled from the electroporation chamber when the electroporation chamber is sealed with the sealing cap. 53. An electroporation system configured to provide flow-through electroporation of a sample, comprising a modular casing having multiple compartments for holding and positioning multiple electroporation system components, the electroporation system components including one or more pumps configured to move the sample through the system; an electroporation compartment configured to receive a flow-through electroporation cartridge configured to hold a sub-volume of the sample within an electroporation chamber for electroporation of the sub-volume; and a tube having an inlet end and an outlet end, the tube routed through the casing to fluidly connect the multiple electroporation system components. 54. The electroporation system of paragraph 53, further comprising a bag compartment configured to receive and support an input bag and / or an output bag. 55. The electroporation system of paragraph 54, wherein the bag compartment comprises an insert slidably connected to the bag compartment such that the bag compartment can be selectively withdrawn from or inserted into the casing. 56. The electroporation system of paragraph 55, wherein the bag compartment includes one or more magnetic latches for holding the bag compartment in a sealed position within the casing. 57. The electroporation system of any one of claims 53 to 56, further comprising a cooling module in thermal contact with the electroporation chamber and configured to regulate the temperature of the electroporation chamber. 58. The electroporation system of paragraph 57, wherein the cooling module comprises a ceramic block. 59. The electroporation system of paragraph 57 or paragraph 58, wherein the cooling module is cooled by thermoelectric cooling. 60. The electroporation system of any one of claims 53 to 59, further comprising a mixer reservoir positioned downstream of the inlet and upstream of the electroporation cartridge, the mixer reservoir comprising a mixing element configured to mix portions of the sample contained in the mixing reservoir. 61. The electroporation system of paragraph 60, wherein the mixing element includes a mixing blade. 62. The electroporation system of paragraph 60 or 61, wherein the mixer reservoir includes a mixer magnet assembly mechanically coupled to the mixing element, and the mixer magnet assembly is positioned so as not to contact the portion of the sample contained within the mixer reservoir. 63. The electroporation system of paragraph 62, further comprising a mixer driver having a magnet magnetically coupled to the mixer magnet assembly and configured to indirectly drive rotation of the mixer magnet assembly via a magnetic connection to the mixer magnetic assembly. 64. The electroporation system of paragraph 62 or 63, wherein the mixer reservoir includes a cover and the mixer magnet assembly is positioned on or near the cover. 65. The electroporation system of any one of claims 60 to 64, further comprising a sample input assembly configured to assist in the transfer of sample between the input and the mixer reservoir, the sample input assembly including: a main section of tubing disposed between the input and the mixer reservoir; and an intermediate section of tubing pneumatically coupled to the main section of tubing and extending from there to an end that has access to air, thereby allowing air to pass through the main section of tubing when the pressure in the main section of tubing is sufficiently reduced. 66. The electroporation system of paragraph 65, wherein the end of the intermediate section is coupled to an air reservoir having a variable volume, and the sample input assembly is configured to detect a threshold decrease in the variable volume, thereby determining that the sample has been transferred to the mixer reservoir. 67. The electroporation system of paragraph 66, wherein the sample input sensor assembly comprises a syringe having a barrel and a plunger disposed within the barrel, the variable volume being defined by the position of the plunger within the barrel, and a threshold decrease in the variable volume being detected as a result of movement of the plunger. 68. An electroporation system according to any one of claims 53 to 67, further comprising a chamber sealing assembly operably coupled to the electroporation chamber and configured to regulate pressure within the electroporation chamber during electroporation, thereby limiting bubble formation. 69. The electroporation system of paragraph 68, wherein the chamber sealing assembly comprises one or more linear actuators configured to advance the plunger toward or retract the plunger from the electroporation chamber, thereby regulating the pressure within the electroporation chamber. 70. The electroporation system of any one of claims 53 to 69, further comprising a pre-cooling assembly positioned upstream of the electroporation chamber and configured to cool a sub-volume of the sample prior to electroporation of the sub-volume. 71. The electroporation system of paragraph 70, wherein the pre-cooling assembly comprises a cooling block and a section of tubing positioned within or adjacent to the cooling block. 72. An electroporation system according to paragraph 70 or 71, wherein the cooling block of the pre-cooling assembly is cooled by thermoelectric cooling. 73. The electroporation system of any one of claims 70 to 72, wherein the pre-cooling assembly includes a flexible biasing element that biases the cooling block against a section of the tube positioned adjacent to the cooling block. 74. The electroporation system of any one of claims 53 to 73, further comprising at least one flow sensor, the at least one flow sensor being positioned between the mixer reservoir and the electroporation chamber. 75. The electroporation system of item 74, wherein the flow sensor is an ultrasonic sensor. 76. The electroporation system of paragraph 74 or paragraph 75, wherein the flow sensor includes an actuatable trigger that, when actuated, places a corresponding section of tubing within the flow sensor to detect flow through the section of tubing. 77. An electroporation system according to any one of paragraphs 53 to 76, further comprising one or more flow indicators attached to a corresponding section of the tubing in a position outside the casing to allow visualization of flow through the section of the tubing. 78. An electroporation system according to any one of claims 53 to 77, wherein the casing includes one or more handles. 79. An electroporation system according to paragraph 78, wherein one or more handles include a handle having a catch configured to engage with the instrument panel to attach the instrument panel to the casing. 80. An electroporation system according to any one of paragraphs 53 to 79, further comprising an electroporation cartridge mounting feature coupled to the electroporation cartridge, the mounting feature including a flexible biasing element that biases the electroporation cartridge towards a cooling module integrated within the casing. 81. An electroporation system according to any one of claims 53 to 80, further comprising a capping mechanism configured to engage with the electroporation cartridge, the electroporation cartridge including a first electrode and a second electrode, each disposed at opposite ends of the electroporation chamber, at least one of the first electrode or the second electrode being engageable with the capping mechanism and capable of being moved from a capped position for electroporation to an uncapped position for venting as a result of actuation of the capping mechanism. 82. The electroporation system of paragraph 81, wherein the electroporation cartridge includes a spring mechanism that allows overtravel of the capping mechanism relative to displacement of the electrode moved as a result of actuation of the capping mechanism. 83. An electroporation system according to any one of claims 53 to 81, wherein the electroporation chamber comprises a chamber inlet and a chamber outlet, the chamber outlet being coupled to an outlet plunger movable from an advanced position that prevents the subvolume from exiting the electroporation chamber to a retracted position that allows the subvolume to exit the electroporation chamber. 84. An electroporation system according to any one of claims 53 to 83, wherein the electroporation cartridge comprises one or more bellows structures each configured to accommodate a moving component of the electroporation chamber. 85. The electroporation system of any one of claims 53 to 84, wherein the electroporation chamber comprises a flow-through electroporation cartridge similar to any one of claims 44 to 52. 86. An electroporation system according to any one of claims 53 to 85, further comprising an electroporation assembly electrically coupled to the electroporation chamber, the electroporation assembly comprising a conductivity sensor for measuring conductivity across the electroporation chamber, and the electroporation assembly being communicatively coupled to a controller having one or more processors and one or more hardware storage devices. 87. The one or more hardware storage devices, when executed by one or more processors, at least: determining, with a conductivity sensor, the conductivity of a subvolume within an electroporation chamber; determining a voltage drop across the electroporation chamber based on the determined conductivity; and 87. The electroporation system of claim 86, having computer-executable instructions stored thereon that configure the controller to: charge a capacitor in an electroporation circuit that includes the electroporation chamber to a voltage level that exceeds a determined voltage drop across the electroporation chamber to compensate for other voltage drops between the capacitor and the electroporation chamber. 88. The electroporation system of paragraph 86 or 87, wherein one or more hardware storage devices have stored thereon computer-executable instructions that, when executed by one or more processors, configure the controller to at least perform one or more of the following: determine the conductivity of a sub-volume in the electroporation chamber with a conductivity sensor; determine a predicted temperature rise of the sub-volume based on the determined conductivity, the set pulse voltage, and the set pulse duration; and, if the predicted temperature rise causes the temperature of the sub-volume to be higher than a predetermined threshold temperature, send an arc risk alert, and / or retrieve the sample sub-volume to preserve it, and / or adjust cooling accordingly to reduce the temperature of the electroporation chamber. 89. The electroporation system of paragraph 88, wherein the computer-executable instructions further configure the controller to determine an initial temperature of the subvolume within the electroporation chamber by correlating the determined conductivity to temperature. 90. The electroporation system of any one of claims 86-89, wherein one or more hardware storage devices have stored thereon computer-executable instructions that, when executed by one or more processors, configure a controller to at least determine the conductivity of a sub-volume within the electroporation chamber with a conductivity sensor, and eject the sub-volume from the electroporation chamber if the determined conductivity falls below a predetermined threshold, indicating the presence of one or more gas bubbles in the electroporation chamber. 91. An electroporation system according to any one of claims 53 to 90, further comprising a controller having one or more processors and one or more hardware storage devices, the one or more hardware storage devices having stored thereon computer-executable instructions that, when executed by the one or more processors, configure the controller to repeatedly increase the charger voltage based on a previous input voltage and a corresponding previously measured actual voltage applied to the electroporation chamber. 92. A system further comprising a controller having one or more processors and one or more hardware storage devices, the one or more hardware storage devices having stored thereon computer-executable instructions that, when executed by the one or more processors, configure the controller to determine a step volume to be moved by the system between each electroporation event, including at least determining the number N of revolutions of the drive pump required to move a sample volume sufficient to fill a tubing disposed between the flow sensor and the electroporation chamber and to completely fill the electroporation chamber, the number N corresponding to the volume between the flow sensor and the outlet of the electroporation chamber; 92. An electroporation system according to any one of claims 74 to 91, wherein calibration is performed by causing the drive pump to move the sample backward to the upstream position of the flow sensor using a fixed number k, where k corresponds to the volume between the upstream position of the flow sensor and the inlet of the electroporation chamber; determining the number x of rotations of the drive pump required to move the sample from the upstream position of the flow sensor to the flow sensor, where x corresponds to the volume between the upstream position of the flow sensor and the flow sensor, and a number (kx) corresponding to the volume between the flow sensor and the inlet of the electroporation chamber; and determining the volume between the inlet and outlet of the electroporation chamber to be N-(kx) and setting this volume as the step volume. 93. An electroporation system according to any one of claims 53 to 92, further comprising a safety door configured to mechanically open the electroporation circuit to prevent voltage discharge while the safety door is open. 94. An electroporation system according to any one of claims 53 to 93, wherein the capacitor circuit includes one or more discharge resistors to discharge the capacitor when the capacitor circuit is not electrically connected to the electroporation cartridge. 95. A method for determining a step volume to be moved by the system between each electroporation event, further comprising: a controller having one or more processors and one or more hardware storage devices; and a method for determining a step volume to be moved by the system between each electroporation event, the method comprising: Perform a first fill of the electroporation chamber by monitoring the electrical resistance of the sample fluid in the electroporation chamber during the first fill as it decreases from several thousand ohms to a stable value in the range of approximately 600-800 ohms, and terminating the first fill when the resistance reaches the stable value. N obtained from a peristaltic pump rev Fill the electroporation chamber for the second time based on the calculated values ​​such as After the second filling, attach a cap to the upper electrode and measure the electrical resistance. If the electrical resistance is within the stable range determined from the first fill, the second fill is complete and you can proceed to electroporate the sample. If the electrical resistance is not within the stable range determined from the first fill, remove the top electrode cap and perform additional rev Fine filling is carried out in the amount After the fine filling, a cap was attached to the upper electrode and the electrical resistance was measured. If the electrical resistance is within the stable range determined from the first fill, the second fill is complete and you can proceed to electroporate the sample. If not, repeating the steps of finely filling and measuring the electrical resistance until the electrical resistance is within the stable value determined from the first filling; Subsequent fills (third fill, fourth fill, etc.) are rev +n rev 92. The electroporation system of any one of claims 74 to 91, wherein calibration is performed by rotating the pump x times (x is equal to the number of microfilling attempts). 96. The system of paragraph 95, in which the stable value of electrical resistance is approximately 700 ohms. 97. The system of paragraph 95, wherein the step of total filling the electroporation chamber as the second fill is further based on a calculation using one or more parameters such as the inner diameter of the tubing, the number of rollers of the pump, the diameter of the electroporation chamber, and / or the height of the electroporation chamber.

[0281] summary The terms and expressions used in this specification are used as terms of description and not of limitation, and are not intended to exclude equivalents of the features shown and described or portions thereof, but it is understood that various modifications are possible within the scope of the invention described in the sections. While the present invention has been specifically disclosed above, in part, using preferred embodiments, exemplary embodiments, and optional features, it should be understood that those skilled in the art may anticipate modifications and variations of the concepts disclosed herein, and such modifications and variations are considered to be within the scope of the invention as defined by the accompanying sections. The specific embodiments provided herein are examples of useful embodiments of the present invention, and various changes and / or modifications of the features of the present invention as shown herein, as well as further applications of the principles shown herein that may occur to those skilled in the relevant art, can be made to the exemplified embodiments without departing from the spirit and scope of the invention as defined by the sections, and should be considered to be within the scope of this disclosure.

[0282] It will also be understood that systems, devices, articles of manufacture, kits, methods, and / or processes according to certain embodiments of the present disclosure may include, incorporate, or otherwise comprise properties or features (e.g., components, members, elements, parts, and / or portions) described in other embodiments disclosed and / or described herein. Thus, various features of particular embodiments may be compatible with, may be combined with, may be included in, and / or may be incorporated into other embodiments of the present disclosure. Thus, the disclosure of a particular feature in connection with a particular embodiment of the present disclosure should not be construed as limiting that feature to the particular embodiment. Rather, it will be understood that other embodiments may include such features, members, elements, parts, and / or portions without necessarily departing from the scope of the present disclosure.

[0283] Furthermore, unless a feature is described as requiring another feature in combination, any feature herein can be combined with any other feature of the same or different embodiments disclosed herein. Moreover, various well-known aspects of example systems, methods, devices, etc. have not been described in particular detail herein to avoid obscuring aspects of the example embodiments. However, such aspects are contemplated herein.

[0284] All references cited in this application are incorporated herein by reference in their entirety, to the extent that they do not contradict the disclosure of this application. It will be apparent to those skilled in the art that methods, devices, device elements, materials, procedures, and techniques other than those specifically described herein may be adapted to practice the invention broadly disclosed herein without resort to undue experimentation. All art-known functional equivalents of the methods, devices, device elements, materials, procedures, and techniques specifically described herein are intended to be encompassed by this invention.

[0285] When a group of materials, compositions, ingredients, or compounds is disclosed herein, it is understood that all individual members of that group and all subgroups thereof are separately disclosed. When a Markush group or other grouping is used herein, it is intended that all individual members of the group and all possible combinations and subcombinations of the group are individually included in the disclosure. All formulations or combinations of ingredients described or exemplified herein can be used to practice the invention, unless otherwise specified. Whenever a range, e.g., a temperature range, time range, or composition range, is given herein, it is intended that all intermediate ranges and subranges, as well as all individual values ​​included in the given range, are included in the disclosure.

[0286] All changes that come within the meaning and range of equivalence of the terms are to be embraced within their scope. [Example]

[0287] The following examples are provided to illustrate the practice of various exemplary embodiments disclosed herein. While a general paradigm or method flow is illustrated in the following examples, it will be understood by those of skill in the art that acts of the disclosed methods may be modified, omitted, or substituted with alternative or additional acts known in the art. Accordingly, the following examples are exemplary in nature and are not intended to unnecessarily limit the scope and / or content of the disclosure provided herein.

[0288] Example 1 To illustrate aspects of the functionality of the disclosed cartridges, systems, and methods for electroporating cells, exemplary cell preparation and processing methods were performed as shown in the exemplary embodiments of Figures 28, 29A, and 30A.

[0289] Referring to Figure 28, a flow diagram of an exemplary method for preparing cells for transformation via a batch or flow-through electroporation system and associated cartridge (disclosed above) is shown. As shown, an exemplary pre-electroporation method can include obtaining a cell culture. This can include, for example, growing an immortalized cell culture stock and / or isolating primary cells from a patient and culturing them in vitro to stabilize the cells prior to electroporation, growing the cells to confluence, and / or expanding the cells by serial passaging. This can be performed in an incubator with appropriate growth medium, as known in the art.

[0290] The method of Figure 28 further includes a mild enzymatic treatment, as known in the art, to release cells attached to the surface of the growth chamber. The acts of this method can be performed in a sterile environment, such as a biological safety cabinet or other enclosed, ventilated laboratory work space, to reduce the possibility of contamination.

[0291] The method of Figure 28 further illustrates the acts of removing the growth medium and washing the cells with a wash solution (e.g., phosphate-buffered saline (PBS)), followed by resuspension and titration to the desired cell density. These acts can be accomplished by any method using any materials known and used in the art for such purposes. Notably, however, Figure 28 illustrates a standard approach involving centrifugation and pipette-based washing and resuspension. Cells to be electroporated can be resuspended to the desired concentration by diluting the washed cells with a calculated volume of electroporation buffer (e.g., from cultured cells of known concentration and / or using a cytometer or other cell counting mechanism / system). A "payload," or electroporation target, as that term is defined herein, can be added to the total volume of resuspended cells or to a desired aliquot of an appropriate amount for batch or flow-through processing, as described herein.

[0292] In the preparation protocol shown in Figure 28, it is noted that for some primary and / or immortalized cell lines, cell viability decreases approximately 15 minutes after the cells are removed from growth medium and / or resuspended in electroporation buffer. Therefore, it is noted that addition of washed and / or resuspended cells to an electroporation system and / or cartridge for transformation using the above-described systems and methods is preferably performed within 15 minutes of washing and / or resuspending the cells. It is understood that this time period may vary between different cell and buffer types, and between different processing conditions.

[0293] Referring now to Figure 29A, an exemplary protocol for batch processing and transformation of cells prepared, for example, by the exemplary method outlined in Figure 28 above is shown. As shown in the exemplary electroporation protocol in Figure 29A, an aliquot or total amount of cells prepared according to the method outlined and discussed with respect to Figure 28 is loaded into an electroporation (EP) cartridge along with the desired payload or electroporation target. The EP cartridge is inserted into an electroporation system described herein configured to batch process the sample, electroporation parameters are set and run on the system, and the EP cartridge is removed from the system. The electroporated cells can then be transferred to complete medium or other recovery medium and incubated for a period of time (e.g., 24-72 hours). The electroporated cells can then be examined for viability and electroporation efficiency using appropriate biochemical, optical, or molecular readouts (e.g., Western blot for protein concentration / expression, flow cytometry for expression of transfected fluorescent proteins, qPCR for molecular analysis, etc.).

[0294] For example, as shown in Figure 29B, primary T cells activated using CD3 / CD28 Dynabeads™ were cultured for 4 days and then prepared for electroporation according to the protocol disclosed and illustrated in Figures 28 and 29A. Washed primary cells were diluted to 50x10 in R buffer. 6The cells were resuspended to a concentration of several cells / mL and loaded with a Cas9+ gRNA payload targeting TRAC-1. Electroporation was completed using a batch processing method and the associated EP cartridge with electroporation parameters of 2300V / 3ms for every four pulses. After electroporation, the cells were transferred to complete medium and incubated for 48 hours. The electroporated cells were then stained with TCRα / β antibodies and evaluated for residual expression by flow cytometry compared to non-electroporated control cells (negative control). As shown in Figure 29B, the transduction efficiency enabled by the disclosed system was within the expected range compared to commercially available platforms. The cells were also not significantly affected by electroporation, as confirmed by Sytox viability staining, which was also confirmed by flow cytometry.

[0295] Referring now to Figure 30A, an exemplary protocol for flow-through processing and transformation of cells prepared, for example, by the exemplary method outlined in Figure 28 is shown. As shown in the exemplary electroporation protocol in Figure 30A, resuspended cells are transferred or resuspended in an input bag along with the desired payload or electroporation target. The input bag is attached to the input port of the flow-through consumable, as described above. The flow-through consumable cartridge is then loaded into an electroporation system, such as the flow-through electroporation system described above. Electroporation parameters are set and run (e.g., using an associated computing system and / or user interface on the electroporation system), and the output bag containing the electroporated cells is removed from the consumable cartridge using, for example, a tube sealer. The electroporated cells can then be transferred to complete medium or other recovery medium and incubated for a period of time (e.g., 24-72 hours). Electroporated cells can then be examined for viability and electroporation efficiency using appropriate biochemical, optical, or molecular readouts (e.g., Western blot for protein concentration / expression, flow cytometry for expression of transfected fluorescent proteins, qPCR for molecular analysis, etc.).

[0296] For example, as shown in Figure 30B, primary T cells activated using CD3 / CD28 Dynabeads™ were cultured for 4 days and then prepared for electroporation according to the protocol disclosed and illustrated in Figures 28 and 30A. Washed primary cells were diluted to 50x10 in R buffer. 6The cells were resuspended to a concentration of several cells / mL and loaded with a Cas9+ gRNA payload targeting TRAC-1. Electroporation was completed using a flow-through processing method and associated flow-through consumables with electroporation parameters of 2300V / 3ms for every four pulses. After electroporating each sample using automated batch processing (e.g., the "flow-through" method), cells were harvested and any inter-sample variability was assessed. Each isolated sample was transferred to complete medium and incubated for 48 hours. Next, electroporated cells were stained with TCRα / β antibody and evaluated for residual expression by flow cytometry compared to non-electroporated control cells (negative control). The results for each sample using automated batch processing or flow-through processing are shown in Figure 30B. As clearly demonstrated by the data, the transformation efficiency enabled by the disclosed system was within the expected range compared to commercially available platforms. Cells were also not significantly affected by electroporation, as confirmed by Sytox viability staining, which was also confirmed by flow cytometry.

[0297] Comparisons of cell viability and electroporation efficiency were performed between single-batch processed samples and flow-through or automated batch processed samples of the same cultures of cells prepared as described above with respect to Figures 28, 29A, and 30A. As shown in Figure 31, the flow-through or automated batch processed samples have higher viability and electroporation efficiency than the prior art system, while the single-batch processed samples have similar transformation efficiency and cell viability.

[0298] The disclosed electroporation system and method have also been shown to function using immortalized cell cultures. Referring now to FIG. 32, a graph is shown demonstrating the viability and transformation efficiency of an exemplary flow-through system and method using Jurkat cells. In particular, Jurkat cells established for four passages after thawing were prepared for electroporation according to the protocols outlined in FIG. 28 and FIG. 30A. Electroporations were completed using exemplary flow-through consumables, and individual electroporations were collected to assess sample-to-sample variability.

[0299] Washed Jurkat cells were diluted to 50 × 10 in R buffer. 6 The cells were resuspended to a concentration of 100 cells / mL and given a payload of 4.6 kB GFP plasmid. Electroporation was completed using a flow-through processing method and associated flow-through consumables with electroporation parameters of 1700 V / 20 ms per single pulse. After electroporation of each sample using the automated batch / flow-through method, cells were collected to assess any inter-sample variability. Each isolated sample was transferred to complete medium and incubated for 24 hours. The electroporated cells were then assessed for GFP expression via flow cytometry compared to a non-electroporated control. Results for each sample using the automated batch or flow-through method are shown in Figures 32 and 33 (negative control data not shown in Figure 32). As clearly demonstrated by the data, the transformation efficiency enabled by the disclosed system is within the expected range compared to commercially available platforms, and cells were not significantly affected by electroporation, as confirmed by Sytox viability staining, which was also confirmed by flow cytometry.

[0300] Example 2 To illustrate aspects of the functionality of the disclosed electroporation cartridges, instruments, systems, and methods for electroporating cells, exemplary cell preparation and processing methods were performed as illustrated by the exemplary embodiments described herein.

[0301] Cell Source and Culture Conditions: Peripheral blood mononuclear cells (PBMCs) were isolated from leukopaks of healthy donors using the standard Ficoll-Paque method and cryopreserved. After thawing, PBMCs were activated with CD3 / CD28 Dynabeads™ and cultured in OpTmizer™ medium containing either 2% human serum or 5% immune cell serum replacement and maintained at 37°C and 5% CO2. Thus, activated primary human T cells were generated.

[0302] Electroporation: Three days after activation, cells were prepared for electroporation by centrifugation and resuspended in standard electroporation buffer or gene editing buffer. Ribonucleoproteins (RNPs) were formed by combining Invitrogen™ TrueCut™ Cas9 Protein v2 with Invitrogen™ TrueGuide™ synthetic gRNA (Thermo Fisher Scientific). The prepared cells and RNPs were combined, incubated for 5 minutes, and donor DNA template was added. Then, electroporation was performed using a newly developed large-scale electroporation system in a single-use cartridge or the disclosed flow-through cartridge, as described below. Immediately after electroporation, cells were returned to complete medium and cultured for 48–72 hours. Analysis was performed using locus-specific antibody targets on an Invitrogen™ Attune™ NxT flow cytometer (Thermo Fisher Scientific).

[0303] In one exemplary embodiment, activated primary human T cells were generated and electroporated with Cas9 RNP-targeted introduction of homology-directed repair transfection of either the Rab11a or TRAC locus. The activated primary human T cells were prepared as described in the method above (under the heading "Cell Source and Culture Conditions"). The activated primary human T cells were then resuspended in gene editing buffer under the following conditions: Cell number = 2x10 7 The concentrations were: c / mL, Cas9 = 80 μg / mL, gRNA = 20 μg / mL, and dsDNA = 80 μg / mL. Cells were immediately electroporated using a newly developed large-scale electroporation system with electroporation condition A (i.e., 1700 V / 10 ms / 1 pulse) or electroporation condition F (i.e., 2300 V / 3 ms / 4 pulses). Cas9:gRNA RNPs targeting either the Rab11a or TRAC locus and a linear dsDNA template (1.4 kb) encoding GFP with 100 bp homology arms were supplied in a single-use cartridge as described herein. Knock-in efficiency (KI efficiency in Figure 34) and cell viability were analyzed 48 hours after electroporation using flow cytometry. The results are shown in Figure 34.

[0304] FIG. 34 shows the cell viability, transformation efficiency, and knock-in efficiency of activated primary human T cells transformed as described above, demonstrating the successful use of an exemplary single-use consumable and electroporation system and method for electroporation according to one or more embodiments of the present disclosure.

[0305] In another exemplary embodiment, activated primary human T cells were generated and electroporated with Cas9:gRNA RNP-targeted transfer of the TRAC locus to generate CAR-T cells. The activated primary human T cells were prepared as described in the method above (under the heading "Cell Source and Culture Conditions"). The activated primary human T cells were then resuspended in gene editing buffer under the following reagent conditions: Cell number = 2.5x10 7 c / mL, Cas9 = 100 μg / mL, gRNA = 25 μg / mL, and dsDNA = 80 μg / mL. Cells were electroporated using electroporation condition F (2300 V / 3 ms / 4 pulses) in both a 1 mL single-use electroporation cartridge of the present disclosure (referred to as "small-scale EP" in Figure 35) and a 1 mL flow-through electroporation cartridge of the present disclosure (referred to as "large-scale EP" in Figure 35) and supplied with a Cas9:gRNA RNP targeting the TRAC locus and a linear dsDNA template (2.8 kb) encoding an Anti-CD19CAR with 100 bp homology arms. Knock-in efficiency (KI efficiency in Figure 35) and cell viability were analyzed 96 hours after electroporation using flow cytometry. The results are shown in Figure 35.

[0306] Figure 35 shows the transduction efficiency, knock-in efficiency, and cell viability during the generation of CAR-T cells using flow-through and single-use cartridges according to one or more embodiments of the present disclosure, and the electroporation systems and methods disclosed herein.

Claims

1. Electroporation system, as follows: An electroporation compartment configured to receive an electroporation cartridge having an electroporation chamber; A cooling module configured to make thermal contact with the electroporation chamber when the electroporation cartridge is received into the electroporation compartment, and configured to regulate the temperature of the electroporation chamber; An electroporation assembly configured to electrically couple with an electroporation chamber when an electroporation cartridge is received into an electroporation compartment, the electroporation assembly comprising a conductivity sensor configured to generate and supply electrical pulses to the electroporation chamber and to measure the conductivity between the ends of the electroporation chamber; and A controller including a processor and hardware storage device, wherein the controller is communicatively coupled to an electroporation assembly. The electroporation system, including the above.

2. When a hardware storage device is executed on it by a processor, the following is communicated to the controller: The conductivity of the sample in the electroporation chamber is determined by a conductivity sensor. Based on the determined conductivity, the voltage drop across both ends of the electroporation chamber is determined, and To compensate for other voltage drops between the capacitor and the electroporation chamber, the capacitor in the electroporation circuit with the electroporation chamber is charged to a voltage level that exceeds the determined voltage drop across the electroporation chamber. The electroporation system according to claim 1, which stores computer executable instructions configured to perform the following:

3. When one or more hardware storage devices are run on by one or more processors, the controller must have at least the following: The conductivity sensor is used to determine the conductivity of the sample in the electroporation chamber. Based on the determined conductivity, set pulse voltage, and set pulse duration, the predicted temperature rise of the sample is determined, and If the temperature of the sample rises above a predetermined threshold temperature due to the predicted temperature rise, one or more of the following actions will be performed: sending an arc risk alert, retrieving the sample from the electroporation chamber, and / or lowering the temperature of the electroporation chamber. The electroporation system according to claim 1, which stores computer executable instructions configured to perform the following:

4. When one or more hardware storage devices are run on by one or more processors, the controller must have at least the following: Determining the conductivity of a sample in an electroporation chamber using a conductivity sensor; and If the determined conductivity falls below a predetermined threshold indicating the presence of one or more bubbles in the electroporation chamber, the sample is discharged from the electroporation chamber. The electroporation system according to claim 1, which stores computer executable instructions configured to perform the following:

5. The electroporation system according to claim 1, wherein the electroporation assembly includes one or more discharge resistors for discharging a capacitor configured to generate an electrical pulse when the capacitor is not electrically connected to the electroporation cartridge.

6. The electroporation system according to claim 1, further comprising a safety door configured to mechanically open the electroporation circuit to prevent voltage discharge while the safety door is open.

7. The electroporation system according to claim 1, further comprising a graphical user interface having a function to receive input from a user in order to control the operation of the electroporation system.

8. The electroporation system according to claim 1, wherein the cooling module includes a ceramic block.

9. The electroporation system according to claim 1, wherein the cooling module is cooled via thermoelectric cooling.

10. The electroporation system according to claim 1, further comprising a pre-cooling assembly configured to cool the sample before it is introduced into the electroporation chamber.

11. The electroporation system according to claim 1, further comprising a capping mechanism configured to engage with an electroporation cartridge, the electroporation cartridge comprising a first electrode and a second electrode, each positioned at opposite ends of an electroporation chamber, and at least one of the first electrode or the second electrode being engageable with the capping mechanism and being able to move between a capped position for electroporation and an uncapped position for venting as a result of the operation of the capping mechanism

12. The electroporation system according to claim 1, further comprising at least one flow sensor.

13. The electroporation system according to claim 12, wherein the flow sensor is an ultrasonic sensor.

14. The electroporation system according to claim 1, wherein the electroporation cartridge is a single-use electroporation cartridge.

15. The electroporation system according to claim 1, wherein the electroporation cartridge is a flow-through electroporation cartridge.

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