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

Abstract

This disclosure provides electroporation cartridges for single-use electroporation, as well as electroporation cartridges, electroporation instruments and systems for automated batch processing, and methods of electroporation using these devices and systems. In some embodiments, the electroporation cartridge includes: an electroporation chamber defined by an elongated body; a first electrode at a proximal end; and a second electrode at a distal end of the chamber. The electroporation system of this disclosure includes one or more components including: a pulse generator; a chamber for placing a flow-through or single-use electroporation cartridge; components for storing cells; cooling and precooling mechanisms; a modularly insertable housing having a chamber for holding and arranging the electroporation system and reagent components; one or more pumps for moving samples through the system; and a processor and a controller.

Description

[0001] This invention patent application is a divisional application of the invention patent application filed on October 26, 2020, with national application number 202011154566.1 and entitled "System, method and apparatus for electroporation containing cellular fluid". Technical Field

[0002] This disclosure generally relates to systems, apparatus, and methods for treating cells by a transient electric field. More specifically, this disclosure relates to systems, apparatus, and methods for automated electroporation of cell-containing fluids, including single and automated batch electroporation systems, apparatus, and methods. Background Technology

[0003] Since at least the 1970s, scientists have used electroporation as a technique for inserting molecules into animal or plant cells. By exposing cells to a transient electric field (especially a short-duration, high-voltage field), the cell membrane becomes permeable to molecules in the surrounding medium, allowing the cell to take up target macromolecules (typically proteins and nucleic acids). When the voltage and duration of exposure to the electric field are properly controlled, electroporated cells are able to restore membrane permeability and normal function. However, overexposure to the electric field—whether for a prolonged period or at excessively high voltages—can permanently disrupt the cell's electrical potential and / or membrane integrity, leading to cell death.

[0004] Traditionally, cell electroporation has been performed using specialized cuvettes containing electrodes positioned relative to each other to create a uniform electric field between them. For example, electroporation cuvettes known in the art comprise two flat plate electrodes attached to the opposing walls of a rectangular cuvette or chamber. A suspension of cells to be electroporated is combined with an electroporation target and placed in the cuvette, where high-voltage and short-duration electric field pulses are applied through the electrodes. Most commercially available electroporation cuvettes of this type are limited in capacity and can only process small amounts of cell suspension at a time (typically less than one milliliter).

[0005] However, this configuration has been preferred and retained over time due to its high efficiency and ability to maintain a more standardized distribution of current through the affected medium by generating a uniform electric field. A uniform electric field helps normalize electroporation efficiency by reducing current hotspots that could damage or destroy cells and by reducing current colds that lead to low electroporation efficiency. However, the strength of the uniform electric field depends on the potential difference or voltage between the electrodes and the distance between them. Increasing the distance between the electrodes weakens the electric field. While this can be compensated for by increasing the voltage, the fragile nature of living cells has traditionally set limits on the voltage and / or distance that can be increased between the electrodes while maintaining a sufficiently strong uniform electric field to effectively electroporate cells.

[0006] For example, to maintain a uniform electric field between two electrodes as the physical distance between them increases, the voltage must be increased. Increasing the voltage causes the electrodes to generate more heat that 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, along with the initial temperature of the sample, rapidly leads to sample temperatures exceeding 60°C or even localized vaporization of portions of the cell-containing sample. As expected, the increased temperature negatively impacts cell viability, especially when those temperatures are prolonged or within ranges incompatible with living cells. Heating and discharging through aqueous solutions (i.e., almost all cell-containing fluids and / or electroporation media) can also cause bubble formation through localized vaporization of water molecules and / or through the electrolysis of water molecules to form oxygen and hydrogen. The presence of bubbles can cause conductivity failure and lead to arcing, which particularly reduces electroporation performance and can potentially damage or destroy the sample. Accordingly, the volume of cell-containing fluid that can be electroporated has traditionally been limited.

[0007] Due to the intensive, manual process of loading and unloading cuvettes, scaling up this type of electroporation model by limiting the volume of the cuvettes has previously been impractical. Furthermore, maintaining sterility is essential for almost all applications of electroporation of large-volume cells, and the repeated loading of cuvettes and the aggregation of cells for electroporation are particularly impractical and prone to contamination. While this electroporation method is convenient and simple, and has met the needs of many researchers performing small-scale electroporation of cells, additional methods are needed, particularly those that can conveniently facilitate large-volume electroporation of cells while maintaining a sterile environment.

[0008] Electroporation of large volumes of cells in a closed, sterile system will enable the use of electroporation for cell-based therapies in humans. In attempts to address this issue, several continuous-flow electroporation systems have been created, typically consisting of parallel electrodes with a cell-containing fluid flowing continuously and steadily between them until the entire volume of the cells has been subjected to a high-voltage electric field. However, the problem is that repeatedly applying high-voltage pulses to the electrodes of a continuous-flow system leads to excessive heat generation. Some systems have compensated for this using cooling mechanisms to prevent the electrodes and cell suspension from reaching excessively high temperatures.

[0009] However, these continuous flow systems suffer from a lack of efficiency and reliability. For example, the hydrodynamic flow rate of cellular fluid through the electroporation chamber of a continuous flow device does not result in each cell passing through the chamber and traveling between the electrodes at the same rate. The flow velocity away from the chamber wall is higher than the velocity near the chamber wall. Cells flowing towards the center of the fluid flow between the electrodes will pass between the electrodes in less time and can receive an inefficient number of pulses, while cells flowing near the wall may take longer to pass between the electrodes and thus receive too many pulses. Therefore, most continuous flow systems are inherently flawed and inefficient because there exists a subset of cells that either pass through the electrodes too quickly and are not adequately electroporated, or pass through the electrodes too slowly and are overexposed to the high-voltage electric field, becoming damaged or destroyed.

[0010] Between the imprecise and non-uniformity of electroporation through which cells pass through electrodes and the continuous application of an electric field through cell-containing fluids (which leads to bubble formation and excessive heat), continuous-flow electroporation systems have failed to achieve sufficient throughput and efficiency to meet the demands of both research and commercial cell therapies for rapid and efficient cell transfection. Indeed, electroporation has so far been unable to compete commercially with viral transfection, which is the most efficient and widely used technique. However, there are inherent risks associated with using viruses to perform transfection on cells that may eventually be returned to patients.

[0011] Correspondingly, there are many shortcomings and problems that can be addressed to automate the electroporation of cells, and there is a significant need for systems, devices, and methods that can automate the electroporation process with high efficiency, particularly by reducing the possibility of contamination and / or arcing caused by bubble formation in the processed cell-containing fluid. Summary of the Invention

[0012] Implementations of this disclosure address one or more of the foregoing or other problems in the art through systems, apparatus, and methods for electroporating cell-containing fluids. The terms "cell-containing fluid" and "sample" are used interchangeably herein.

[0013] Specifically, an exemplary embodiment includes 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 a opposite distal end of the electroporation chamber. In some embodiments, at least one of the first electrode or the second electrode is movable between a capped position for electroporation and an uncapped position for loading samples for single use or automated batch processing, and / or the electroporation cartridge is configurable between a sealed state and an unsealed state.

[0014] In one aspect, the elongated body of this disclosure may comprise or include one or more of, or be made of, non-conductive plastics, glass, and / or ceramics, and is configured to receive cell-containing fluid to be electroporated within the electroporation chamber defined by the elongated body. For example, the electroporation chamber of this disclosure may comprise or include glass and / or ceramics, or be made of, them. As an additional example, the electroporation chamber may comprise or include polycarbonate and / or other non-conductive, radiation-stabilized plastics, or be made of, them.

[0015] In one aspect, at least a portion of the electroporation chamber of this disclosure tapers gradually between the first electrode and the second electrode. When present, the tapering portion of the electroporation chamber does not substantially interfere with the generation of a uniform electric field between the first electrode and the second electrode.

[0016] Additionally or alternatively, the electroporation chamber of this disclosure has a uniform cross-section along the length of the reaction chamber. In some instances, the uniform cross-section may extend 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. For example, the electroporation chamber of this disclosure may be a cylindrical cavity defined by the elongated body, such that the uniform cross-section is circular.

[0017] In one aspect, the electroporation cartridge of this disclosure includes a proximal sidewall defined between a proximal opening of the elongated 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 position distal from the inflection point.

[0018] In one aspect, the first electrode includes a spherical extension. In another aspect, the spherical extension may have a substantially flat distal surface. However, preferably, the spherical extension has a distal surface with a convex or angled profile and is operable to displace one or more air bubbles associated with the cell-containing fluid to be electroporated within the electroporation chamber while the first electrode is secured within the electroporation chamber or the electroporation chamber is additionally sealed. In any embodiment, the spherical extension can be separated from the base portion of the first electrode by a narrow rod.

[0019] In one aspect, the electroporation cartridge of this disclosure includes a sealing member disposed between the first electrode and the proximal surface of the elongated body, the sealing member being operable to form a fluid-tight connection between the first electrode and the elongated body. The first electrode may further include a first electrode flange, and the elongated body may include a proximal body flange. The proximal body flange may be oriented in a plane substantially parallel to the first electrode flange, wherein the sealing member is disposed between the first electrode flange and the proximal body flange to form the fluid-tight connection therebetween.

[0020] In one aspect, the first electrode is operable to: configure an electroporation cartridge between a sealed state and an unsealed state, and this operation can be performed without an additional removable capping workpiece.

[0021] In one aspect, the first electrode itself is a cap. In another aspect, the first electrode itself is a removable cap.

[0022] In one aspect, the electroporation cartridge of this disclosure includes a removable cap secured to the first electrode. The removable cap includes a coupling member for selectively securing the first electrode to the elongated body.

[0023] 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 may include a protrusion extending from the distal end of the elongated body into the electroporation chamber and may have a complementary shape, for example, to the inner surface of the elongated body defining the electroporation chamber. Additionally or alternatively, the second electrode may include a first sealing member disposed between the second electrode and the distal surface of the elongated body, the first sealing member being operable to form a fluid-tight connection between the second electrode and the distal surface of the elongated body. For example, the second electrode may include an electrode flange, and the elongated body may include a distal body flange oriented in a plane substantially parallel to the electrode flange, and the sealing member may be positioned between the electrode flange and the distal body flange to form a fluid-tight connection therebetween. Additionally or alternatively, the second electrode may include or be associated with a second sealing member deployed around the protrusion of the second electrode and positioned distal from the proximal surface of the second electrode, the second sealing member being operable to form a fluid-tight connection between the protrusion and the inner surface of the elongated body defining the electroporation chamber.

[0024] In some aspects, the proximal surface of the second electrode includes a flat, uniform surface, and / or the proximal surface of the second electrode may be orthogonal to the longitudinal axis of the electroporation chamber.

[0025] In one aspect, the electroporation cartridge of this disclosure includes a retaining pin associated with and configured to secure the second electrode to the elongated body. For example, the second electrode defines a channel configured in size and shape to receive the retaining pin and can be aligned with a pair of holes defined by the sidewall of the elongated body to receive the retaining pin, thereby securing the second electrode in a fixed position relative to the elongated body. The channel may be formed to pass through the central region of the protruding portion of the second electrode distal to the first sealing member and / or the second sealing member.

[0026] In one aspect, 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.

[0027] In one aspect, the electroporation cartridge of this disclosure includes a volume-reducing sleeve configured in size and shape to fit within the electroporation chamber. The volume-reducing sleeve defines a secondary electroporation chamber having a smaller volume than the electroporation chamber and a distal opening configured to abut against a second electrode when secured within the electroporation chamber.

[0028] In one aspect, the volume reduction sleeve includes a vent hole deployed adjacent to the proximal end of the volume reduction sleeve, the vent hole being configured to allow air to pass through it during introduction into or removal from the electroporation chamber of the present disclosure of the volume reduction sleeve, and to prevent the formation of a vacuum between the sub-electroporation chamber and the electroporation chamber, thereby allowing cell-containing fluid of electroporation to fill the sub-electroporation chamber upon introduction of the volume reduction sleeve and to exit the sub-electroporation chamber upon removal of the volume reduction sleeve.

[0029] The volume-reducing sleeve of this disclosure may further include a radial sealing member configured to secure the volume-reducing sleeve within the electroporation chamber of this disclosure. The radial sealing member may form 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-reducing sleeve.

[0030] The first electrode can be configured to selectively associate with the volume-reducing sleeve and form a fluid-tight seal with the volume-reducing sleeve.

[0031] In some embodiments, the electroporation cartridge of this disclosure may have a space between the outer surface of the volume-reducing sleeve and the inner sidewall of the elongated body to form a fluid overflow space, which is configured to receive the overflow volume displaced by the first electrode when the electroporation chamber is sealed.

[0032] In one aspect, the electroporation cartridge includes a fluid overflow space associated with a proximal region of the electroporation chamber and configured to receive an overflow volume displaced by the first electrode when the electroporation chamber is sealed.

[0033] In one aspect, the electroporation cartridge of this disclosure is a flow-through electroporation cartridge. A flow-through electroporation cartridge may include a port associated with the first electrode, defining a lumen within the first electrode such that the lumen is fluidly connected to the electroporation chamber. Alternatively, the flow-through electroporation cartridge of this disclosure may include a port associated with a proximal portion of the elongated body, configured to: vent displaced air from the electroporation chamber when it is being filled, and / or introduce filtered or purified air into the electroporation chamber when it is being emptied. In some instances, the flow-through electroporation cartridge includes a chamber inlet and a chamber outlet, both fluidly connected to the electroporation chamber. One or more of the chamber inlet or chamber outlet may be deployed above the proximal surface of the second electrode, and / or the lumens of the chamber inlet and / or chamber outlet are substantially parallel to the proximal surface of the second electrode. Additionally or alternatively, one or more of the chamber inlet or chamber outlet may be associated with a plug and / or a valve to control the inward flow of the cell-containing fluid to be electroporated into the electroporation chamber, and / or to control the outward flow of the cell-containing fluid to be electroporated from the electroporation chamber.

[0034] The electroporation cartridge disclosed herein may include a fluid overflow space associated with the first electrode and / or the elongated body, the fluid overflow space being configured to receive overflow volume displaced from the electroporation chamber when the electroporation chamber is filled or when the electroporation chamber is sealed by the sealing cap.

[0035] An exemplary electroporation system configured to provide flow-through electroporation of a sample includes a modular housing having multiple chambers for holding and arranging multiple electroporation system components. The electroporation system of this disclosure may include: one or more pumps configured to move a sample through the system; and an electroporation chamber configured to receive a flow-through electroporation cartridge configured to hold a sub-volume of sample within the electroporation chamber for electroporation of the sub-volume. The electroporation system of this disclosure may also include conduits routed through the housing to fluidly connect the multiple electroporation system components between an inlet and an outlet.

[0036] In one aspect, the electroporation system of this disclosure includes a compartment configured to receive and support an input bag and / or an output bag. The compartment may include an insertion portion slidably connected to the compartment to allow selective extraction from or containment within the housing, and may include one or more magnetic latches for holding the compartment in a closed position within the housing. In another aspect, the electroporation system includes one or more hooks on which one or more bags are suspended externally from the housing.

[0037] In one aspect, the electroporation system of this disclosure includes one or more features for regulating the temperature of the sample (e.g., according to a predetermined target temperature) via cooling and / or heating. For example, the electroporation system may include a cooling module that is in thermal contact with the electroporation chamber and configured to regulate the temperature of the electroporation chamber. In some embodiments, the cooling module may include a ceramic block that is cooled via thermoelectric cooling. Other embodiments of the cooling modules used in the systems and apparatus of this disclosure may additionally or alternatively utilize air cooling, liquid cooling, or other temperature regulation mechanisms known in the art. As described below, while these components are described herein as “cooling modules” based on their typical function, they may also be configured to provide heat in applications where such heating is desired.

[0038] In one aspect, the electroporation system of this disclosure includes a mixer container deployed downstream of the inlet and upstream of the electroporation cartridge, the mixer container including a mixing element configured to provide mixing to a portion of the sample contained within the mixing container. The mixing element may be formed as mixing blades or other mixing devices that avoid contact between magnetic portions and the sample fluid.

[0039] In one aspect, the mixer container includes a mixer magnet assembly mechanically coupled to the mixing element, the mixer magnet assembly being deployed without contacting the portion of the sample contained within the mixer container. For example, the mixer container may have a cover, and the mixer magnet assembly may be deployed at or near the cover. The electroporation system of this disclosure may also 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 with the mixer magnet assembly.

[0040] In one aspect, the electroporation system of this disclosure may include or comprise a sample input assembly configured to facilitate the transfer of a sample between the input and the mixer container. In some embodiments, the sample input assembly includes: a main conduit section disposed between the input and the mixer container; and an intermediate conduit section coupled to the main conduit section in a manner allowing air to be passed from the intermediate conduit section to the main conduit section, the intermediate conduit section extending from the main conduit section to a terminal end. The terminal end of the intermediate conduit section has an air inlet (e.g., an air container or filter opening to the atmosphere), and the intermediate conduit section thereby allows air to be passed to the main conduit section when there is a sufficient pressure drop in the main conduit section. For example, when the contents of the input reservoir coupled to the inlet are empty or nearly empty, continuous pumping will cause a pressure drop in the main conduit section, thereby drawing air from the intermediate conduit section into the main conduit section.

[0041] In one aspect, the electroporation system of this disclosure includes a chamber sealing assembly operatively coupled to the electroporation chamber and configured to regulate the pressure within the chamber during electroporation and thereby limit bubble formation. The chamber sealing assembly may include one or more linear actuators configured to advance a plunger toward or retract a plunger from the chamber, thereby opening and closing corresponding ports to regulate fluid flow through the chamber and pressure within the chamber.

[0042] In one aspect, the electroporation system includes a precooling assembly deployed upstream of the electroporation chamber and configured to regulate the temperature of a sub-volume of the sample prior to electroporation. In some embodiments, the precooling assembly may include or comprise a cooling block and a conduit segment deployed within or adjacent to the cooling block. In some embodiments, the cooling module may be cooled, for example, via thermoelectric cooling. In other embodiments, the cooling block may additionally or alternatively utilize air cooling, liquid cooling, or other temperature regulation mechanisms known in the art. As described herein, while these components are described based on their typical function as a “precooling module / assembly,” they may also be configured to provide heat in applications where such heating is desired. The electroporation system of this disclosure may also include a flexible biasing element that biases the cooling block against the conduit segment deployed within or adjacent to the cooling block.

[0043] In one aspect, the electroporation system of this disclosure may include one or more flow sensors configured to detect flow rate (or absence thereof) through a specific section of the conduit. The flow sensors may be deployed between the mixer container and the electroporation chamber. In some non-limiting embodiments, for example, the flow sensor may be an ultrasonic sensor.

[0044] In one aspect, the housing of the electroporation system of this disclosure is configured to route one or more pipe sections in a path that provides a visual indication of the flow rate through the pipe sections (e.g., along the exterior of the housing).

[0045] In one aspect, the housing of the electroporation system disclosed herein includes one or more handles. In a non-limiting embodiment, the one or more handles may include a handle having a latch portion configured to engage with a dashboard to attach the dashboard to the housing.

[0046] In one aspect, the electroporation system of this disclosure includes an electroporation cartridge attachment feature coupled to the electroporation cartridge and configured to allow selective attachment and unattachment of the electroporation cartridge to the housing. The attachment feature may include a flexible biasing element that biases the electroporation cartridge toward the cooling module.

[0047] In one aspect, the electroporation system of this disclosure includes or comprises a capping mechanism. The capping mechanism is configured to engage with an inserted electroporation cartridge and is configured to be actuated to move one of the electrodes of the electroporation cartridge between a capped position for electroporation and an uncapped position for discharge. In some embodiments, the electroporation cartridge of this disclosure may include or comprise a spring mechanism that allows the capping mechanism to overtravel relative to the displacement of the electrodes as a result of actuation of the capping mechanism.

[0048] In one aspect, the electroporation cartridge of this disclosure includes one or more bellows structures, each configured to enclose a movable component of the electroporation chamber.

[0049] In one aspect, the electroporation system of this disclosure includes an electroporation assembly electrically coupled to the electroporation chamber of the electroporation cartridge. In some embodiments, the electroporation assembly includes a conductivity sensor for measuring the 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.

[0050] In one aspect, the controller is configured to implement a method for determining the conductivity of a sub-volume within the electroporation chamber of the present disclosure, and accordingly charge a capacitor to increase the repeatability and / or accuracy of electroporation pulses transmitted through a sample volume (e.g., in a single-use electroporation cartridge of the present disclosure) or through a continuous sub-volume (e.g., in a flow-through electroporation cartridge of the present disclosure).

[0051] In one aspect, the controller is configured to implement a method for predicting the risk of arc discharge during electroporation by determining a predicted temperature increase of a sub-volume within the electroporation chamber of the present disclosure. In some embodiments, the controller may determine the conductivity of the sub-volume within the electroporation chamber using the conductivity sensor. In some embodiments, the controller may then (or subsequently) determine a predicted temperature increase of the sub-volume based on the determined conductivity, an expected pulse voltage, and an expected pulse duration. The controller may then send an arc risk alarm if the predicted temperature increase results in a temperature of the sub-volume exceeding a predetermined threshold temperature. In some embodiments, the controller may additionally cause the electroporation system to evacuate the sub-volume from the electroporation chamber to retain the sample.

[0052] In one aspect, the controller is configured to implement a method for predicting the risk of arc discharge during electroporation by determining the presence of bubbles in the electroporation chamber of this disclosure. In this aspect, the controller can determine the conductivity of the sub-volume within the electroporation chamber using the conductivity sensor. Then, if the determined conductivity falls below a predetermined threshold indicating the presence of one or more bubbles in the electroporation chamber, the controller can send an arc risk alarm. The controller can additionally cause the system to evacuate the sub-volume from the electroporation chamber to retain the sample.

[0053] In one aspect, the controller is configured to implement a method for determining a calibration step volume to be moved by the system between each electroporation event, corresponding to the filling volume of the electroporation chamber. In this aspect, the controller can determine the number N of rotations of the drive pump required to fill the pipe deployed between the flow sensor and the electroporation chamber, and the sample volume required to completely fill the electroporation chamber. The number N thus represents the volume between the flow sensor and the outlet of the electroporation chamber. The determination that the electroporation chamber has been filled can be accomplished using the conductivity sensor.

[0054] The controller can then empty the electroporation chamber and then cause the drive pump to return the sample to a point upstream of the flow sensor by a fixed number of rotations k, where k represents the volume between the point upstream of the flow sensor and the inlet of the electroporation chamber. The controller can then determine the number x of rotations x of the drive pump required to move the sample from the point upstream of the flow sensor to the flow sensor, where x represents the volume between the point upstream of the flow sensor and the flow sensor. Therefore, the quantity (kx) represents the volume between the flow sensor and the inlet of the electroporation chamber, and the quantity N-(kx) represents the filling volume of the electroporation chamber.

[0055] In another aspect, the controller is configured to implement a method for determining a calibration step volume to be moved by the system into the flow-through electroporation chamber between each electroporation event, corresponding to the filling volume of the flow-through electroporation chamber. In one embodiment, such a method includes a first filling of the flow-through electroporation chamber until the sample contacts (touches) the top electrode of the electroporation chamber (as described in some embodiments of the first electrode). At this time (i.e., during the first filling), the electroporation system monitors the resistance in the electroporation chamber as decreasing from several thousand ohms to a stable value in the range of approximately 600 to 800 ohms. The first filling is stopped when this stable resistance value is reached.

[0056] For the second fill (and subsequent fills), the coarse fill volume is obtained from a combination of empirical data and theoretical calculations. Accordingly, for the second fill (and subsequent fills), the electroporation system (e.g., a controller within it) determines the number of rotations “N”. rev "," that is, the number of pump rotations required to initially move enough sample volume to completely fill the electroporation chamber (i.e., the number of rotations is counted from the time the sample fluid enters the electroporation chamber from the fixed entry point to the time the sample fluid contacts the top electrode—reaching its sample volume). Additionally, the internal pipe diameter "d" of the pump tubing was determined. i The number of rollers "n" in the pump is determined empirically. In a non-limiting example, the pump may be a peristaltic pump, which in some embodiments may have six rollers (e.g., n=6).

[0057] Determine N rev The value includes (e.g., empirically or otherwise) measuring one or more of the following items, and then combining them with theoretical calculations to obtain N. rev The one or more of said components include: the number of revolutions required for the peristaltic pump to fill the electroporation chamber from the inlet to the point where the electroporation chamber reaches the top electrode (corresponding to a decrease in the resistance of the sample fluid to 600 to 800 ohms); and the inner diameter "d" of the tubing in the pump. i "; number of pump rollers "n"; fluid volume / pump complete rotation ("a" µL); fluid volume / one roller action ("b" µL); minimum diameter of the area in the electroporation chamber where the electroporated sample resides; and / or (obtained using a voltmeter, conductivity sensor, etc.) the resistance or conductivity of the fluid in the electroporation chamber.

[0058] The following steps follow this operation: After capping the top electrode following the second filling (and subsequent fillings), the resistance is measured; if the resistance is within a stable range determined from the first filling (i.e., within the range of approximately 600-800 ohms), the filling is complete, and electroporation of the sample can continue. However, if, after the second filling, the resistance is not within the stable range determined from the first filling (i.e., not within the range of approximately 600-800 ohms), the following steps are performed: the top electrode is uncapped, and fluid is introduced to add n rev Quantity (n of the pump) rev The electroporation chamber is then finely filled. This operation is followed by the following step: after capping the top electrode following the fine filling, the resistance is measured. If the resistance is within a stable range determined from the first filling (i.e., from approximately 600-800 ohms), the second filling is complete, and electroporation of the sample can continue. If not, the second filling is complete, and electroporation of the sample can continue. If not, the fine filling is repeated, and the resistance is stepped upwards until the resistance is within the stable range determined from the first filling (i.e., from approximately 600-800 ohms). For N rev + n rev x (the number of attempts at fine filling) of pump rotations complete subsequent fillings (third filling, fourth filling, etc.). In some embodiments, the stable value of the resistance is approximately 700 ohms (e.g., in the range of 650-750 ohms and any values ​​in between).

[0059] In one embodiment, N rev The following calculations were performed: In one embodiment of the system disclosed herein, the system includes a pump with six rollers and a conduit with an inner diameter of 2.4 mm. 172 µL of fluid was allocated for each complete rotation of the pump, based on empirical data. According to empirically determined data, 28 µL of fluid was allocated for each rotation through one roller distance (60 degrees in this case). Theoretical calculations were then performed to determine the fluid volume that the electroporation chamber could contain. In the case of a nominal diameter of 6.4 mm (2r, where r = radius of the electroporation chamber), with a lower limit of 6.3 mm and an upper limit of 6.5 mm, and a height (h) of 30 mm for the electroporation chamber, the formula πr was used. 2h was determined to be a nominal sample volume of 965 µL, with a lower limit of 935 µL and an upper limit of 995 µL. The height (h) difference, with a design tolerance of 0.2 mm, was considered insignificant due to the maximum 7 µL difference. To reduce sample loss from coarse packing due to the benefits of the electroporation chamber, a lower limit of 6.3 mm for the chamber diameter was used in the calculation. Based on the calculated volume of 935 µL divided by the empirical data of 172 µL, the peristaltic pump rotation number was determined to be 5.4; however, to introduce a convex meniscus, a rotation number of 5.5 is recommended. Therefore, in this case, "N rev "Equals 5.5 rotations. The difference in chamber diameter from 6.3 mm to 6.5 mm results in a sample volume difference of approximately 30 µL for every 0.1 mm diameter change. This is very close to the empirical data of 28 µL for the fluid, which is allocated to each rotational motion by a roller distance of the pump (60 degrees in this case), referred to in the formula as fine filling ("n")." rev Based on 5.5 rotations (N) rev During the initial coarse filling, the instrument will read the conductivity, and if the filling is incomplete, fine filling will begin. rev The second fill (and the random fill) will be determined by the number of attempts at fine fill, "x". rev + n rev It is composed of "x".

[0060] Accordingly, systems, methods, and apparatus for automated electroporation of cellular fluids are disclosed.

[0061] This summary is provided to introduce, in a simplified form, the selection of concepts further described below in the detailed description. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to serve as an indication of the scope of the claimed subject matter.

[0062] Additional features and advantages of this disclosure will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practice of the disclosure. The features and advantages of this disclosure can be achieved and obtained by the tools and combinations particularly pointed out in the appended claims. These and other features of this disclosure will become more fully apparent from the following description and the appended claims, or may be learned by practice of the disclosure as set forth below. Attached Figure Description

[0063] To describe in detail the ways in which the above and other advantages and features of this disclosure can be obtained, the present disclosure, which has been briefly described above, will be described in more detail with reference to specific embodiments shown in the accompanying drawings. It should be understood that these drawings depict only typical embodiments of the present disclosure and should therefore not be considered as limiting its scope. The present disclosure will be described and explained with additional details and nuances using the accompanying drawings, in which: Figure 1A This is an exploded view of an exemplary single-use electroporation cartridge according to some embodiments of the present disclosure; Figure 1B This is a bottom perspective view of an exemplary single-use electroporation cartridge of FIG1, showing a partially assembled and unsealed position according to an embodiment of the present disclosure; Figure 1C yes Figure 1B A front view of a longitudinal section of a partially assembled, unsealed electroporation box cylinder; Figure 1D It shows the assembled assembly and in the capped position. Figure 1A A front view of an exemplary single-use electroporation cartridge; Figure 1E yes Figure 1D A front view of the longitudinal section of the assembled and sealed electroporation box cylinder; Figure 2A This is an exploded front view of an exemplary single-use electroporation cartridge with a volume-reduced sleeve according to some embodiments of the present disclosure; Figure 2B yes Figure 2A Exploded top perspective view of an exemplary single-use electroporation cartridge and volume-reducing sleeve; Figure 2C It is shown as a partially assembled electroporation cartridge in its unsealed position. Figure 2A A front view of an exemplary single-use electroporation cartridge, wherein a volume-reduced sleeve is associated with a removable cap; Figure 2D yes Figure 2C A front view of the longitudinal section of a partially assembled and unsealed electroporation box and a volume-reduced sleeve; Figure 2E It shows the assembled and sealed assembly. Figure 2A A front view of an exemplary single-use electroporation cartridge; Figure 2F yes Figure 2E A front view of the longitudinal section of the assembled and capped electroporation cartridge, showing a volume-reducing sleeve of the electroporation chamber deployed in the chamber defined by the elongated body of the cartridge to form a reduced volume between opposing electrodes. Figure 3This is a front view of a longitudinal section of another single-use electroporation cartridge according to some embodiments of the present disclosure; Figure 4 This is a perspective view of another exemplary single-use electroporation cartridge with an electrode cap according to some embodiments of the present disclosure; Figure 5 This is a cross-sectional view of another exemplary single-use electroporation cartridge having an authentication chip associated with an electrode cap, according to some embodiments of this disclosure; Figure 6A This is a perspective view of another exemplary single-use electroporation cartridge having an authentication chip associated with an electrode cap and a gripping member associated with a chamber body, according to some embodiments of this disclosure. Figure 6B yes Figure 6A A top view of a single-use electroporation cartridge; Figure 6C yes Figure 6A A front view of a single-use electroporation cartridge; Figure 6D Yes (along) Figure 6C (As shown, plane 6D was obtained) Figure 6C A cross-sectional view of a single-use electroporation cartridge; Figure 7A An exploded front view of an exemplary flow-through electroporation cartridge according to some embodiments of the present disclosure is shown, wherein the cartridge body is shown as transparent for ease of viewing and description herein; Figure 7B yes Figure 7A Top perspective view of the decomposed flow-through electroporation cartridge; Figure 7C It shows the assembly. Figure 7A A front view of an exemplary flow-through electroporation cartridge, wherein the cartridge body is surface-masked rather than transparent; Figure 7D yes Figure 7C A front view of the longitudinal section of the assembled flow-through electroporation chamber, the section dividing the port associated with the first electrode, as well as the chamber inlet and outlet, into two parts; Figure 8A This is a top perspective view of another embodiment of a flow-through electroporation cartridge, wherein the flow-through electroporation cartridge shown includes a container integrated with a chamber body and is shown as partially assembled, wherein the top sleeve cap is disassembled away from the chamber body; Figure 8B It is in a partially assembled state. Figure 8A Top perspective view of a flow-through electroporation chamber, with the top sleeve cap associated with the proximal opening of the chamber body; Figure 8C Yes (along) Figure 8B(As shown, plane 8C was obtained) Figure 8B The longitudinal section of the flow-through electroporation cartridge shown in the figure has a top electrode associated with it. The arrows shown in the figure indicate that the overflow fluid is discharged from the electroporation chamber in response to being collected in the associated container. Figure 9 This is another embodiment of a flow-through electroporation cartridge according to some embodiments of the present disclosure; Figure 10A An embodiment of the electroporation process using the exemplary electroporation system of this disclosure is illustrated schematically; Figure 10B An embodiment of a control system is shown that is configured to communicatively couple to one or more components of the electroporation system of this disclosure and is configured to provide one or more computer-implemented process control methods to the system; Figures 11A-11D Example embodiments of an electroporation system or electroporation instrument are shown, wherein, Figure 11A An embodiment of an electroporation instrument is shown; Figure 11B Another embodiment of an electroporation instrument with an outer cover in the open position is shown; Figure 11C One embodiment of an electroporation system is depicted, the electroporation system comprising: an electroporation instrument; and a removably attachable modular housing that may include various electroporation components containing cells and reagents; Figure 11D Another embodiment of an electroporation instrument with an outer cover is shown; Figure 12A An example embodiment of a modular housing for arranging various electroporation components that can be selectively attached and detached for an electroporation instrument is shown. Figures 12B-12E Another example embodiment of a modular housing for arranging various electroporation components that can be selectively attached and detached for an electroporation instrument is shown; Figure 12F and Figure 12G Show Figures 12B-12E Different views of the empty modular housing are shown, and attachment features that enable the housing to be removably attached to the electroporation instrument are illustrated. Figure 13A Another embodiment is shown for arranging a modular housing for selectively attaching and detaching various electroporation components for an electroporation system / instrument. Figure 13B Show Figure 13A Rear view of the modular housing; Figure 13C An embodiment is shown that can be attached to a modular housing of this disclosure to hold solutions / payloads / reagents for manual dispensing into an additional electroporation assembly available in the electroporation cell mixing chamber as needed and when required; Figure 14A and Figure 14B An example of a compartment that can be incorporated into the electroporation system / instrumentation described herein is shown; Figures 15A-15C An example of a sample transfer assembly configured to provide efficient transfer of sample fluid from an input reservoir to a mixer container and to an electroporation chamber, according to an embodiment of the present disclosure, is shown. Figures 15D-15E This illustration shows an example of a sample transfer assembly according to an embodiment of the present disclosure, illustrating the flow path of sample fluid through various tubes of an electroporation system from an input reservoir to an input tube to a cell mixer container and to an electroporation chamber. Figures 15F-15G This illustration shows an example of a sample transfer assembly according to an embodiment of the present disclosure, illustrating the air flow path during the flow of sample fluid from the inlet tube to the electroporation chamber. Figure 15H An example of an air flow control assembly including an air filter, tubing, and valve stem, comprising an electroporation system / housing module, is shown according to an embodiment of the present disclosure. Figure 15I This illustration shows the steps for disassembling an electroporation housing module from an electroporation instrument according to an embodiment of the present disclosure; Figure 16A and Figure 16B An unfolded view of an exemplary mixer container, according to some embodiments, is shown to hold the transferred sample and maintain it in a homogeneous suspension while a continuous sub-volume of the sample is transferred to an electroporation cartridge for electroporation. Figure 17A , Figure 17B and Figure 17C An example embodiment of a pre-cooling module is shown, deployed upstream of the electroporation chamber and configured to cool the sample being transferred before electroporation. Figure 17D An example of a cooling module configured to cool the electroporation chamber itself is shown; Figure 18A and Figure 18B An example of a high-voltage contact pin of the electroporation instrument of this disclosure detached from an example electroporation cartridge is shown, and Figure 18C and Figure 18D An example of a high-voltage contact pin of the electroporation instrument of this disclosure, which is engaged with an electroporation chamber, is shown. Figures 19A-19C An embodiment is shown that is configured to enable the electroporated cartridge attachment feature to be attached to a suitable location in the cooling module; Figures 20A-20CThe operation of a capping mechanism according to some embodiments for moving an electroporation cartridge between a capped state ready for electroporation and an uncapped state ready for filling or emptying the corresponding electroporation chamber is illustrated. Figure 21 The diagram illustrates the operation of a capping mechanism according to some embodiments and its combination with the capping of an electroporation cartridge, demonstrating the function of a spring-based mechanism that allows overtravel of the capping mechanism to ensure that the electrode is fully moved to the desired position despite different cartridge dimensional tolerances. Figures 22A-22E Examples of sealing mechanisms and associated components of the electroporation cartridge according to some embodiments of the present disclosure are shown in more detail; Figure 22F and Figure 22G Exemplary flow-through electroporation cartridges according to some embodiments are shown, including those associated with inlet and outlet ports and in various open and closed configurations, such as... Figure 22E The sealing mechanism described in the document; Figure 22H and 22I An exemplary flow-through electroporation cartridge is shown according to one embodiment, which includes an outlet port and is in an open and closed configuration, such as... Figure 22E The sealing mechanism described in the document; Figure 22J and Figure 22K An umbrella valve is shown in open and closed configurations according to an embodiment of the present disclosure for use as a sealing mechanism; Figure 22L Show Figure 22J and Figure 22K A top view of the entrance, where the corresponding umbrella valve is removed from the view; Figures 22M-22S An example setup is shown for the outlet port of a one-way valve (e.g., a miniature valve) according to one embodiment for pressurizing and sealing an exemplary electroporation cartridge. Figure 23 A method is shown, according to one embodiment, for predicting the risk of arc discharge during electroporation operation based on a predicted temperature change in sample subvolume. Figure 24 A method for preventing arc discharge in an electroporation chamber based on an initial conductivity measurement of a sub-volume, according to one embodiment, is shown. Figure 25 A schematic diagram of an electroporation circuit according to one embodiment is shown; Figure 26 A method for generating repeatable and consistent electrical pulses through an electroporation chamber is illustrated according to one embodiment; Figures 27A to 27D A method for calibrating the filling volume of an electroporation chamber is shown according to one embodiment; Figure 27E A method for calibrating the filling volume of an electroporation chamber is shown according to one embodiment; Figure 28 An exemplary method flow is shown according to one or more embodiments of the present disclosure for preparing cells for conversion via one or more electroporation systems disclosed herein; Figure 29A This illustration shows one or more embodiments of the present disclosure for use, for example, by... Figure 28 An exemplary method flow for the batch processing and transformation of cells prepared by the exemplary method outlined herein; Figure 29B It shows the basis Figure 28 and Figure 29A Exemplary graphs of viability and transformation efficiency of primary cells prepared according to the protocols outlined herein; Figure 30A This illustration shows one or more embodiments of the present disclosure for use, for example, by... Figure 28 Exemplary methods for the flow-through processing and transformation of cells prepared by the exemplary methods outlined herein; Figure 30B It shows the basis Figure 28 and Figure 30A Exemplary graphs of viability and transformation efficiency of primary cells prepared according to the protocols outlined herein; Figure 31 It is a graph showing the results of batch processing and flow-through processing of primary cells and electroporation according to one or more embodiments of the present disclosure; Figure 32 This is a graph illustrating the viability and conversion efficiency of an exemplary flow-through system and method for converting immortalized cell cultured cells via electroporation according to one or more embodiments of the present disclosure; Figure 33 This is a series of graphs providing an exemplary comparison of the transformation efficiency and cell viability of immortalized cell cultures electroporated using the systems and methods disclosed herein against prior art electroporation systems; Figure 34 This is a graph illustrating the viability and transformation efficiency of cells transformed by knock-in genes using an exemplary single-use consumable electroporation system and method according to one or more embodiments of this disclosure; and Figure 35 This is a graph illustrating transformation efficiency and cell viability during the generation of CAR-T cells using the flow-through and single-use cartridges and electroporation systems and methods disclosed herein, according to one or more embodiments of this disclosure. Detailed Implementation

[0064] Before describing the various embodiments of this disclosure in detail, it should be understood that this disclosure is not limited to the parameters of the specifically illustrated systems, methods, apparatuses, products, processes, and / or kits, which may of course vary. Therefore, while specific embodiments of this disclosure will be described in detail with reference to specific configurations, parameters, components, elements, etc., the description is illustrative and should not be construed as limiting the scope of the claimed invention. Furthermore, the terminology used herein is for the purpose of describing embodiments and is not necessarily intended to limit the scope of the claimed invention.

[0065] Furthermore, it should be understood that, for any given component or embodiment described herein, any possible candidates or alternatives listed for that component may generally be used individually or in combination with each other, unless otherwise implicitly or explicitly understood or stated. Moreover, it should be understood that any list of such candidates or alternatives is illustrative only and not restrictive, unless otherwise implicitly or explicitly understood or stated.

[0066] Furthermore, unless otherwise indicated, the numbers of expressions, components, distances, or other measures used in the specification and claims should be understood as being modified by the term "approximately," as defined herein. Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and appended claims are approximate values, which may vary depending on the desired properties sought to be obtained from the subject matter presented herein. At least, and not as an attempt to limit the scope of the claims, each numerical parameter should be understood at least according to the number of significant figures reported and by applying common rounding techniques. While the numerical ranges and parameters that set forth the broad scope of the subject matter presented herein are approximate, the values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains a specific error that necessarily arises from the standard deviation found in their respective test measurements.

[0067] Any headings and subheadings used herein are for organizational purposes only and are not intended to limit the scope of the specification or claims.

[0068] Overview and advantages of exemplary electroporation cartridges

[0069] This disclosure relates to electroporation systems and various components thereof, including several types of electroporation chambers, electroporation devices, electroporation boxes, electroporation modules, module housings, electroporation instruments, and electroporation systems, including systems comprising a computer processor, a user interface, and computer-implemented methods.

[0070] This disclosure relates to electroporation systems and related instruments and equipment (e.g., electroporation cartridges and / or electroporation chambers) useful for single electroporation experiments. This disclosure also relates to electroporation systems and instruments for non-automated electroporation.

[0071] This disclosure relates to high-throughput electroporation systems and related instruments and equipment (e.g., electroporation cartridges, and / or chambers and / or electroporation housings / modules). This disclosure also relates to electroporation systems and instruments for automated high-throughput electroporation.

[0072] As described above, there are many drawbacks and problems that can be addressed in the field of electroporation. Embodiments of this disclosure address many of the drawbacks and problems in the field of automated, high-throughput electroporation and in single electroporation applications. For example, embodiments of this disclosure provide devices, instruments, systems, and methods capable of performing electroporation in a functionally closed, aseptic pathway, offering one or more advantages, including: minimizing or substantially reducing arc discharge; minimizing or substantially reducing bubble formation and associated arc discharge in cell-containing fluids; significantly improving and maximizing transfection efficiency and recovery efficiency of transfected cells; providing a uniform electric field for efficient electroporation, etc.

[0073] In some embodiments, the systems, apparatus, instruments, and methods of this disclosure combine the advantages of continuous flow electroporation with batch or static volumetric electroporation, particularly for the ability to automatically electroporate large volumes of cells in a sterile, closed system while minimizing arc discharge caused by bubble formation in cell-containing fluids and maximizing recovery efficiency.

[0074] This disclosure addresses one or more of the aforementioned problems in the field of automated electroporation. For example, Figures 1A-6D , Figure 10A and 10B , Figures 11A-11D The corresponding descriptions illustrate various electroporation devices (e.g., single-use electroporation cartridges, and electroporation instruments and systems capable of achieving high-efficiency electroporation). In other examples, Figures 7A-9 , Figures 10A-22S The embodiments described herein illustrate various electroporation devices (e.g., flow-through electroporation cartridges, electroporation modules, electroporation instruments and systems enabling highly efficient electroporation of large volumes of cell-containing fluids using automated batch processing). Figures 23-33 The corresponding description provides exemplary methods for utilizing one or more of the electroporation cartridges.

[0075] Advantageously, the disclosed systems, apparatus (e.g., single-use and flow-through electroporation cartridges) and methods reduce bubble formation in a variety of ways, including, for example, pressurizing the electroporation chamber prior to electroporation, thus making it more energy-dense (and therefore more difficult or less likely to) vaporize aqueous cellular fluids or cause water molecules to electrolyze into oxygen and hydrogen. Furthermore, the single-use and flow-through electroporation cartridges of this disclosure additionally direct any bubbles formed during processing toward the distal electrode surface, thereby further reducing the likelihood of arcing. With fewer bubbles forming or accumulating on or near the electrode surface, embodiments of this disclosure allow for less arcing and maximize recovery efficiency per sample.

[0076] The disclosed flow-through devices, modules, and systems further offer the benefit of automating the manual process of loading / unloading samples into and out of electroporation cuvettes—a process many have attempted but failed at. Instead of the continuous processing already performed in existing systems and tending to reduce electroporation efficiency, the systems, cartridges, and methods disclosed herein enable automated batch electroporation, combining the advantages of continuous flow electroporation with those of batch or static volumetric electroporation, while minimizing the inherent disadvantages of each type. For example, the devices and systems disclosed herein provide sterile, closed systems that eliminate (or significantly reduce) the risk of sample contamination previously associated with batch or static processing techniques. Preferably, the devices and systems disclosed herein are made of materials conforming to ISO (International Organization for Standardization) guidelines and suitable for use in cell and gene therapy applications.

[0077] Exemplary electroporation box

[0078] As stated above, there are many drawbacks with electroporation systems known in the art. Specifically, electric arcing during electroporation is undesirable because it affects transfection efficiency and cell viability. Systems and devices are needed to minimize or prevent electric arcing in electroporation cartridges and chambers manufactured for single use. Systems and devices are also needed to minimize or prevent electric arcing during continuous flow or continuous batch electroporation processes.

[0079] One of the main causes of electric arcs is the presence of air bubbles at or near the electrode surface and / or within the path of the current generated by the application of high voltage through the cellular medium during electroporation. Under high voltage application (e.g., electroporation), any air bubble of nearly significant size will cause an arc. Air bubbles are known to form as a result of heat generated by the discharge through the aqueous cellular solution. This intense, frequently repeated heating can also cause bubble formation through the localized vaporization of water molecules. The discharge through the cellular solution that occurs during electroporation can cause the electrolysis of water molecules, forming oxygen and hydrogen—another source of air bubbles during electroporation.

[0080] This disclosure includes embodiments of electroporation cartridges, associated systems, and methods for using them, all capable of reducing bubble formation during electroporation, even in large volumes (e.g., 1 mL or more) that conventionally cause arcing. Figures 1A-1E An exemplary single-use electroporation cartridge is shown, wherein, Figure 1A An exploded view of the components of an exemplary electroporation cartridge is provided. Figure 1B and Figure 1C Showing partially assembled and in the unsealed position Figure 1A The electroporation box tube, and Figure 1D and Figure 1E The diagram shows a surface shading and cross-sectional view of the assembled cartridge in the capped position. The single-use electroporation cartridges shown, along with other single-use electroporation cartridges described herein, may include one or more safety features to ensure they are used only once. For example, single-use electroporation cartridges may include locking features, disconnect features, and / or other mechanical features that prevent the electroporation cartridge from being reused after it has been inserted into and removed from the electroporation system. Single-use of the single-use electroporation cartridge may be additionally or alternatively ensured electronically (e.g., by an electronic tag or code associated with each single-use electroporation cartridge and scanned / recorded by the electroporation system).

[0081] According to one embodiment, such as Figures 1A-1E As shown, the 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 a 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 between a capped position for electroporation and an uncapped position for loading a sample (e.g., as shown). Figure 1B , Figure 1C , Figure 1D and Figure 1E (As shown in the image). Specifically, as shown in the image. Figure 1AAs shown, the elongated body has two open ends—a proximal end 110 defining a proximal opening 112 and a distal end 114 defining a distal opening 116—where a first electrode 106 and a second electrode 108 are respectively mounted. According to one embodiment, the first electrode 106 is associated with a removable cap 107 that allows selective association of the first electrode 106 with the elongated body 104. In some embodiments, the second electrode 108 of the cartridge 100 is inserted into the distal opening 116 of the elongated body 104, which is secured in place by the distal cap 118. In some embodiments, the second electrode 108 is secured to and / or associated with the distal end 114 of the elongated body 104 by the distal cap 118 via a locking feature to prevent the lower cap from being removed. This allows for advantageous operation to reduce potential confusion regarding which end the user will use for adding and / or removing cellular fluid for electroporation, and also provides the benefit of a modular construction that can be partially assembled to form a structurally more robust cartridge.

[0082] like Figure 1C As shown, the second electrode 108 additionally includes a first sealing member 120 disposed between the second electrode 108 and the distal surface 122 of the elongated body 104. The first sealing member is operable to form a fluid-tight connection between the second electrode 108 and the distal surface 122 of the elongated body 104. In some instances, a fluid-tight connection is provided by compressing a sealing member (e.g., an O-ring or other gasket) between the electrode flange 124 and the distal body flange 126 oriented in a plane substantially parallel to the electrode flange 124. In some embodiments, a distal cap 118 locks into the association between the elongated body and the sealing member 120 disposed between the electrode flange 124 and the distal body flange 126, forming a fluid-tight connection therebetween.

[0083] Continue to refer to Figure 1CIn some embodiments, the second electrode 108 may include a protrusion 128 extending from the distal end 114 of the elongated body 104 into the electroporation chamber 102 to define the bottom of the electroporation chamber 102. In some embodiments, the second electrode 108 may additionally include a second sealing member 130 deployed around the protrusion 128 and positioned distal from the proximal surface of the second electrode, wherein it forms a fluid-tight connection with the inner wall of the electroporation chamber 102. The first sealing member 120 forms a fluid-tight seal between the second electrode 108 and the elongated body 104 via compression of the distal cap 118 to seal the distal end of the electroporation chamber 102 from the external environment, and the second sealing member 130 forms a fluid-tight connection between the protrusion 128 of the second electrode 108 and the inner wall of the electroporation chamber 102 to minimize dead zone volume by creating a seal closer to the wetted proximal surface of the second electrode 108.

[0084] In some embodiments (e.g., such as) Figure 1C In the illustrated embodiment, the perimeter of the protruding portion 128 may have a shape complementary to the contour of the inner surface of the elongated body 104 defining 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 may be a flat, uniform surface positioned orthogonal to the longitudinal axis of the electroporation chamber.

[0085] Specific benefits can be obtained by including one or more of the aforementioned structural features in the shape and / or position of the second electrode relative to the electroporation chamber. For example, the generation of a uniform electric field is a factor for successful and efficient electroporation. For a uniform electric field, it is advantageous for the opposing first and second electrodes to be substantially parallel and to have a cross-sectional geometry substantially the same as that of the electroporation chamber. A uniform electric field is most effectively generated within an electroporation chamber having a uniform cross-section (e.g., a constant diameter). Accordingly, in some embodiments, the electroporation cartridge disclosed herein may include an electroporation chamber having a uniform cross-section along the length of the reaction chamber. The uniform cross-section may 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 deployed between the first and second electrodes. As a non-limiting example of the foregoing, the electroporation chamber may 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.

[0086] Alternatively, the uniform cross-section may extend along a length less than the entire length of the electroporation chamber. In these embodiments, at least a portion of the electroporation chamber may taper between the first and second electrodes. Preferably, the taper portion of the electroporation chamber does not substantially interfere with the generation of a uniform electric field between the first and second electrodes. For the purposes of this disclosure, the taper portion of the electroporation chamber does not substantially interfere with the generation of a uniform electric field if the electric field generated between the opposing first and second electrodes is defined by substantially parallel and equally spaced field lines within a 10% tolerance. For clarity, the taper portion within the electroporation chamber may include a narrowing of the proximal sidewall defined between a proximal opening of the elongated body and an inflection point on the sidewall defining the electroporation chamber, such that the proximal sidewall narrows from a first diameter defined by the proximal opening to a smaller second diameter defined at a position distal to the inflection point.

[0087] It should be understood that the presence of a tapered section 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 elongated body and / or electroporation chamber comprises or is made of non-conductive radiation-stabilized plastic, ceramic, and / or glass. For example, the elongated body and / or electroporation chamber may be made of polycarbonate or another non-conductive gamma-stabilized plastic. Alternatively, both glass and ceramic are more electrically insulating and more thermally conductive than polycarbonate; advantageously, both materials can be mass-produced to have zero-draft sidewalls and provide a constant cross-section.

[0088] In some embodiments, the chamber is made of a material that can be sterilized by one or more of the following methods: steam sterilization, flash sterilization, hydrogen peroxide sterilization, vaporized hydrogen peroxide sterilization, gamma ray sterilization, peracetic acid sterilization, ethylene oxide sterilization, chlorine dioxide gas sterilization, electron beam sterilization, etc., without compromising the function of the chamber (e.g., without reducing electroporation efficiency or affecting cell viability).

[0089] As previously described, a uniform electric field can be generated between two opposing electrodes having a cross-section with a shape and size close to that of the uniform cross-section of the electroporation chamber. However, the inventors have found that when various cartridges with these characteristics and geometries are fabricated, air tends to be more easily trapped when the chamber is sealed. The trapped air can cause a failure in the conductivity within the electroporation chamber and lead to arcing, which negatively affects the electroporation process and performance.

[0090] To overcome the air trapping problem, the electroporation cartridge disclosed herein utilizes the surface tension at the liquid-air interface, where the attraction between liquid molecules is stronger than the attraction between liquid molecules and air molecules. This results in the formation of a convex meniscus, and when the top electrode is covered, the liquid shifts 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 may have a bell-shaped or spherical protrusion 132, which is separated from the substrate region 134 by a narrow rod 136, as shown below. Figures 1A-1E As shown. The spherical protrusion 132 may have a smaller diameter than the cross-section of the electroporation chamber 102, so as to form a gap between them. The gap between the spherical protrusion and the chamber body allows small air bubbles to exit the electroporation volume. The narrow rod 136 allows for a larger air volume in the proximal portion of the chamber, which can act as a compressible volume during electroporation to minimize the pressure accumulation in the chamber caused by partial vaporization of the sample.

[0091] The bell-shaped or spherical shape of the distal end of the first electrode can provide additional advantages during electroporation. Bubbles can form during electroporation due to the electrolysis and / or vaporization of water. The spherical extension can be operable to displace one or more bubbles generated during electroporation such that they are removed from the electrode surface before coalescing into bubbles of sufficient size to induce an arc. For example, the spherical extension can have an arcuate or convex surface that facilitates any bubbles rising from the electroporation volume along the surface of the spherical extension and rising to the sample-air interface near the rod.

[0092] Furthermore, it was advantageously found that at higher pressures, the intensity of vaporization can be reduced, resulting in smaller and / or fewer bubbles forming during electroporation. By sealing the electroporation chamber prior to electroporation, the energy release and electrolysis that occur during electroporation will cause the electroporation chamber to behave like a pressure chamber. Any increase in pressure within the chamber will delay the formation of bubbles of a significant size that could lead to an electric arc.

[0093] Accordingly, in some embodiments (e.g., Figures 1A-1E In the illustrated embodiment, the first electrode 106 is associated with a sealing member 138 operable to form a fluid-tight connection between the first electrode and the elongated body 104. In some embodiments, a removable cap 107 associated with the first electrode 106 may provide axial compression for maintaining a fluid-tight seal. In some embodiments, the removable cap 107 is threaded to the elongated body 104, but it should be understood that other forms of connection (e.g., friction fit, snap-fit, etc.) are contemplated herein. Figures 1A-1EAs shown, each of the covers 107 and 118 represents a flange extending beyond the outer surface of the electrode. For example, when placed on a flat surface, these flanges can provide additional balance and stability to the device. In the absence of the flanges shown, the narrow footprint of the housing and the centerline of its center of gravity would likely make the housing unstable.

[0094] Because the first electrode 106 is removed by the user during normal operation, there is a tendency for the sealing member to be lost or detached from the electrode when decoupled from the elongated body. To prevent this, the electrode may include a retaining feature 140 of the access rod 136, configured to allow the sealing member 138 to extend over the retaining feature 140, but then be retained, thereby adjacent to the sealing surface of the first electrode 106. This sealing surface forms a functionally closed system at the upper end of the chamber when the cap 107 is associated with the upper end of the chamber.

[0095] In some embodiments, the first electrode 106 may additionally include a cap retaining feature 142 to prevent the end cap from separating from the electrode once installed. The cap retaining feature 142 may act as a barb for snap-fit ​​engagement, and may be threaded to the cap, or thereby retained by any other means known in the art. Clearly, in some embodiments, it may be advantageous to secure the first electrode to the cap in a manner that allows the cap to rotate independently of the first electrode, thereby causing the associated sealing member to receive only axial compression.

[0096] In some embodiments, electrodes 106, 108 are made of, or plated with, a conductive material that will not negatively affect cells by passively or during electroporation introducing harmful or toxic elements. For example, plating the electrodes in pure gold can provide the electrodes with beneficial conductive properties that prevent the introduction of harmful or toxic elements into the electroporation medium. Furthermore, it should be understood that, depending on the electroporation protocol, electrodes 106, 108 may be connected to a high-voltage circuit and may act as an anode or cathode, or alternate between the two. Alternatively, other non-toxic and / or non-reactive metals or materials, as known in the art, may be used.

[0097] In some embodiments, the volume within the electroporation chamber of the present disclosure is larger when compared to prior art electroporation cuvettes. In some embodiments, the electroporation chamber has an internal volume from about 10 mL to about 1 mL. In some embodiments, the electroporation chamber has an internal volume from about 1 mL to about 100 μl.

[0098] In some embodiments, the exemplary electroporation chamber of this disclosure may have a volume of less than about 5 mL, preferably less than about 3 mL, or in some embodiments a volume of less than about 4 mL, less than about 2 mL, or less than about 1 mL. In some embodiments, where the internal volume of the electroporation chamber is from about 10 mL to about 1 mL, the distance between the first electrode and the second electrode 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) within these volumes and within the preferred voltage range for electroporating a majority of cell samples.

[0099] In another exemplary embodiment, the volume of the electroporation chamber is approximately 1 mL or between approximately 100 μL and 1 mL. In some embodiments, within this volume and within a preferred voltage range for electroporating a majority of cell samples, the distance between the first and second electrodes is between approximately 20 mm and 40 mm (e.g., between approximately 22 mm and 38 mm, between approximately 25 mm and 35 mm, and preferably between approximately 30 mm). It is advantageous to configure the electroporation chamber to this size because a standard 1 mL pipette can easily enter the body of the 1 mL electroporation chamber and thus aspirate almost 100% of the electroporated sample, and it is unnecessary to go beyond the specialized equipment commonly used for moving these volumes of fluid in laboratory or clinical settings.

[0100] Volume reduction sleeve

[0101] In some instances, the sample volume is less than 1 mL. If the distance between the two electrodes remains constant, the diameter of the chamber must be drastically reduced to accommodate the smaller volume. The problem is that existing standard pipette tips cannot fill or empty such small-volume chambers.

[0102] This disclosure includes embodiments for the purposes described above. Figures 1A-1E The described adapter for the electroporation cartridge. In principle, the elongated body is fitted with a volume-reducing sleeve that forms a fluid-tight connection with the second (bottom) electrode and defines an internal secondary electroporation chamber with a smaller volume than that defined by the original elongated body 104. In this way, the above electroporation cartridge can be easily adapted to smaller volume samples while retaining the benefits and advantages of the original design.

[0103] Figures 2A-2F The illustration shows a sleeve with a reduced volume configuration. Figures 1A-1EExemplary embodiments of the electroporation cartridge are provided. As shown in these figures, the volume-reducing sleeve defines a secondary electroporation chamber of approximately 200 μL in volume. However, it should be understood that this volume is exemplary in nature, and other volumes are contemplated herein and can be fabricated by adjusting the cross-sectional perimeter of the volume-reducing sleeve. For example, a 1 mL elongated body can be used to support volumes from 100 μL to 1 mL using sleeves of different sizes. Specifically, smaller volume sleeves (e.g., 100 μL sleeves) can be fabricated by reducing the cross-sectional perimeter of the sleeve. Alternatively, larger volume sleeves (e.g., 200 μL, 250 μL, 400 μL, 450 μL, or 500 μL sleeves) can be fabricated by increasing the cross-sectional perimeter of the volume-reducing sleeve.

[0104] Now refer to 2A- Figure 2F , Figure 2A and Figure 2B The diagram shown is an exploded view of an exemplary single-use electroporation cartridge and a volume-reducing sleeve. Figure 2C and Figure 2D Showing the assembled and unsealed positions Figure 2A and Figure 2B The box and sleeve, Figure 2E and Figure 2F Showing the assembly and capping positions Figure 2A and Figure 2B The box and sleeve.

[0105] exist Figures 2A-2F In the embodiment shown, the sleeve assembly 200 replaces... Figures 1A-1E The standard upper electrode assembly 150 (including a removable cap 107, a first electrode 106, and a sealing member 138) of the 1 mL single-use cartridge shown and described herein. The lower electrode assembly 155 (including...) Figures 1A-1E The distal cap 118, the second electrode 108, and the sealing members 120, 130 remain unchanged. Essentially, the internal volume of the secondary electroporation chamber 202 defined by the sleeve 204 is 200 μL (or other defined volumes less than 1 mL as disclosed herein), and preferably mimics the geometry of a 1 mL chamber.

[0106] Reference Figure 2C and Figure 2D The lower electrode assembly 155 is shown coupled to the elongated body 104 to seal and isolate the distal portion of the elongated body from the environment, as described above. The sleeve assembly 200 is in... Figure 2C and Figure 2DThe sleeve electrode 206 is assembled, and obviously, the sleeve electrode 206 can be associated with the removable cap 207 in the same or similar manner as described above. Furthermore, 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 connection with the corresponding electrode and the elongated body flange, as described above.

[0107] In some embodiments, the sleeve 204 may additionally include a radial sealing member 210 that seals against the inner surface of the elongated body 104. The radial sealing member 210 forms a fluid-tight seal with the sidewall defining the electroporation chamber 102 to prevent cellular fluid from the secondary electroporation chamber 202 from leaking through the distal opening of the volume-reduced sleeve and entering the electroporation chamber 102. The distal end of the sleeve 204 may include a groove 216 for receiving and / or aligning the radial sealing member 210. In embodiments, the bottom surface of the sleeve is flat to minimize dead zone volume by occupying any area where fluid might be trapped.

[0108] During operation, the user places a sample of the desired volume into the elongated body 104, as they would normally use... Figures 1A-1E The cartridge 100 is then inserted, followed by placing the sleeve 204 into the elongation body 104. When inserted into the elongation body 104, the sleeve 204 displaces any additional fluid into the hollow center of the sleeve. The sleeve is designed such that a volume of 200 μL completely wets the lower surface of the sleeve electrode 206. The sleeve assembly 200 includes a vent 212 to allow air to escape from the secondary electroporation chamber 202 during insertion. When the sleeve 204 is removed after electroporation, the vent 212 additionally allows the electroporated sample to remain inside the elongation body 104. The vacuum created in the chamber during sleeve 204 removal pulls the electroporated sample out of the secondary electroporation chamber 202 and into the elongation body 104. This design allows users to aspirate small volumes (e.g., 200 μL) and dispense them into the elongation body 104 using a standard pipette without special techniques. This also allows for high-voltage electroporation in small volumes while maintaining a "capillary" geometry. In some embodiments, slots may also appear near vent holes to ensure that the vent holes are not obstructed. These slots can be useful during sleeve removal when the vent holes are very close to the inner surface of the elongated body.

[0109] The volume-reducing sleeve 200 may be made of, or comprise, non-conductive plastic, glass, or ceramic, and may include any other structural features described above with respect to the elongated body and / or the electroporation chamber 102 defined therefrom. Additionally, the sleeve body may be injection molded and may have external ribs for reinforcing and guiding the sleeve during insertion / removal.

[0110] In some embodiments, the sleeve 204 is secured to the sleeve electrode 206 by adhesive, welding, or geometric (physical) locking features to form a one-piece sleeve assembly 200. In this embodiment, the sleeve 204 is introduced into the elongated body 104 when the proximal cap 214 is secured to the elongated body 104. Similar to the first electrode 106, the sleeve electrode 206 may be made of or plated with a conductive material that does not negatively affect the cell by passively or during electroporation introducing harmful or toxic elements. For example, plating the electrode in pure gold can provide the electrode with beneficial conductive properties that make it impossible to introduce harmful or toxic elements into the electroporation volume. Furthermore, it should be understood that, depending on the electroporation protocol, the sleeve electrode 206 may be connected to a high-voltage circuit and may act as an anode or cathode or alternate between the two. It should be further understood that the sleeve electrode 206 may include the same or similar structural features described above with respect to the spherical protrusion 132, rod 136 and base region 134 of the first electrode 106 (appropriately scaled to match the reduced cross-section of the sleeve) to mitigate bubbles and advantageously reduce the possibility of arcing during electroporation.

[0111] Now refer to Figure 3 The diagram shows a longitudinal section of another embodiment of a single-use electroporation chamber 100' with additional features. For example, the electroporation chamber 102 of the chamber body 104 is sealed at its lower portion by two sealing members—a first proximal sealing member 120 and a reserved 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 reserved sealing member 130, which also forms a fluid-tight seal with the inner surface of the chamber body 104, is provided as a backup seal to prevent any leakage from the first proximal sealing member 120.

[0112] The housing 100' additionally includes a retaining pin 119 for securing the second electrode to the elongated body 104. This can advantageously prevent displacement of the second electrode when pressure is applied to the chamber 102. To accommodate the retaining pin 119, in some embodiments, the second electrode 108 defines a channel configured in size and shape to receive the retaining pin and can be aligned with a pair of holes defined by the sidewalls of the elongated chamber body 104, which are also configured to receive and / or retain the retaining pin 119, thereby securing the second electrode in a fixed position relative to the chamber body 104.

[0113] In some embodiments, Figure 3 The cartridge 100' may additionally include a fluid overflow space 125 operable to receive overflow volume displaced from the electroporation chamber when the electroporation chamber is sealed by a sealing cap and / or a first electrode. A sealing member 138 similar to that described above may also be present to form a fluid-tight connection between the first electrode 106 and the elongated body / chamber body 104.

[0114] Now refer to Figure 4 Another embodiment of the exemplary single-use cartridge 100'' may include a fluid overflow container 125', which may be attached to the proximal outlet of the associated electroporation chamber 104'. The fluid overflow container 125' may include a port 127 aligned with a filtered air source and / or associated conduit to facilitate the formation and breaking of a vacuum seal within the electroporation chamber 104' between iterative cycles of filling, electroporating, and emptying cells within the chamber 104'.

[0115] like Figure 4 As additionally illustrated in the cartridge 100'', the top electrode 106 can operate as a selectively removable cap, operable to position the cartridge 100'' in a capped and uncapped position. In the uncapped position, the electrode 106 moves away from the proximal end of the electroporation chamber 104', allowing the associated distal sealing member 138 to disengage from the proximal end of the electroporation chamber 104', thereby disrupting any fluid-tight seal formed between them. The top electrode 106 can 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 a fluid-tight seal between them. In this example embodiment, the top electrode 106 can be used without an associated removable cap to form, disrupt, and reform a fluid-tight seal with the electroporation chamber, repeatedly placing the electrode in contact with the cellular fluid to be electroporated.

[0116] Although Figure 4 Not shown, but it should be understood that single-use or flow-through devices may similarly include a top electrode operable to seal and deseal the electroporation chamber without the use of an associated removable capping workpiece.

[0117] Now refer to Figure 5 The image shows a cross-section of an exemplary single-use electroporation cartridge 450. The components and functions of the cartridge 450 can be the same as described above. Figure 3 Similar to the cartridge 100', an authentication chip 452 associated with the electrode cap 454 is added. The authentication chip 452 can be any form of authentication or use restriction device known in the art, and can endow the disclosed cartridge and system with any of the desired functions.

[0118] For example, the certification chip may be or include non-volatile memory, which can be used to embed manufacturing characteristics and operating parameters, data storage, security, or to manage the restricted use or reuse of the associated cartridge. This can beneficially protect against the prohibition of using uncertified aftermarket consumables and / or ensure the authenticity and usability of cartridges manufactured by the original equipment manufacturer (OEM). The non-volatile memory may also provide added functionality that allows for factory calibration of the cartridge, enabling it to deliver a given operating protocol to its associated electroporation system during use. In this way, the cartridge can specify one or more operating time parameters for the electroporation system and / or can be optimized for use with various cell types or electroporation targets.

[0119] Authentication chips can take various forms and, in some instances, can depend on the type of sterilization protocol (if any) used in manufacturing and / or packaging. Typically, gamma radiation (a common sterilizing agent in the manufacture of medical or laboratory-grade equipment) is directly incompatible with semiconductor devices that traditionally incorporate floating-gate memory technologies used in many non-volatile memories, such as erasable programmable read-only memories (EPROMs), electrically erasable programmable read-only memories (EEPROMs), and flash memory. In embodiments where sterilization via gamma radiation is desired, alternative non-floating-gate technologies, including those user-programmable non-volatile memory devices known in the art, can be used.

[0120] Other forms of authentication chips may be used and are within the scope of this disclosure. For example, simple electronic devices may be used as a reliable method for limiting the use of a cartridge to a single use or a predetermined number of uses. In an exemplary use, the cartridge utilizing circuitry within the authentication chip that incorporates a fuse can be associated with an electroporation system. After a given electroporation protocol has been run via the associated cartridge, the electroporation system can send a high 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 that keeps the associated electroporation system inactive, preventing the system from performing the necessary circuitry for its function, etc.

[0121] The alternative authentication chip also includes a radio frequency identification (RFID) tag. The RFID tag can be associated with each cartridge, and when the cartridge is loaded into a compatible electroporation system with an RFID reader, the RFID tag can transmit information to the electroporation system associated with a given cartridge. In this way, the RFID tag can advantageously provide both usage restriction and counterfeiting prevention capabilities. For example, the RFID tag may include the type of cartridge it is associated with, the number of times the cartridge can / has been used, a key, or manufacturer-specific authentication, etc.

[0122] Continue to refer to Figure 5The authentication chip 452 can be housed within the space 456 formed by the electrode cap 454. Access to the chip can be achieved via a contactless communication protocol or via one or more pin ports formed within the electrode cap 454 (e.g., for communication with an electroporation system).

[0123] Figures 6A-6D Various views of another exemplary single-use electroporation cartridge 460, containing an authentication chip 465 housed within an electrode cap 462, are shown. Figure 6D As shown in the cross-section, the authentication chip 465 resides within a recess formed by the lower portion 478 of the electrode cap 462 and is held therein by the upper portion 476 of the electrode cap 462. In some embodiments, the upper portion 476 may be secured to the lower portion 478 in a manner that prevents tampering with or additional removal of the authentication chip 465 from the electrode cap 462 after manufacturing.

[0124] Figures 6A-6D The housing 460 is shown as a plurality of pin ports 464 formed in the upper portion 476 having an electrode cover 462. The number and orientation of the pin ports 464 can be varied, and in some embodiments, may correspond to the number and / or orientation of the pins 464 associated with the connector / bus of the electroporation system that contacts and communicates with the authentication chip. For example, Figures 6A-6D The authentication chip shown includes six pin ports 464, which correspond to and match the six pins of the connector / bus of the electroporation system (not shown). In some embodiments, such as when the authentication chip is an RFID tag, the number of pin ports may be fewer, or may be absent. Therefore, it should be understood that the electrode cap can be configured in any way to accommodate the desired authentication chip and communicator / bus associated with the paired electroporation system.

[0125] Continue to refer to Figures 6A-6DIn some embodiments, the body 466 of the cartridge 460 may include one or more stabilizers 468. The illustrated embodiment depicts two stabilizers 468. In some embodiments, the stabilizer 468 itself may be sized and shaped to facilitate easier manipulation of the cartridge 460, and may include one or more gripping members 470 to assist a user in safely gripping and manipulating the cartridge 460. For example, when the cartridge 460 is loaded into an electroporation system, a user may grip the stabilizer 468 (e.g., at the gripping member 470) and easily manipulate the cartridge 460 into place. Similarly, the stabilizer 468 may be used to remove the cartridge 460 from the electroporation system after use. The gripping member 470 may include one or more protrusions extending from the surface of the stabilizer 468 as shown, or may include other contours in the surface of the stabilizer 468. In some embodiments, the gripping member may additionally or alternatively include different materials (e.g., rubber or silicone) designed to increase friction between the user's grip and the stabilizer.

[0126] In some embodiments, the stabilizer may additionally include a foot 472 located at the same height as the base (or second electrode) of the cartridge 460, thereby providing an effectively wider base to support the cartridge 460. The foot 472 may be actuated to stabilize the cartridge 460 when placed, for example, on a tabletop or other flat surface, preventing the cartridge 460 from tipping over due to side impacts or otherwise increasing the stability of the cartridge in an upright position.

[0127] like Figure 6B As shown, the stabilizer may additionally include a signal or symbol 474 to passively transmit the orientation or directionality of the cartridge 460 when inserted into or otherwise associated with the electroporation system. Although Figure 6B Symbol 474 is depicted as an arrow, but it should be understood that, as is known in the art, other signals, symbols or names may be placed on it.

[0128] In some embodiments, the electroporation cartridge, for example, Figure 1- Figure 5 , Figures 6A-6D In (and as described later in the instruction manual) Figure 9 and Figures 22A-22S The electroporation cartridges described and illustrated in this disclosure are designed to form convex menisci when fluid is dispensed into them. In some embodiments, forming convex menisci in the electroporation cartridges of this disclosure (as opposed to concave menisci formed in several prior art electroporation cartridges) prevents air bubbles from being trapped during capping or sealing of the chamber. In some embodiments, forming convex menisci in the electroporation cartridges of this disclosure (as opposed to concave menisci formed in several prior art electroporation cartridges) substantially reduces air bubbles from being trapped during capping or sealing of the chamber.

[0129] Some embodiments of the electroporation cartridges described herein can be implemented within the continuous batch processing system described in more detail below. Figures 7A-7D Such embodiments are disclosed in the literature. Figure 7A and Figure 7B An exploded view of an exemplary flow-through electroporation cartridge 300 is shown, wherein, Figure 7C and Figure 7D The image shows a flow-through electroporation cartridge 300 in an assembled configuration. (Example) Figures 7A-7D As shown, the flow-through electroporation cartridge 300 includes the components mentioned above. Figures 1A-1E The electroporation cartridge is the same or similar component described. However, it is not a single (or limited batch) electroporation cartridge requiring manual handling between individual electroporation events. Figures 7A-7D The flow-through electroporation cartridge 300 shown can be used to repeatedly electroporate continuous samples without user intervention. These flow-through electroporation cartridges can be used to process tens to hundreds of milliliters of samples in a single or even multiple different runs.

[0130] like Figures 7A-7D As shown, 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. Similar to other electrodes disclosed herein, the flow-through electrode 306 is made of or plated with a conductive material (e.g., gold) that does not negatively affect cells by introducing harmful or toxic elements. The flow-through electrode 306 is operable for connection to a high-voltage circuit and can act as an anode, cathode, or alternate between the two. In some embodiments, the flow-through electrode 306 additionally includes a bell-shaped or spherical protrusion with a slightly convex curvature to assist in the exit of the bubble from the electroporation volume by moving the bubble radially outward and away from the electrode surface, thereby reducing the likelihood of arcing during electroporation.

[0131] In some embodiments, the flow-through electrode 306 may have additional physical features to accommodate continuous flow functionality. For example, the flow-through electrode 306 may form one or more holes or through-holes 308 perpendicular to the axial direction of the flow-through electrode 306. The through-holes 308 may be blind holes or lumens 310 that traverse the axial direction of the flow-through electrode 306 (see...). Figure 7D The through-holes 308 and lumen 310 can function as outlets to allow filtered air to exit and enter the chamber, respectively, when fluid is filled and removed from the electroporation chamber 302. When the chamber 302 is filled with the fluid to be electroporated, air in the chamber 302 exits through these through-holes 308 and lumen 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 to prevent a vacuum from forming within the chamber 302. In some embodiments (e.g., Figures 7A-7DIn the illustrated embodiment, the upper section of the flow-through electrode 306 is shaped as a barb or port 312 to help maintain sterility. A tube may be mounted on this port, or fitted with a micron filter, to allow the chamber to vent to the atmosphere. Port 321 may be connected to a sterile air source or air filter. During electroporation, pressure buildup can be dissipated through the through-hole 308 and lumen 310. Similar to other electrodes disclosed herein, in some embodiments, the flow-through electrode 306 is associated with a cap so that it can rotate independently and that the sealing member 314 receives only (or primarily) axial compression.

[0132] The flow-through electroporation cartridge 300 may additionally include a chamber inlet 316 and a chamber outlet 318, which are 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 can be used to move the fluid into the electroporation chamber 302 by the action of one or more pumps in the system. Conversely, the chamber outlet 318 may be fluidly connected to an output container for electroporated cells, and can be used as an exit point for electroporated cells from the electroporation chamber 302 by the action of one or more pumps associated with the system. The chamber inlet 316 and chamber outlet 318 may be associated with plugs and / or valves, respectively, to control the inward flow of cell-containing fluid to be electroporated into the electroporation chamber, and / or to control the outward flow of cell-containing fluid from the electroporation chamber. In some embodiments, the chamber inlet / outlet may be located above the proximal surface of the second electrode, and / or the lumen of the chamber inlet / outlet may be substantially parallel to the proximal surface of the second electrode.

[0133] Now refer to Figures 8A-8C This illustration shows another embodiment of a flow-through electroporation cartridge 400 having an inlet and an outlet and a container 402 integrated with the electroporation chamber body 404. In exemplary operation, the fluid to be electroporated is filled into the chamber body 404 using a standard pipette 404, and an electrode adapter 406 can fit over its opening. The electrode 106 can fit into the adapter to seal the chamber 408 (see example...). Figure 8C Fluid may overflow from the chamber, creating a convex meniscus at the top portion of chamber 404. Subsequent insertion of the electrodes allows the overflowing fluid to gush out of the adjacent opening and into container 402, ensuring proper wetting of the electrodes (e.g., as shown in the image). Figure 8C As shown, it is obtained along plane A. Figure 8B (Cross-section of the flow-through electroporation cartridge). Figure 8C The arrows shown indicate that the overflow fluid is discharged from the electroporation chamber in response to being collected in the associated container.

[0134] Now refer to Figure 9The diagram shows an exemplary flow-through electroporation cartridge 400' with an inlet and an outlet. The components and functions of the cartridge 400' are the same as described above. Figures 8A-8C Similar to the cartridge 400, an authentication chip 452 associated with the electrode cap 454 is added. The authentication chip 452 can be any form of authentication or use restriction device known in the art, and can endow the disclosed cartridge and system with any number of desired functions. Several examples of authentication chips described in the preceding sections can also be used here. Figures 22A-22S The previous descriptions included several aspects and additional embodiments of the electroporation box and how it operates for refilling and electroporating batches of cells.

[0135] Overview of electroporation systems

[0136] The following provides an initial overview of an exemplary electroporation system and a corresponding procedure for electroporating samples. It should be understood that the embodiments described herein provide efficient electroporation of relatively high-volume samples and offer additional benefits such as high electroporation efficiency, high cell viability, and a safe and relatively simple user experience. As will be seen in the more detailed description below, the system can be functionally closed to isolate all contact points from the surrounding environment, thus limiting potential contamination and also enhancing safe operation of the device. Furthermore, as described below, the disclosed system is capable of recovering samples in the event of system errors, excessively high temperature readings, arc risk readings, and / or other warning events. In the event of such events, for example, a relevant sub-volume of the sample can be pumped out of the electroporation chamber and returned to the mixing container, or even returned to the sample input bag from which it originated.

[0137] Figure 10A The process of an exemplary flow-through electroporation system 500 is illustrated schematically. Figure 10A This document aims to provide an overview of exemplary processing flows that can be performed using the system described herein. Various electroporation system components that may be included within system 500 will be described in more detail below. It should be understood that various alternative embodiments of different electroporation system components may be implemented through, for example... Figure 10A The overall diagram shows a combination of various methods for constructing an electroporation system.

[0138] In the illustrated system 500, an input bag 502, including a sample, may be fluidly attached to a first pump 504. The first pump 504 is configured to drive a fluid flow rate from the input bag 502 to a mixer container 508. A first flow sensor 506 may be positioned between the pump 504 and the mixer container 508 to enable the system to determine when fluid is flowing through a corresponding pipe section. The mixer container 508 is fluidly attached to a second pump 510. The second pump 510 is configured to drive a fluid flow rate from the mixer container 508 to an electroporation cartridge 516. In some embodiments, the electroporation cartridge may be a flow-through electroporation cartridge, for example, any flow-through electroporation cartridge described above (see, for example, those described above). Figures 7A-9 and Figures 22A-22Q (as shown in the image).

[0139] 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) may be deployed between the second pump 510 and the electroporation cartridge 516. As described in more detail below, it is advantageous to provide the pre-cooling module upstream of the electroporation cartridge 516 to assist in reducing the temperature of the passing sample (e.g., toward a target temperature) to a lower temperature more suitable for electroporation and less conducive to bubble formation (or otherwise regulate the temperature of the passing sample via heating, if desired).

[0140] like Figure 10A As shown, the electroporation cartridge 516 is fluidly connected to a third pump 518, which is configured to drive sample fluid out of the electroporation cartridge 516 and toward an output (e.g., an output bag 526). A third flow sensor 520 may be deployed between the third pump 518 and the output bag 526.

[0141] In operation, the electroporation cartridge 516 is operated to provide electroporation pulses to portions of the sample contained therein. The terms "sub-volume" and "sample sub-volume" will be used herein to refer to discrete portions of the sample contained within the electroporation cartridge 516 at any given time, to distinguish them from the larger total volume of the sample intended to be processed by the system 500. The system 500 is advantageously operated to move a series of consecutive sub-volumes through the electroporation cartridge 516 without requiring the electroporation cartridge 516 to be removed from the electroporation of one sub-volume to the electroporation of the next.

[0142] This system 500 advantageously enables efficient electroporation of relatively large sample volumes. For example, a single "run" can process a total sample volume of approximately 5 mL to approximately 25 mL, or even up to approximately 50 mL, by electroporating several consecutive sub-volumes until the total sample volume has been electroporated. Even sample volumes larger than 50 mL can be reasonably handled with limited additional settings, the limitation being that most standard sample bags are not designed to be that large. Therefore, the system can handle even larger volumes, provided that the input and output reservoirs are configured to accommodate such volumes.

[0143] As shown, system 500 may also include one or more air filters or other air venting devices to provide air venting and assist sample fluid movement 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. A vent line and / or air filter 521 may also be connected to an intermediate conduit section 524 extending from the main conduit section between first pump 504 and mixer container 508. As explained in more detail below, this feature can be used to determine and / or control when sample transfer from input bag 502 to mixer container 508 has occurred.

[0144] While specific examples of the foregoing components will be described in more detail below, it should be understood that alternative embodiments of the electroporation components may be additionally or alternatively 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, inline pumps, other types of pumps, or other suitable fluid transfer mechanisms known in the art. Furthermore, although three pumps are shown in this particular example, other embodiments may include fewer or more pumps.

[0145] By way of another example, while the flow sensor may be described as an ultrasonic sensor, other embodiments may include one or more rotor flow meters, spring and piston flow meters, turbine / paddle sensors or other positive discharge flow meters, vortex flow meters, pitot tubes, Hall effect sensors, or other suitable flow sensors known in the art. Furthermore, although three flow sensors are shown in this particular example, other embodiments may include fewer or more flow sensors. Additionally, while the input and output reservoirs are described as bags in the following example, other suitable sample reservoirs may also be utilized, provided that their contents can be efficiently transferred from the input reservoir to the electroporation system and then exit into the output reservoir.

[0146] Furthermore, while the cooling and pre-cooling module components described herein are described in the context of cooling, it should be understood that, at least in some embodiments, they may also be operated to heat the sample and / or sample sub-volumes. For convenience, since typical operation will benefit from cooling rather than heating, these components will be referred to herein as “cooling” or “pre-cooling” modules. However, this should not be construed as limiting their ability to provide heating to complement or replace their ability to provide cooling.

[0147] Furthermore, while the specific example described herein illustrates a single system with a single input bag, other implementations may utilize more than one system and / or more than one input bag per system to process larger volumes through parallel operations.

[0148] Figure 10B A control system 600 (used interchangeably herein with the terms "computer system" and "computing system") that can be included as part of an electroporation system is shown. For example, the control system 600 forms... Figure 10A A portion of one or more components of the electroporation system 500 shown may be communicatively coupled to the one or more components or to any other electroporation system described herein.

[0149] 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 commands to the one or more electroporation components. As shown, the controller 601 may be communicatively coupled to a pump 604, a flow sensor 606, a mixer driver 608 (for controlling mixing in a mixer container), a pre-cooling module 614, a cooling module 615, and an electroporation assembly 617.

[0150] The controller 601 operates to provide control over various linked electroporation components. For example, the controller 601 may be configured to: control the actuation, direction, and / or speed of the pump 604; receive flow data from the flow sensor 606; control the actuation, direction, and / or speed of the mixer driver 608 to thereby correspondingly control mixing in the mixer container; and control the temperature in the pre-cooling module 614 and / or the cooling module 615.

[0151] The electroporation assembly 617 includes various components configured to interact with the electroporation cartridge. As shown, the electroporation assembly 617 may include a conductivity sensor 630 configured to measure the conductivity of the electroporation chamber across the electroporation cartridge. As explained in more detail below, a controller may utilize information from the conductivity sensor to control one or more electroporation parameters (e.g., whether to deliver a pulse and the calibration voltage used to deliver the pulse).

[0152] The electroporation assembly 617 also includes components constituting an electroporation circuit configured to generate and deliver electrical pulses to the electroporation chamber. These components include a charger 632 configured to act as a voltage source for charging the capacitor 634, one or more discharge resistors 636, and / or other safety components configured to allow the capacitor to safely discharge when it is not immediately discharged for normal operation. As is known in the art, additional or alternative circuit components may be included for generating and delivering electrical pulses through the electroporation chamber.

[0153] The electroporation assembly 617 may also include a capping mechanism 638 configured to mechanically engage with the electroporation cartridge when it is properly inserted into the electroporation assembly 617. As explained in more detail below, in some embodiments, the capping mechanism 638 operates to move the electroporation cartridge between a capped state where electroporation can occur and an uncovered state where discharge and fluid movement into and out of the electroporation chamber can occur. A sealing mechanism 640 may operate in conjunction with the capping mechanism 638 to seal and unseal the electroporation chamber as the system moves through the electroporation chamber from one sub-volume to the next.

[0154] Various exemplary electroporation components will now be described in more detail. Any of the following embodiments can be incorporated into the electroporation system 500 and / or control system 600.

[0155] Figures 11A-11D An example embodiment of the electroporation system of this disclosure is shown. Figure 11A and Figure 11B An exemplary electroporation system / instrument 605' according to one embodiment is shown, which includes one or more components of electroporation system 500 and one or more components of electroporation system 600 (the components of system 500 and system 600 are shown but not numbered). Figure 11A The illustrated embodiment includes: a door that can close while the electroporation is in progress; and a user interface panel 606' that allows the user to select electroporation parameters, operating protocols, monitor the progress of the operation, receive any error messages to correct potential problems, etc., via a graphical user interface and computer-implemented methods.

[0156] In one embodiment, Figure 11A and Figure 11B The electroporation system / instrument 605' may include one or more components, including: at least one or more pumps; at least one or more sensors for sensing liquids (e.g., such as, but not limited to, ultrasonic sensors); at least a cell mixer mechanism and / or a region for removably inserting a cell mixer; a region 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 electrical pins (e.g., but not limited to high-voltage electrical pins for electrical contact with electrodes located in the inserted electroporation chamber); an authentication chip reader (configured to read a chip on the electroporation chamber); one or more bubble sensors; one or more pre-cooling chambers or modules; and a modular housing for inserting various electroporation device components (e.g., but not limited to dashboards or modular housings, such as, Figures 12A-12G and Figures 13A-13C The following are described in the text: housings or placers (700, 701, 703, 800, or 803); pressure sensors; locking mechanisms for clamping in modular housings and / or electroporation chambers; handles for easy insertion and removal of modular housings and / or electroporation chambers; stopcocks and placement mechanisms; air filters; tubes for transferring sample flow from one component to another; tubes for air flow; one or more stopcocks for controlling air and / or liquid flow; sterile air sources and / or air filters; outlets; valves; rotary mechanisms for opening or closing chambers or inlets and outlets for cells; pressure sensing mechanisms; conductivity sensors; hooks or pullers for attaching sample inlet bags and sample outlet bags (bags containing cells in fluid before and after electroporation, respectively); and for sensing, controlling, and operating one or more of the aforementioned components (some of these components are shown on inner surface 607, but...). Figure 11A and Figure 11B Components of the processor system (e.g., computer controller and processor) not explicitly labeled or described in the specification. Some of the components listed above may be located inside the housing of 605', while others may be located on the inner surface of the system / instrument 605' (e.g., 607).

[0157] In some embodiments, one or more pumps of system 605' may include, for example: a first pump (e.g., a peristaltic pump) for transferring a sample (e.g., a sample including cells to be electroporated from an input bag / container) to a cell mixer; and a second pump (e.g., a peristaltic pump) for transferring a processed sample (e.g., a sample including electroporated cells from an electroporation chamber to an output bag / container) to a cell mixer.

[0158] In some embodiments, system 605' may include a pre-cooling module located just before the cells enter the electroporation chamber to cool the cells to an optimized temperature. In some embodiments, an additional pre-cooling module may be located in an area where the electroporation cartridge is removably inserted into the instrument. The pre-cooling module is described in the preceding sections.

[0159] Figure 11C An example electroporation system is shown, which includes: Figure 11A Electroporation instruments; and removably attachable modular housings (which may be similar to 700, 701, 703, 800, 801, 803, etc.) for arranging various electroporation components (as described below). Figures 12A-12G , Figures 13A-13C and Figure 15A-15I (Further details on the modular housing are provided in the document.) The modular housing is designed to accommodate the use of a flow-through electroporation chamber to process large volumes of samples for electroporation by passing small batches of samples through a single electroporation.

[0160] Electroporation instruments and systems 605' and / or 600' typically have an outer cover to allow the user to close the system while electroporation is being performed. Figure 11B An embodiment with a sliding outer cover in the open position is shown. Figure 11D Another embodiment of an electroporation instrument with different outer covers that can be swung upwards to open is shown. Various other types of outer covers can be used. The outer covers can provide safety against the high voltages used by the user and can provide a sterile environment.

[0161] In some embodiments of the electroporation system 605' or 600', for example, when using a single-use electroporation chamber, a modular housing with multiple components is not required. In a system configured for a single electroporation event (as opposed to batch processing / continuous flow), 605' or 600' may 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 the electroporation chamber having appropriate electrical contacts (e.g., high-voltage pins) available for providing electrode pulses to electrodes inside the electroporation chamber, a locking mechanism for clamping within 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 for receiving the electroporation chamber and connected to electrode contacts and other components inside the electroporation instrument to perform electroporation.

[0162] Electroporation system and modular housing

[0163] Figure 12AAn example embodiment of an electroporation system 700 including a modular housing 703 and a dashboard 701 is shown. In some embodiments, the housing 703 is attached to the dashboard 701 and can be detached from the dashboard 701 (see [link]). Figure 12A Many traditional electroporation systems require multiple consumable components and are complicated to set up and operate due to easily tangled tubing and bags that can obstruct other components. The modular housing 703 is designed to advantageously arrange different electroporation components and tubing in one place in an organized and easily manageable manner. This, along with other design features of the system described herein, helps to reduce errors and maximize electroporation efficiency. For example, the design of the electroporation housing described herein advantageously reduces the number of user contact points where errors or problems may occur.

[0164] like Figure 12A As shown, housing 703 is selectively attachable / removable from instrument panel 701. Housing 703 may include one or more attachment features that engage with instrument panel 701 and allow selective attachment. Housing 703 may be attached to one side of instrument panel 701, as shown. Additionally or alternatively, instrument panel 701 may be configured to enclose all or part of housing 703.

[0165] Figure 12A (as well as Figure 11C (It includes an instrument panel / modular housing similar to 701 / 703)) An exemplary arrangement of various electroporation components is shown. An input bag 702 is fluidly coupled to a first pump 704 via a conduit. The first pump 704 drives sample fluid from the input bag 702 through a first flow sensor 706 and into a mixer container 708. A second pump 710 then drives sample fluid from the mixer container 708 through a second flow sensor 712, through a pre-cooling module 714, and into the electroporation chamber of the electroporation cartridge 716. After electroporation of the sample subvolume, a third pump then drives fluid out of the electroporation chamber, through a third flow sensor (not shown in this view), and toward an output chamber (e.g., an output bag) (not shown in this view).

[0166] As shown, both housing 703 and instrument panel 701 may separately include one or more hooks or other attachment features for supporting input bag 702 and, optionally, output bag. In the illustrated embodiment, input bag 702 may be attached to housing hook 707 during the movement of housing 703 and loading onto instrument panel 701. After attachment, input bag 702 may be moved to instrument hook 709 to move it out of the field of view of other electroporation components and / or better position the bag for transfer and electroporation processing.

[0167] The modular housing of this disclosure, containing the components of system 700, can be removably placed into electroporation instrument 600' or system 605' (e.g., Figures 11A-11D Those described herein are enclosed within an outer housing or cover 720, which can be manually opened and closed to access the dashboard (e.g., upward away from the system base, or slid upward, or slide / open to the side) to expose and allow access to these components, such as Figure 11A As shown. In some embodiments, the outer cover 720 is rotatable about a hinge to expose and allow access to the internal components of the system 700.

[0168] Figures 12B-12E A slightly different embodiment of the electroporation system 800 is further shown, which has a housing 803 configured for attachment to a corresponding instrument panel 801. Figure 12B The rear side of housing 803 is shown. Figure 12C The front side of housing 803 is shown. Figure 12D The diagram shows the pipes for the input and output bags attached to the housing 803, and... Figure 12E The housing 803 is shown attached to the instrument panel 801.

[0169] As with system 700, system 800 includes a housing 803 and conduit that runs through the housing 803 from an inlet 805 (configured for attachment to an input bag 802) to an outlet 825 (configured for attachment to an output bag 826). The conduit connects to a mixer container 808, a precooling module 814, one or more flow sensors 812, and an electroporated cartridge 816. The conduit also passes through one or more cut-out sections of the housing (flow indicators 844 and 846) that allow for flow visualization. Housing 803 may also include one or more side handles 852 and one or more top handles 850 to assist in moving and manipulating housing 803, for example, from a lab bench to a dashboard 801.

[0170] The housing 803 includes an arrangement of compartment 842 that corresponds to the arrangement of pumps (e.g., pumps 804 and 810, other pumps not shown) on the instrument panel, such that when the housing 803 is attached to ( Figure 12E When the instrument panel 801 is shown, the pump is received into compartment 842. Figure 12C As best shown, housing 803 also includes mixer compartment 827, which is configured to provide space adjacent to mixer container 808 for receiving a corresponding mixer drive attached to instrument panel 801.

[0171] like Figure 12CAs can be seen, system 800 may also include an air container 848, which is pneumatically coupled to an intermediate duct section 824 deployed between inlet 805 and mixer container 808. As described in more detail below, air container 848 is configured to supply air to the intermediate duct section after the sample has been drawn into the mixer container.

[0172] Figure 12F and Figure 12G Different views of housing 803 are shown to illustrate one embodiment of attachment features that enable housing 803 to be attached to instrument panel 801. In this embodiment, side handle 852 is flexible and includes a latch 856 that can engage with a corresponding structure of instrument panel 801. Handle 852 can be biased toward a neutral, upright position and is configured to flex when gripped and pulled outward. Figure 12G As shown, a user can position his / her fingers on the gripping surface 854 and flex the handle 852 outward by pulling on the gripping surface 854. Upon releasing the handle 852, the handle 852 will move back to the neutral position, allowing the latch 856 to move inward to engage with the dashboard. Other embodiments may additionally or alternatively include other attachment features known in the art, including latches, alignment pins, hooks, clamps, etc.

[0173] Figures 13A-13C An embodiment of another exemplary modular housing that can be attached to an electroporation instrument / system (e.g., 605' or 600') is shown. Figure 13A A front view is shown of a modular housing 700' for arranging various electroporation components that can be selectively attached to and detached from an electroporation instrument. Figure 13B The diagram illustrates the additional components. Figure 13A Rear view of the modular housing 700'.

[0174] As with system 700, system 700' includes: a housing 703'; and a conduit that runs through housing 703' from an inlet pipe 739 configured for attachment to an input bag (not shown), wherein the input bag is to be placed on an input bag hook 707. The inlet pipe 739 connects to a cell mixer container 708 and then continues through a first pump (not shown, as it is located on the surface of the instrument / system (e.g., 605' / 600')) to a tube 737 (which is the tube exiting from the mixer container 708), visible through a slot in the modular housing 708' for a mixer pump (not shown), depicted as mixer tube 737, which then continues through a pre-cooling module (not shown, as it is located on the surface of the instrument / system (e.g., 605' / 600')) as a pre-cooling pipe 736; however, a perspective view of the pre-cooling module 714' is visible from the front. Figure 17C (In the image), although a slot is shown in the housing for the pre-cooling module 714', the tubing continues to an electroporation cartridge (e.g., 300, 400, or 400') (which is placed / received in an electroporation cartridge holder 878), and then to an outlet tube 735 (configured for attachment to an output bag (not shown)). The output bag is to be attached to an output bag hook 707'. In some instances, the tubing passes through one or more cut-out sections of the housing that allow flow. The housing 703' may also include one or more side handles 752', which may have grippers (e.g., 709') and one or more upper handles 750' to assist in moving and manipulating the housing 703' (e.g., from a lab bench to an instrument / system 605' or 600' or a dashboard on the instrument / system (e.g., 701 or 801)).

[0175] like Figure 13A and Figure 13B As depicted in the embodiments, the cell mixer container 708 is positioned at an angle within a modular housing. The inventors have shown an angled cell mixer 708 to provide a surprisingly significant reduction in sample loss by reducing or preventing sample trapping within the cell mixer 708. In some embodiments, the cell mixer 708 is tilted at an angle ranging from approximately 5 degrees to approximately 20 degrees and any value in between. In some embodiments, the cell mixer 708 is tilted at an angle of approximately 10 degrees (e.g., approximately 8 degrees, approximately 8.5 degrees, approximately 9 degrees, approximately 9.5 degrees, approximately 10 degrees, approximately 10.5 degrees, approximately 11 degrees, approximately 12 degrees, etc.).

[0176] Reference Figure 13A , Figure 13B and Figure 13C System 700' may include an air container (not explicitly shown) pneumatically coupled to an intermediate piping section; and an air inlet pipe 738 disposed between the sample inlet pipe 739 and the mixer container 708. The air container, configured to supply air to the intermediate piping section after a sample has been drawn into the mixer container, is described in detail elsewhere in this application.

[0177] Figure 13B The illustration depicts the placement of an air inlet pipe 738 on a mixer container 708. The air inlet pipe 738 leads through several air filters 722 to a stopcock (not explicitly shown), which has a stopcock adapter 730 that holds the stopcock onto a modular housing 700'. The operation of the stopcock and stopcock adapter 730 is described in the previous section. Icon 731 can be used as a visual indicator for the user of the airflow direction. The air inlet pipe connected to the air filters 722 allows air exchange to different zones to allow fluid flow and / or prevent pressure buildup in the mixing chamber or electroporation chamber.

[0178] Figure 13C An embodiment of the modular housing 700' is shown, wherein an additional input chamber 740 (e.g., a payload chamber) with its own additional input tube 741 is provided for adding one or more additional components to a sample. In addition to the inlet of the sample inlet tube 739, the input tube 741 may also have an inlet into the mixer container 708. In one example, the additional material may include a payload (e.g., material to be electroporated into cells (e.g., nucleic acids, DNA, RNA, proteins, drugs, or any other molecules or materials desired to be electroporated into cells), which is to be added later during the setup and assembly of the electroporation system prior to performing electroporation and is not exposed to cells or other contents in the cell bag). In some non-limiting embodiments, the additional input chamber 740 may be a syringe containing additional material (e.g., ...) that can be manually injected into the cell mixer when needed and as required. Figure 13C (As depicted). However, other compartments (e.g., input bags, storage containers, etc.) can be used as compartment 740.

[0179] The electroporation cartridge 300, 400, or 400' is inserted into a slot on the instrument via the cartridge housing 878. (Previously...) Figures 19A-19C The description of housing 878 and its functions is provided.

[0180] Bag compartment

[0181] Figure 14A and Figure 14B An example of a bag chamber 858 that can be incorporated into the electroporation system described herein is shown. During use, the sample reservoir bag should be supported in an upright position with the ports facing downwards to minimize residual fluid (with respect to the inlet bag) and bubble accumulation (with respect to the outlet bag). Preferably, the bag is also able to fit within a small footprint to maintain the size of the suppression device and minimize obstruction of view of other components of the shielding system. Furthermore, it is beneficial, but not necessary, to close the bags during system use to prevent them from being manipulated by the user during high-voltage electroporation operations.

[0182] In the illustrated compartment 858 (the remainder of the housing is removed for clarity), an insertion portion 860 is slidably connected to the compartment 858 to allow it to be selectively withdrawn from or retracted within the housing. For example, the insertion portion 860 may be connected to the compartment 858 via a track and rail system 862. The insertion portion 860 may include a hook 864 or other suitable attachment feature for supporting an input bag and a support 866 for supporting an output bag. The compartment 858 may include one or more latches (e.g., magnetic latches), clamps, stops, etc., for holding the insertion portion 860 in a desired position.

[0183] Input sample transfer

[0184] Figures 15A to 15C An example assembly configured to provide efficient transfer of sample fluid from the input bag to the mixer container 808 is shown. The sample input can vary throughout the process based on sample type, sample volume, sample fluid properties (e.g., viscosity), and bag type. Therefore, it is advantageous to be able to accurately determine when the sample fluid has been completely transferred from the input bag to the mixer container. Furthermore, it is desirable to minimize sample loss due to residual amounts remaining in the bag and / or in the tubing between the bag and the mixer container 808.

[0185] The transfer assembly shown is configured to indicate when the input bag is substantially emptied of sample fluid, and accordingly allows the controller to configure process control. The transfer assembly is also configured to minimize sample waste by clearing the piping between the sample inlet 805 and the mixer container 808 at the end of the sample transfer process.

[0186] Figure 15A The sample transfer assembly is shown before the end of the sample transfer process, and... Figure 15B and Figure 15C The sample transfer assembly is shown at the end of the sample transfer process. Figure 15A In this process, air has not yet entered the main pipe section 829 from the air container 848 through the intermediate pipe section 824. That is, although the sample fluid is being transferred from the inlet 805 to the mixer container 808 through the main pipe section 829, the air container 848 will remain full.

[0187] like Figure 15B and Figure 15C As shown, once the input bag is nearly emptied, the continuous operation of pump 804 further reduces the pressure within the pipeline, thereby drawing air away from intermediate pipeline section 824 and into main pipeline section 829. This additional bolus of air helps flush / clean the main pipeline section 829 of residual sample, thus maximizing the amount of sample delivered to mixer container 808.

[0188] The air container 848 can also be associated with a sensor (e.g., a proximity sensor) that senses when the volume of the air container has decreased, thereby signaling the progress or completion of the sample transfer process. In the illustrated embodiment, the plunger 849 moves as the air container 848 is depleted, and the movement of the plunger 849 thus indicates the pressure within the main conduit section 829, and thereby indicates the progress of the sample transfer process.

[0189] Other embodiments may additionally or alternatively utilize other means for determining the progress or completion of the sample transfer process. For example, Figures 11A to 11B The electroporation system 600' or 605' shown omits the syringe and plunger arrangement of system 800. Instead, system 700 includes an intermediate conduit section that extends from the main conduit section and terminates in an air filter open to the atmosphere. The airflow within the intermediate conduit section can be controlled by adjusting the degree of opening of the intermediate conduit section (e.g., by using a retraction mechanism to open / close the intermediate conduit section). (See also: ...) Figure 15A and Figure 15B As with other systems, when the pressure in the main pipeline section becomes sufficiently low near the end of the sample transfer process, air can be drawn into the main pipeline section through the intermediate pipeline section. As indicated above, system 700 also includes a first flow sensor 706, which is deployed between the input and the mixer container 708 to assist in determining when the sample transfer has been completed.

[0190] Figures 15D-15E Shown by, as Figures 13A-13C An example embodiment of the sample flow rate of the modular housing shown in System 700'. Similar component numbers have... Figures 13A-13C Similar functionality to that in [the context of the game]. Figure 15D and Figure 15E The arrows depicted in the different sections of the tube are drawn through the front view of the modular housing 700'. Figure 15D ) and rear view ( Figure 15E The diagram illustrates the flow rate of a sample (e.g., a sample comprising cells in a fluid containing a material to be electroporated) in the modular housing system 700'. For example, as depicted by the arrows, the sample may enter the inlet pipe 739 and then through the inlet into the mixer container 708 (see...). Figure 15D and Figure 15E (The arrow leads into 708), and exits through the exit in 708 (see...) Figure 15E The sample continues through mixing tube 737, through mixing pump (not shown), and through pipe 736 through precooling chamber (not shown), and into inlet leading to electroporation chambers 300, 400, or 400'. Additional payloads can be loaded via input device 740 (e.g., syringe) to manually dispense the payload into the sample in mixer container 708. After electroporation of the sample, the electroporated sample exits electroporation chambers 300, 400, or 400' via outlet, see [link to relevant documentation]. Figure 15E The arrow in the outlet tube 735. The electroporated sample continues through the outlet tube 735, where... Figure 15E In one embodiment, the process continues toward the top of the electroporation chamber and housing 700' to access the outlet pocket (not shown) that can be suspended on the hook 707'.

[0191] Figures 15F-15G Showing through as ( Figures 13A-13CAn example embodiment of the airflow through the modular housing system 700' is shown in system 700'. Arrows depicted in different sections of the pipe illustrate the airflow through the modular housing system 700', and show air entering the air inlet pipe 738 (see...). Figure 15F ). Figure 15G Arrows indicate the airflow through pipes 738 and 734 (the pipes exiting from electroporation chambers 300, 400, or 400') during filling and subsequent filling of the chamber via air filter 722 and via stopcock 731 located after stopcock retainer 730.

[0192] Figure 15H Additional details of the stopcock mechanism for airflow are shown, with shaded arrows depicting the rotation of the stopcock. The instrument's actuator 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 operate, where air pushes the sample / liquid from tube 737 to the subsequent stage (which is the mixer container 708) and from there via mixer tube 737 to the mixer pump. Stopcock adapter 730 acts as a retainer to hold stopcock 731 on housing module 700'. Stopcock adapter 730 also serves as an easier inlet for the user to properly load it into the stopcock actuator on the instrument (e.g., 605' or 600').

[0193] Figure 15I The document describes three steps for removing the modular housing 700' from the instrument or system 600' or 605, and includes the following steps: removing the electroporation chamber by first removing the knob of the housing retainer 878; then removing the stopcock adapter 730; and then removing the modular housing 700' using the handle and grip (if present) to remove it from the system 605' or instrument 600'.

[0194] Mixer container

[0195] Figure 16A and Figure 16B An unfolded view of an exemplary mixer container 808 is shown. The mixer container 808 is advantageously used to maintain a (e.g., cell-containing) sample fluid in a homogeneous suspension while continuous sub-volumes of the sample are electroporated and moved to an output bag. Including the mixer container 808 advantageously allows for the use of a wide variety of input bag types. Because bags can vary in volume, shape, stiffness, etc., attempts to transfer directly from the bag itself to the electroporation cartridge can result in non-uniform cell density between sub-volumes, cell sedimentation, blockage of the tubing, and other undesirable problems. These problems are circumvented by moving the sample volume to the mixer container 808.

[0196] like Figure 16A As shown, the mixer container 808 may include a mixer element 868. In contrast to freely moving elements (e.g., magnetic stir bar), the mixer element 868 is preferably configured as blades (as shown), impellers, etc. Centrifugal stirrers are also less preferred because they tend to concentrate cells in the outer portion of the circulating fluid.

[0197] Preferably, the magnetic element does not contact the sample fluid. The mixer element 868 therefore preferably does not include magnetic materials, but is formed of medical-grade polymers or other suitable materials. Mixer element ( Figure 16B (Not shown) can be coupled to a mixer magnet assembly located in the cover 811. When the housing is attached to the dashboard, the mixer driver (not shown, part of the dashboard) is received in the mixer compartment 827. The mixer driver includes one or more magnets that are magnetically coupled to the mixer magnet assembly and thereby indirectly drive the rotation of the mixer magnet assembly via a magnetic connection.

[0198] The mixer container 808 shown includes a container inlet 869 and a container outlet 870. As shown, the container inlet 869 is preferably located in the upper section of the mixer container 808 so that the resulting sample fluid can flow downwards along the inner surface of the mixer container 808 before being collected at the bottom. This configuration tends to limit the formation of bubbles upon entering the mixer container 808.

[0199] Cooling mechanism

[0200] Figure 17A and Figure 17B An embodiment of the pre-cooling module 814 is shown. In this embodiment, the pre-cooling section 831 of the pipe upstream of the electroporation chamber is contacted with the cooling block 871 via one or more attachment clamps 876. Other attachment modes may be used, and clamp 876 is an example embodiment. The cooling block 871 may be a ceramic block, or may comprise any other material capable of effective thermal communication and heat transfer with the pre-cooling section 831 of the pipe. The cooling block 871 may be cooled according to methods known in the art (preferably via thermoelectric cooling). Other embodiments may additionally or alternatively utilize air cooling, liquid cooling, or other temperature regulation mechanisms known in the art.

[0201] In the illustrated embodiment, the pre-cooling section 831 of the conduit is deployed in an annular or meandering arrangement. This advantageously provides greater contact between the pre-cooling section 831 of the conduit and the cooling block 871, allowing for greater heat transfer. However, it has been found that an annular or meandering arrangement can also lead to bubble formation, which can negatively affect the subsequent electroporation of the sample subvolume. Therefore, some embodiments keep the pre-cooling section 831 of the conduit substantially straight (e.g., Figure 12A The pre-cooling module 714 and in the embodiment Figure 12B (In the pre-cooling module 814 of the embodiment).

[0202] like Figure 17B As shown, the precooling module 814 may also include a flexible biasing element 872. In this embodiment, the flexible biasing element 872 is deployed on one side of the cooling block 871 opposite to the precooling section 831 of the pipe. The flexible biasing element 872 includes features that bias against the cooling block 871 and tend to press the cooling block 871 against the precooling section 831 of the pipe, thus helping to maintain good thermal contact between the cooling block 871 and the precooling section 831 of the pipe.

[0203] Figure 17C Show Figure 12A A perspective view of an embodiment of the pre-cooling module 714 shown. A section of pipe carrying the sample, substantially straight (e.g., Figure 12A 831 or Figures 13A-13C The sample (737) moves through the pre-cooling module 714 to cool the sample before electroporation.

[0204] Figure 17D An example of a cooling module 873 configured to cool the electroporation chamber of the present disclosure is shown. The cooling module 873 shown is configured in size and shape to receive the corresponding electroporation chamber portion of the electroporation cartridge of the present disclosure. Figure 17D Upper electrode contact 874 and lower electrode contact 875 are also shown, positioned to contact the corresponding upper and lower electrodes of the inserted electroporation cartridge. As with the pre-cooling module, the cooling module 873 may comprise a ceramic block or other suitable heat transfer material and may be operated via thermoelectric cooling and / or other cooling methods known in the art (e.g., air cooling and / or liquid cooling).

[0205] Figure 18A and Figure 18B An example of the high-voltage contact pin of the electroporation instrument of this disclosure is shown detached from the example electroporation cartridge. Figure 18A A rear sectional view depicting an electroporation instrument or system, showing the area where an electroporation cartridge (not explicitly shown) can be removably placed. Figure 18A The image shows cam 921 in the engaged portion. Figure 18B The corresponding area is shown in the top cross-sectional view, and the high-voltage pin 920 of the system is depicted, wherein the electroporation cartridge can be removably placed at the end of the high-voltage pin. Spring 922 and sensor 923 are also depicted. The linear actuator stroke is approximately 10 mm. Figure 18B The high-voltage pin 920 is shown disengaging from the electroporation cartridge when the cam 921 is engaged. Figure 18C and Figure 18D An example of a high-voltage touch pin of the electroporation instrument of this disclosure is shown, which is coupled to the electroporation chamber. Figure 18C A rear sectional view depicting an electroporation instrument or system, showing the area where an electroporation cartridge (not explicitly shown) can be removably placed. Figure 18C The image shows the cam 921 in the disengaged portion, which corresponds to the engagement of the high-voltage pin 920 with the electroporation cartridge. Figure 18D The corresponding area is shown in the top cross-sectional view, and the high-voltage pin 920 of the system engaging with the electroporated cartridge (the cartridge is not depicted) is depicted. The linear actuator stroke is approximately 2 mm.

[0206] Electroporation box attachment features

[0207] Figure 19A , Figure 19B and Figure 19C An embodiment of an electroporated cartridge attachment feature configured to allow the electroporated cartridge to be attached in a suitable location within a cooling module 815 is shown. In the illustrated embodiment, the electroporated cartridge 816 includes a cartridge body 877 and a retainer 878. The cartridge body 877 is adapted to be attached to the electroporated cartridge 816 via one or more retaining rings 883. The retainer 878 is then attached to the cartridge body 877. The retainer 878 includes two latching portions 879 that extend beyond the cartridge body 877 when the retainer 878 is attached to the cartridge body 877 and can engage with corresponding structures of the cooling module 815 to thereby hold the cartridge body 877 and the electroporated cartridge 816 in place within the cooling module 815.

[0208] As shown, the retainer 878 may also include a flexible biasing element 872 configured to bias the cartridge body 877 and the electroporated cartridge 816 toward the cooling module 815. The flexible biasing element 872 advantageously takes into account differences in component tolerances and ensures compensation for small differences so that the electroporated cartridge 816 is in effective thermal contact with the cooling module 815 in any case.

[0209] The attachment feature is also configured to allow selective disengagement and removal of the electroporated cartridge. An inward press on the proximal section 880 of the retainer 878 causes the latch 879 to buckle outward, allowing removal from the cooling module 815. Lugs 881 of the retainer 878 engage in corresponding slots 882 in the cartridge body 877, enabling the user to pull the entire cartridge out of the cooling chamber when disengaging the latch 879.

[0210] Electroporation chamber sealing mechanism

[0211] Figures 20A to 20CThe diagram illustrates the operation of a capping mechanism 838 for moving the electroporation cartridge between a capped state in preparation for electroporation and an uncapped state in preparation for filling or emptying the electroporation chamber. The capping mechanism 838 functions as a linear actuator, engaging with a cap 885 of the electroporation cartridge 816. The cap 885 is further coupled to an upper electrode 884 of the electroporation cartridge, such that movement of the capping mechanism 838 controls upward and downward movement of the upper electrode 884.

[0212] Figures 20A to 20C Also shown is a chamber inlet plunger 886 controlled by a chamber inlet actuator 887 and a chamber outlet plunger 888 controlled by a chamber outlet actuator 889. Figure 20A The diagram shows the upper electrode 884 in the unsealed position, the inlet plunger 886 in the retracted open position, and the outlet plunger 888 in the forward closed position. Figure 20A This indicates the relative positions of these components during the fill operation.

[0213] Figure 20B The upper electrode 884 in the closed position, the inlet plunger 886 in the forward closed position, and the outlet plunger 888 held in the forward closed position are shown. Figure 20B This indicates the relative positions of these components when they are in a sealed state in preparation for electroporation.

[0214] Figure 20C The diagram shows the upper electrode 884 in the unsealed position, the inlet plunger 886 held in the forward closed position, and the outlet plunger 888 moved to the retracted open position. Figure 20C This indicates the relative positions of these components during a venting operation (e.g., after electroporation of a sub-volume within the electroporation chamber).

[0215] Figure 21 A more detailed view of the capping mechanism ( Figures 20A-20C The operation of element 838 (shown in the cross section) and its engagement with cap 885. As shown, cap 885 includes: spring 890 disposed in spring chamber; and extension member 891 mechanically associated with spring 890 such that linear movement of extension member 891 can be transferred to spring 890, and that force stored in spring 890 can be transferred to extension member 891. For example, as shown, extension member 891 may include a flange sized to extend outward and engage with coil of spring 890, while the remainder of extension member 891 extends through internal lumen of spring 890.

[0216] An elongated member 891 extends from the cap 885 and is mechanically coupled to the upper electrode 884. When the cap 885 moves in response to actuation of the capping mechanism 838, and if there is no resistance, the linear motion causes the elongated member 891 and thus the upper electrode 884 to move accordingly. However, once the electrode 884 reaches the top or bottom, the elongated member 891 can no longer move. At this point, continued movement of the cap 885 deforms the spring 890. That is, after the upper electrode 884 has reached the upper or lower terminal position, the spring 890 allows overtravel of the cap 885. Despite actuation drift and / or potential tolerance differences from one electroporation cartridge to the next, the mechanically allowed overtravel ensures that the electrode 884 moves to the correct position.

[0217] As shown, the upper limit of electrode 884 can be defined by a hard stop 892. The upper portion of the electroporation chamber can similarly serve as a hard stop indicating the lower limit of electrode 884. Some embodiments may include one or more spring pins 893 deployed at the upper portion of the electroporation chamber to define a useful "homing point" or position during initial calibration of the instrument. The spring pins 893 are configured such that, assisted by a certain amount of force from the overtravel of spring 890 from the cap 885, the downward force during the initial downward movement of the upper electrode 884 overcomes and presses the spring pins 893 downward, allowing electrode 884 to reach the fully capped position.

[0218] Electroporation chamber sealing mechanism

[0219] Figure 22A and Figure 22B An example of the sealing mechanism 840 is shown in more detail. Providing an effective seal to the electroporation chamber 895 advantageously minimizes bubble formation and associated arc discharge during electroporation. The ability to maintain a higher relative pressure within the electroporation chamber 895 reduces fluid vaporization (i.e., raises the boiling point of the fluid) and also reduces the growth rate of oxygen and hydrogen bubbles formed as a result of the electrolysis of water in the sample fluid.

[0220] The outlet plunger 888 is shown here for illustrative purposes, but the inlet plunger 886 can be configured similarly. When the plunger 888 is in the retracted position, as... Figure 22A As shown, the outlet port 894 is exposed, allowing fluid to exit the electroporation chamber 895. On the other hand, when the plunger 888 is in the forward position, as... Figure 22B As shown, the outlet port 894 is sealed to prevent fluid from flowing out of the electroporation chamber.

[0221] The external 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 advantageously serves as a moving part of the enclosed device and helps to isolate the internal environment to minimize the possibility of particles entering the electroporation chamber 895.

[0222] Figure 22B A detailed view of the outlet plunger 888 shows that the plunger may include a rubber cap 897 for forming a watertight seal with the outlet passage. Alternative embodiments may additionally or alternatively include other sealing components (e.g., one or more O-rings). Movement of the inlet plunger 886 and the outlet plunger 888 can be controlled by corresponding linear actuators / drives 887, 889 (see...). Figures 20A-20C ).

[0223] Figure 22C Another view of the plunger 888 is shown, illustrating a rubber cap 897 at a first end and an attachment 898 at a second end. The attachment is configured to engage with a corresponding outlet actuator 889, such that the movement of the actuator can be mechanically transferred to the plunger 888. The attachment 898 can be configured as a hole, as shown here, or alternatively as a retaining ring, clamp, magnetic coupling, or other suitable mechanical linkage.

[0224] Figure 22D An alternative embodiment of a plunger 1088 with a threaded portion 1098 and configured to convert rotary motion into linear motion is shown. The plunger 1088 may have an O-ring seal 1097, as shown, or alternatively may have a rubber cap (e.g., in other plunger embodiments) or other suitable sealing member. Additional or alternative components for providing controlled linear motion of the plunger may be included. For example, some embodiments may include a track and rail assembly, a worm gear assembly, or a pneumatic or hydraulic actuator.

[0225] Figures 22E-22G Show Figure 22C and Figure 22D Alternative embodiments of the plunger. As shown, the sealing mechanism may include a rotatable sealing mechanism 1000 having a semi-cylindrical head 1002 that is rotatable 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 may engage a rotary piston 1014a operable by the electroporation system, and rotate the head 1002a to an open position (e.g., as shown). Figure 22F and Figure 22G(As shown). Fluid can enter the electroporation chamber 1020 via inlet port 1016 and through inlet 1018a. The opposing sealing mechanism 1000b can be engaged by a complementary rotary piston 1014b and rotated to a closed position (e.g., as shown). Figure 22G As shown, this allows fluid to fill the electroporation chamber 1020. Once filled, the sealing mechanism 1000a can be rotated so that the head 1002a blocks the inlet 1018a. Electroporation can then occur as described herein. By rotating the head 1002b of the sealing mechanism 1000b associated with the outlet 1018b to the open position, the electroporated cells can be removed from the electroporation chamber 1020. The electroporated cells can then be removed through the outlet port 1016b.

[0226] 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, thereby preventing sample leakage and protecting the sterility of consumables. Figure 22F The box 1010 shown may also include the above-mentioned... Figure 5 and Figures 6A-6D The described authentication chip is 1012.

[0227] The electroporation system disclosed herein may include a linear piston, a rotary piston, or a combination thereof. For example, as Figure 22H As shown, the linear piston 1022 is associated with the inlet 1018, while the rotary piston is associated with the outlet 1016. Figure 22H In this configuration, a linear piston is actuated to block the inlet, and a rotary piston is actuated to move the head 1002 to a closed position on the outlet 1016. In this configuration, cells can be retained within the chamber 1020 for electroporation. To discharge from the chamber 1020 and release cells from it, the rotatable sealing mechanism 1000 can be rotated so that the head is oriented in the open position, thereby opening the outlet 1016. Figure 22I As shown.

[0228] In some embodiments, the port (e.g., shown in association with a rotatable sealing mechanism) Figures 22F-22I The inlet and outlet ports (of the chamber) can be angled to allow for more efficient flow into and / or out of the chamber. Figure 22I The right angle formed between the inlets (which can cause bubbles to form and impede or prevent the flow through them) is opposite. Figure 22I The outlet 1016 is angled, which allows the fluid to flow through the outlet more efficiently and reduces the formation of bubbles between the interfaces.

[0229] In some embodiments, the inlet may be connected to a valve (e.g., Figure 22J and Figure 22KThe umbrella valve 1026 shown is associated with this. Under pressure in the first direction (e.g., from the fluid flow rate), as... Figure 22J As indicated by arrow C, the umbrella valve moves to the open position 1024a by flexibly deforming the head portion 1028 away from the hole 1030 formed by the inlet 1032. Figure 22J and Figure 22K As shown, umbrella valve 1026 allows fluid to flow through the inlet in a single direction. When pressure is applied against the head portion 1028 (as shown...), Figure 22K As shown by arrow D in the diagram, the head portion is pressed against the top hole 1030, and the valve is in the closed position 1024b, maintaining a seal at the hole 1030 of the inlet 1032.

[0230] Figure 22L A top view is shown of an exemplary inlet 1032 having a plurality of peripheral holes 1030 formed around a central hole 1034. The size and shape of the central hole 1034 are such that it receives a parasol valve, wherein, when in the closed position, the head portion of the parasol valve extends over the peripheral holes 1030. It should be understood that, when in the closed position, the holes 1030 may have any number or configuration, as long as they are covered and sealed by complementary head portions of the parasol valve.

[0231] Figure 22M An example setup is shown for pressurizing and sealing an outlet port fitted with a check valve (e.g., a miniature valve). Figure 22M As shown, the rotary and translational piston 1014, attached to 1002, is associated with the inlet 1018, while the rotary piston 1014 is associated with the outlet 1016. Figure 22N In this configuration, a rotary and translational piston is actuated to block inlet 1018, and a rotary and translational piston 1014 is actuated to move head 1002 to a closed position on outlet 1016. In this configuration, cells can be retained within chamber 1020 for electroporation. To discharge chamber 1020 and release cells from chamber 1020, a rotatable sealing mechanism 1000 of rotary piston 1014 can be rotated so that the head is oriented in an open position, thereby opening outlet 1016. 1022 is a rubber stopper assembled to 1014.

[0232] Figure 22M The rotatable sealing mechanism 1000 of the rotating and translating piston 1014 is also called a latch. The latch or rotatable sealing mechanism 1000 interacts with an adapter attached to a rotary electric motor. Figure 22N As shown, pin 1000' (e.g., a dowel pin) can act as a cam to guide the movement of rotary piston 1014. According to one embodiment, during operation driven by a rotary electric motor on the instrument, the latch or rotatable sealing mechanism 1000 rotates. Figure 22OAs shown, piston 1014 is latched to latching part 1000 via “I” feature 1015. As latching part 1000 rotates, latching part 1000 drives piston 1014.

[0233] like Figure 22P and Figure 22Q As shown, when the protrusion 1017 is aligned with the pin 1000', the inlet 1018 is blocked, and the electroporation chamber 1020 is sealed. Figure 22R and Figure 22S As shown, the latch 1000 rotates 180 degrees to open the inlet 1018. The piston 1014 is guided to a linear position by the groove and pin 1000'. The piston 1014 and the head 1002 move away from the chamber surface of the electroporation chamber 1020.

[0234] It should be understood that in some embodiments, the electroporation chamber of the cartridge is under pressure (even slightly above atmospheric pressure) during electroporation, and therefore, an umbrella valve (if present) is preferably used on the inlet side, wherein the increased pressure in the chamber will press against the head portion of the valve to facilitate a closed state. If an umbrella valve is used at the outlet, the pressure required to open the valve should ideally be greater than the pressure applied to the valve during electroporation.

[0235] Arc detection and prevention

[0236] Arc discharge negatively impacts both cell viability and transfection efficiency. Generally, samples intended for electroporation are valuable, and therefore, minimizing waste and / or yield loss is desirable. The primary cause of arc discharge is bubble formation. The systems and methods of this disclosure advantageously include features that reduce the occurrence of arc discharge or at least detect the risk of arc discharge and allow for sample recovery before a portion of the sample is wasted as a result of arc discharge.

[0237] Figure 23 A method 900 for predicting the risk of arc discharge during electroporation operations is shown. Method 900 can be executed by a controller 601 (see...). Figure 10B A computer-implemented method that uses data received from one or more communicatively coupled system components and / or executes instructions sent to one or more communicatively coupled system components.

[0238] In the initial step of method 900, the controller determines the initial temperature of a sub-volume within the electroporation chamber of the electroporation cartridge (step 902). The determination of the sub-volume temperature is preferably performed non-invasively. That is, the initial temperature determination is preferably performed without using a temperature probe. It has been found that the use of a temperature probe interferes with the uniformity of the electric field within the electroporation chamber. It has also been found that temperature probes and associated sensing circuitry have a limited lifespan in the high-voltage environment of the chamber. External infrared temperature sensors are also less preferred because these measurements will be affected by the chamber sidewalls.

[0239] In a preferred embodiment, the initial temperature of the sub-volume is determined indirectly by a predetermined correlation between conductivity and temperature. For a given known chamber geometry, the temperature has been estimated by measuring conductivity and using a predetermined correlation for transformation, providing a temperature with an accuracy of ±2°C.

[0240] If this operation has not yet been performed, the controller can then determine the conductivity of the sub-volume within the electroporation chamber of the electroporation cartridge via a conductivity sensor (step 904). The controller can then determine the predicted temperature increase of the sub-volume based on the determined conductivity, the expected pulse voltage, and the expected pulse duration (step 906). The risk of arc discharge is related to the voltage level, temperature, and the total energy of bubble formation and transfer into the sub-volume. As the temperature increases, it is more likely that bubbles will be generated as a portion of the sub-volume undergoes a phase transition from liquid to gas.

[0241] Using known electrical principles, the total energy delivered by a given electrical pulse can be calculated from the voltage used to deliver the pulse, the resistance of the sub-volume between the electrodes (determined by a conductivity sensor), and the expected pulse duration. The law of conservation of energy can then be applied by assuming that all electrical energy applied to the sub-volume is converted into heat (Joule heating effect). The specific heat capacity of the sub-volume can be measured, or alternatively assumed to be substantially similar to that of water. Using the specific heat capacity, the predicted temperature increase can be readily determined.

[0242] The controller can then determine the predicted result temperature of the sub-volume based on the determined initial temperature and predicted temperature (step 908). The controller can then determine whether the result temperature is higher than a predetermined threshold (step 910). The threshold may be, for example, approximately 60°C or approximately 70°C. If determined to be "yes", the controller can send an arc risk alarm (step 912), which may include an alarm sent to an input / output device that the user can interact with, a shutdown command, a process pause that must be manually vetoed, etc. Optionally, the controller can be configured to evacuate the sub-volume back to the mixer container (step 914) to allow further cooling or other intervention steps to occur.

[0243] Conversely, if it is determined that the predicted temperature will not exceed the threshold, the controller can then allow the system to continue the electroporation scheme (step 916).

[0244] Figure 24 A method 940 for preventing arcing in an electroporation chamber is shown. As with method 900, method 940 can be implemented by a controller 601 (see Figure 10). In the illustrated method, the controller determines the conductivity of a sub-volume within the electroporation chamber of the 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 will reduce conductivity to a considerable extent, making them easily detectable by measuring conductivity.

[0245] If the result is "yes", the conductivity of the sub-volume is below a threshold, and the controller can send a bubble detection signal (step 946). As with step 912 of method 900, this can include sending alarms, shutdown commands, process pauses that must be manually rejected, etc., to an input / output device that the user can interact with. Optionally, the controller can be configured to evacuate the sub-volume back to the mixer container to retain the sample (step 948).

[0246] Conversely, if it is determined that the conductivity is not lower than the threshold, the controller can then allow the system to continue the electroporation scheme (step 950).

[0247] Electroporation pulse optimization

[0248] Figure 25 A schematic diagram of an electroporation circuit 930 is shown, comprising a charger 932 electrically connected to an electroporation chamber 995 and a capacitor 934. High-voltage electroporation systems can utilize voltage pulses in the range of approximately 500V to approximately 2,500V, with the expectation of achieving electrical pulses based on such high voltages with repeatability and accuracy.

[0249] Calibration of charger 932 and / or capacitor 934 can reduce some variability. However, as indicated in schematic circuit 930, there will be an inherent amount of inherent circuit resistance 938. This may be attributed, for example, to the resistance of circuit protection components, discharge resistors, or other safety features of the circuit and / or high-voltage switches, for example, when in the open position.

[0250] Furthermore, the resistance of the electroporation chamber 995 will vary depending on the sub-volume properties and temperature. For example, for a 1 mL chamber volume, the resistance can typically range from approximately 500 ohms to 2,000 ohms. This variation in resistance from one sub-volume to another can lead to inconsistent electrical pulses, which in turn result in inconsistent electroporation results.

[0251] Figure 26A method 960, which can be implemented by a computer system (e.g., controller 601) for generating repeatable and consistent electrical pulses through an electroporation chamber, is illustrated. In method 960, the controller can cause the system to determine the conductivity of a sub-volume within the electroporation chamber of the electroporation cartridge via 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 in the electroporation circuit between the capacitor and the electroporation chamber (step 966). This represents the inherent fixed circuit resistance.

[0252] The controller can then charge the capacitor to a voltage level higher than 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 through the electroporation chamber to determine the total circuit resistance, and then charge the capacitor accordingly. Taking into account both the fixed portion of the circuit resistance and the variable resistance of the electroporation chamber allows for a finer-tuned voltage charge, ensuring that the delivered actual electrical pulses remain more consistent from one sample subvolume to the next.

[0253] The method may also optionally include the step of repeatedly tilting the charger voltage based on the previous input voltage and the corresponding previously measured actual voltage applied to the electroporation chamber (step 970). The charger output tolerance can vary by up to ±100V; therefore, instead of simply operating the charger based on the expected voltage level, the controller can apply an iterative voltage compensation method to continuously reduce voltage errors (e.g., to approximately ±5V) without sudden adjustments (which could risk drastic changes).

[0254] The iterative voltage compensation method can be continued by first selecting a target pulse voltage (e.g., 2500V) and offsetting the input voltage by a predetermined amount (e.g., 200V). Then, the actual pulse voltage delivered to the electroporation chamber is measured. In subsequent iterations, the input voltage varies according to the following conditions: Charger input V = (Previous input V / Previous measured output V) x (Target V - Offset) The "offset" can be ramped down 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, and then 5V, and then eventually decrease to 0. These offsets are merely exemplary, and other implementations can ramp down the offset faster or slower depending on application needs and / or preferences. Furthermore, depending on, for example, whether the initially expected charger error results in a charge greater than or less than the target, the offset can be applied as a positive or negative offset.

[0255] Calibration of electroporation chamber filling

[0256] The high-throughput electroporation system described herein comprises many different components that operate in combination with each other. Accordingly, multiple mechanical tolerances will accumulate on top of each other, which can lead to volume variations in the electroporation chamber as it traverses different consumable sets and / or even different sub-volumes within the same consumable set. As mentioned above, the negative consequences of improperly filling the electroporation chamber include underfilling, which causes arcing, and overflow, which results in production losses.

[0257] Figure 27A , Figure 27B and Figure 27C A method for calibrating the filling volume of an electroporation chamber is illustrated graphically. The mixer container 708, the second pump 710, the second flow sensor 712, the pre-cooling module 714, and the electroporation chamber 716 are shown. Figure 27D A flowchart of method 980 is shown. Method 980 can be implemented by a computer system (e.g., controller 601).

[0258] In the first step, the controller can determine the number N of rotations of the drive pump required to fill the pipe deployed between the flow sensor and the electroporation chamber and to completely fill the sample volume of the electroporation chamber (step 982). Figure 27A This is illustrated graphically. The quantity N represents the volume between the flow sensor and the outlet of the electroporation chamber. The filling of the electroporation chamber can be determined using a conductivity sensor. That is, once the conductivity sensor measures the conductivity indicating the fluid flow from the lower electrode to the upper electrode, the electroporation chamber can be determined to be filled.

[0259] The controller can then evacuate the electroporation chamber (step 984). At this point, the controller can cause the drive pump to return the sample to the point upstream of the flow sensor by a fixed number of revolutions k (step 986). Figure 27B This situation is illustrated graphically. The fixed quantity k represents the volume between the upstream point of the flow sensor and the inlet of the electroporation chamber. The controller can then determine the number of pump rotations x required to move the sample from the upstream point of the flow sensor to the point where the flow sensor detects it (step 988). Figure 27C This situation is illustrated graphically. The quantity x represents the volume between the upstream point of the flow sensor and the flow sensor itself. The quantity (kx) thus represents the volume between the flow sensor and the inlet of the electroporation chamber. The volume between the inlet and outlet of the electroporation chamber (i.e., the volume of the electroporation chamber) is therefore equal to N-(kx). The controller can then set this volume as a step volume (step 990) such that the pump performs N-(kx) repetitions between each consecutive sub-volume electroporation.

[0260] Figure 27EAnother method for filling the flow-through electroporation chamber of this disclosure is shown. As shown, in the first step (1050), the first filling of the chamber will stop once the sample fluid touches the top electrode and closes the circuit. This is, for example, when the resistance reading drops from several thousand to about 700 ohms (or in the range of 600-800 ohms).

[0261] In the second step (1060), for the second filling of the chamber and for subsequent fillings, according to the nominal value (e.g., N) rev After coarse filling is completed, the top electrode is capped to read the resistance value.

[0262] At this stage, in the third step (1070), the resistance value (or conductivity) is read. If the resistance or conductivity is within the range determined from the first or any previous filling value (e.g., 1 x 700 ohms), the second filling is complete, and we can proceed to electroporation of the sample (step 1090).

[0263] However, if it exceeds the range determined from the first or any previously filled value (e.g., greater than 1.X times 700 ohms), then proceed to step 1080, which includes: unsealing the top electrode and pressing the additional n rev The volume is then finely filled. Subsequent filling will bring the pump to N. rev + n rev The value of x (where x = number of attempts) in the value.

[0264] Accordingly, in some embodiments of the electroporation system disclosed herein, a controller is included having one or more processors and one or more hardware storage devices, wherein computer-executable instructions are stored on the one or more hardware storage devices, which, when executed by the one or more processors, configure the controller to determine the step volume to be moved between each electroporation event. Calibration is performed by performing at least the following operations: performing a first filling of the electroporation chamber by monitoring the resistance in the electroporation chamber during a first filling period to decrease from several kiloohms to a stable value in the range of approximately 600-800 ohms. The first filling is stopped when this stable voltage range is reached.

[0265] Then, this operation is followed by the following steps: based on the calculated value (e.g., N from the peristaltic pump). rev (For example, the number of revolutions of the peristaltic pump to fill the chamber until a stable resistive volume is reached), coarsely filling the electroporation chamber for the second filling. The step of coarsely filling the electroporation chamber for the second filling is also based on using N... rev Calculation of one or more empirical values ​​other than the standard value (e.g., the inner diameter of the pipe, the number of rollers in the pump, the diameter of the electroporation chamber and / or the height of the electroporation chamber).

[0266] For N rev Rotate the sample several times to coarsely fill the electroporation chamber for the second filling; after the second filling, cap the top electrode and measure the resistance; if the resistance is within the stable range determined from the first filling, the second filling is complete, and we can continue electroporating the sample; if the resistance is not within the stable range determined from the first filling, uncap the top electrode and proceed with an additional n... rev The first fine filling is performed; after capping the top electrode following the first fine filling, the resistance is measured; if the resistance is within a stable range determined from the first filling, the second filling is complete, and we can continue electroporating the sample; otherwise, the fine filling is repeated, and the resistance is measured in steps as described above until the resistance is within a stable range determined from the first filling, for N. rev + n rev x (the number of fine-fill attempts) of pump rotations complete subsequent fillings (third fill, fourth fill, etc.). In some embodiments, the stable resistance is approximately 700 ohms.

[0267] In some of these aspects, the controller is configured to implement a method for determining a calibration step volume to be moved through the system into the flow-through electroporation chamber between each electroporation event, the calibration step volume corresponding to a first volume of the flow-through electroporation chamber. In one embodiment, such a method may include a first filling of the flow-through electroporation chamber until the sample contacts (touches) the top electrode of the electroporation chamber (the first electrode described in some embodiments). At this time (i.e., during the first filling), the electroporation system monitors the resistance in the electroporation chamber as decreasing from several kiloohms to a stable value in the range of approximately 600-800 ohms. The first filling is stopped when this stable resistance value is reached.

[0268] For the second fill (and subsequent fills), the coarse-fill sample volume is derived from a combination of empirical data and theoretical calculations. Accordingly, for the second fill (and subsequent fills), the electroporation system (e.g., a controller within it) determines the number of rotations “N”. rev "," that is, the number of pump rotations required to move a sufficient sample volume to completely fill the electroporation chamber (i.e., the number of rotations calculated from the moment the sample fluid enters the electroporation chamber from a fixed entry point until the sample fluid contacts the top electrode (reaching its sample volume). Additionally, the inner pipe diameter "d" of the pump tubing is determined empirically. i The number of rollers in the pump “n”. In a non-limiting example, the pump may be a peristaltic pump, which in some embodiments may have six rollers (e.g., n = 6).

[0269] Determine N revThe values ​​include measurements of one or more of the following: the number of peristaltic pump rotations required to fill the electroporation chamber from the entry point until it reaches the top electrode (this corresponds to the sample fluid resistance decreasing to 600-800 ohms); the inner diameter "d" of the tubing within the pump. i The number of pump rollers, "n"; the fluid volume per complete pump revolution ("a" µL); the fluid volume per roller movement, "b" µL; the minimum diameter of the electroporation chamber region containing the electroporated sample; and / or the resistance or conductivity of the fluid within the electroporation chamber (using a voltmeter, conductivity sensor, etc.), then N is calculated theoretically. rev .

[0270] Next, the step is to measure the resistance after capping the top electrode following the second fill (and subsequent fills); if the resistance is within the stable range determined from the first fill (i.e., in the range of approximately 600-800 ohms), the filling is complete, and electroporation of the sample can proceed. However, if, after the second fill, the resistance is not within the stable range determined from the first fill (i.e., not in the range of approximately 600-800 ohms), then uncapping the top electrode and performing an additional n... rev Quantity (n of the pump) rev The process involves finely filling the electroporation chamber with a fluid. After fine filling, the top electrode is capped, and the resistance is measured. If the resistance is within a stable range determined by the first filling (i.e., in the range of approximately 600-800 ohms), the second filling is complete, and electroporation of the sample can continue. If not, the fine filling and resistance step size described above are repeated until the resistance is within a stable range determined by the first filling (i.e., in the range of approximately 600-800 ohms). Subsequent fillings (third filling, fourth filling, etc.) are performed for N... rev + n rev This is accomplished by x (the number of attempts at fine filling) of pump rotations. In some embodiments, the stable value of the resistance is approximately 700 ohms, for example, in the range of 650-750 ohms and any value in between.

[0271] In one example embodiment, N revThe calculations are as follows: In one embodiment of the system disclosed herein, a peristaltic pump with six rollers and a conduit with an inner diameter of 2.4 mm are used. Empirical data suggests dispensing 172 µL of fluid per complete pump rotation. Based on empirically determined data, 28 µL of fluid is dispensed per rotational motion obtained by one roller pitch (60 degrees in this case) of the pump. Theoretical calculations are then performed to determine the volume of fluid that the electroporation chamber can hold. The nominal diameter is 6.4 mm (2r, where r = radius of the electroporation chamber), with a lower limit of 6.3 mm and an upper limit of 6.5 mm, along with a height of 30 mm (h) for the electroporation chamber, using the formula πr 2 h was determined to be a nominal sample volume of 965 µL, with a lower limit of 935 µL and an upper limit of 995 µL. Variations in height (h) (with a design tolerance of 0.2 mm) were considered negligible, as the maximum variation they caused was 7 µL. To minimize sample loss due to coarse packing caused by overfilling, a lower limit of 6.3 mm for the cavity diameter was used in the calculations. Based on the empirical data of dividing the calculated 935 µL volume by 172 µL, the number of peristaltic pump rotations was determined to be 5.4; however, to accommodate the convex meniscus, a maximum rounding to 5.5 rotations is recommended. Therefore, in this case, “N” rev "This equals 5.5 rotations. The difference in chamber diameter from 6.3 mm to 6.5 mm results in approximately 30 µL of sample volume for every 0.1 mm diameter change. This is very close to empirical data for dispensing 28 µL of fluid per rotational motion obtained by one roller pitch (60 degrees in this case) from the pump, referred to in the formula as fine filling." rev When the coarse fill is 5.5 revolutions (N) rev When the filling is incomplete, the instrument will read the conductivity. If the filling is incomplete, it will begin fine filling of the "n" symbol. rev "Based on the number of attempts "x", for fine fill, the second fill (and subsequent fills) will be determined by "N". rev + n rev Composed of "x".

[0272] The computer system disclosed herein

[0273] It should be understood that computer systems are increasingly taking on a wide variety of forms. In this specification and claims, the terms “controller,” “computer system,” or “computing system” are broadly defined as any device or system, or a combination thereof, comprising at least one physical and tangible processor and physical and tangible memory capable of having computer-executable instructions that can be executed by the processor. By way of example, and not as a limitation, the terms “computer system” or “computing system” as used herein are intended to include personal computers, desktop computers, laptop computers, tablets, handheld devices (e.g., mobile phones, PDAs, pagers), microprocessor-based or programmable consumer electronics, minicomputers, mainframes, multiprocessor systems, network PCs, distributed computing systems, data centers, message processors, routers, switches, and even devices not traditionally considered computing systems (e.g., wearable devices (e.g., glasses)).

[0274] Memory can take any form and can depend on the nature and form of the computing system. Memory can be physical system memory, which includes volatile memory, non-volatile memory, or a combination of both. The term "memory" may also be used herein to refer to non-volatile mass storage (e.g., physical storage media).

[0275] A computing system also has several structures thereon, generally referred to as "executable components." For example, the memory of a computing system may include executable components. The term "executable component" is a name used by those skilled in the art in the field of computing for a structure that may be software, hardware, or a combination thereof.

[0276] For example, when implemented in software, those skilled in the art will understand that the structure of an executable component can include software objects, routines, methods, etc., executable by one or more processors on a computing system, regardless of whether the executable component exists in the heap of the computing system or on a computer-readable storage medium. The structure of the executable component exists on a computer-readable medium in such a form that, when executed by one or more processors of the computing system, it is operable to cause the computing system to perform one or more functions (e.g., the functions and methods described herein). This structure can be directly computer-readable by the processor—as if the executable component were binary. Alternatively, the structure can be structured to be interpretable and / or compileable (whether in a single-level or multi-level context)—to generate a binary that is directly interpretable by the processor.

[0277] The term "executable component" is also well understood by those skilled in the art to include structures implemented exclusively or almost exclusively within hardware logic components, such as field-programmable gate arrays (FPGAs), application-specific integrated circuits (ASICs), program-specific standard products (ASSPs), system-on-a-chip (SoCs), complex programmable logic devices (CPLDs), or any other special-purpose circuitry. Accordingly, the term "executable component" is used for structures well understood by those skilled in the art of computing, regardless of whether they are implemented in software, hardware, or a combination thereof.

[0278] The terms “component,” “service,” “engine,” “module,” “control,” “generator,” etc., may also be used in this specification. As used in this specification, and in this case, these terms—whether expressed with or without modifiers—are intended to be synonymous with the term “executable component,” and therefore have a structure well understood by one of ordinary skill in the art of computing.

[0279] While not all computing systems require a user interface, in some embodiments, the computing system includes a user interface for use in passing information to / from a user. The user interface may include output and input mechanisms. The principles described herein are not limited to precise output or input mechanisms, as this will depend on the nature of the device. However, output mechanisms may include, for example, speakers, displays, haptic outputs, projections, holograms, etc. Examples of input mechanisms may include, for example, microphones, touchscreens, projections, holograms, cameras, keyboards, styluses, mice, or other pointing inputs, any type of sensor, etc.

[0280] Accordingly, the embodiments described herein may include or utilize dedicated or general-purpose computing systems. The embodiments described herein also include physical and other computer-readable media for carrying or storing computer-executable instructions and / or data structures. Such computer-readable media may be any available media accessible to a general-purpose or dedicated computing system. Computer-readable media storing computer-executable instructions are physical storage media. Computer-readable media carrying computer-executable instructions are transmission media. Therefore, 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.

[0281] Computer-readable storage media include RAM, ROM, EEPROM, solid-state drives (“SSDs”), flash memory, phase-change memory (“PCM”), CD-ROMs or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other physical and tangible storage medium that can store desired program code in the form of computer-executable instructions or data structures and can be accessed and executed by a general-purpose or special-purpose computing system to perform the functions disclosed in this invention. For example, computer-executable instructions may be embodied on one or more computer-readable storage media to form a computer program product.

[0282] The transmission medium may include networks and / or data links that can carry desired program code in the form of computer-executable instructions or data structures and can be accessed and executed by general-purpose or special-purpose computing systems. Combinations of the above should also be included within the scope of computer-readable media.

[0283] Furthermore, upon arrival at various computing system components, program code in the form of computer-executable instructions or data structures can be automatically transferred from the transmission medium to the storage medium (or vice versa). For example, computer-executable instructions or data structures received via a network or data link can be buffered in RAM within a network interface module (e.g., a "NIC") and then eventually transferred to the computing system RAM and / or less volatile storage at the computing system. Therefore, it should be understood that storage media can be included in computing system components that also (or even primarily) utilize the transmission medium.

[0284] Those skilled in the art will further understand that a computing system may also include communication channels that allow the computing system to communicate with other computing systems via, for example, a network. Accordingly, the methods described herein can be practiced in networked computing environments with many types of computing systems and computing system configurations. The disclosed methods can also be practiced in distributed system environments, where both local and / or remote computing systems linked together via a network (either via hardwired data links, wireless data links, or a combination of hardwired and wireless data links) perform tasks. In a distributed system environment, processing, memory, and / or storage capacity can also be distributed.

[0285] Those skilled in the art will also understand that the disclosed methods can be practiced in a cloud computing environment. A cloud computing environment can be distributed, but is not required to be. When distributed, a cloud computing environment can be internationally distributed within an organization and / or make components owned across multiple organizations. In this specification and the appended claims, “cloud computing” is defined as a model that enables on-demand network access to a shared pool of configurable computing resources, such as networks, servers, storage, applications, and services. The definition of “cloud computing” is not limited to any other numerous advantages that can be obtained from this model when properly deployed.

[0286] Cloud computing models can be composed of various features, such as on-demand self-service, widespread network access, resource pooling, rapid elasticity, and measurable services. They can also take the form of various service models, such as Software as a Service (“SaaS”), Platform as a Service (“PaaS”), and Infrastructure as a Service (“IaaS”). Furthermore, cloud computing models can be deployed using different deployment models, such as private clouds, community clouds, public clouds, and hybrid clouds.

[0287] List of abbreviations for the defined terms

[0288] To aid in understanding the scope and content of this written description and the appended claims, a few 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.

[0289] As used herein, the terms “approximately,” “about,” and “substantially” refer to a quantity or condition that is close to a specific stated quantity or condition that still performs the desired function or achieves the desired result. For example, the terms “approximately,” “about,” and “substantially” can refer to a quantity or state that deviates from a specifically stated quantity or condition by less than 10%, or less than 5%, or less than 1%, or less than 0.1%, or less than 0.01%.

[0290] As used herein, the term "electroporation" is intended to encompass the process of exposing a cell to an electric field (typically, a short-duration, high-voltage electric field) to induce an electroporation target to be taken up from the surrounding electroporation medium into the electroporated cell. The cell can be any living cell, and it should be understood that the electroporation systems and methods disclosed herein can be used with both prokaryotic and / or eukaryotic cells. As known to those skilled in the art, the process of electroporating a target into a prokaryote (e.g., bacteria) is referred to as "transformation," while the process of electroporating a target into a eukaryote (e.g., primary cells or cell lines) is typically referred to as "transfection." For the purposes of this disclosure, unless otherwise specifically stated, the terms "transformation" and "transfection" are interchangeable and are independent of the type or species of organism being transformed. Accordingly, the term "electroporation" or its forms are intended to encompass the transformation / transfection of living cells (prokaryotic or eukaryotic) with an electroporation target.

[0291] As used herein, the term "electroporation target" is intended to be understood as any molecule, compound, or substance intended to be introduced into target cells via electroporation. By way of example, and not limitation, an electroporation target may include a protein, peptide, nucleic acid, drug, or other compound. Proteins may include purified, folded, or unfolded proteins having native, mutant, or engineered sequences, and peptides are understood to include any string of amino acids and may include portions of a protein sequence. Nucleic acids include those sequences derived from biological or environmental sources and may be one or more of genes, regulatory sequences, intergenetic sequences, genomic DNA, plasmid DNA, cDNA, or any of the various known forms of RNA. As described herein, an electroporation target may take any of the foregoing forms, but in a preferred embodiment, the electroporation target constitutes a nucleic acid for transfecting primary cells or cell lines.

[0292] As used herein, the term "primary cell" is intended to refer to cells isolated directly from the tissues or fluids of an organism using standard cell culture techniques, which have a finite lifespan and are limited in their ability to expand in vitro without intervention. Primary cells are typically not associated with homogeneous genotype and phenotypic traits. In contrast, the term "cell line" is intended to include those cells that have acquired homogeneous genotype and phenotypic traits (e.g., through continuous passage over a long period). As is 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 through genetic mutation or artificial modification.

[0293] As used herein, the term "sealing member" is intended to include any structural element or mechanism known in the art that facilitates, forms, or performs actions 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 cartridge to function as a functionally closed environment. O-rings or similar gaskets provided within the scope of this disclosure may include or be made of any suitable material known in the art, including, by way of example and not limitation, non-conductive materials (e.g., rubber or silicone).

[0294] Various aspects of this disclosure, including devices, systems, and methods, may be illustrated with reference to one or more embodiments or implementations that are essentially exemplary. As used herein, the term "exemplary" means "serving as an example, instance, or illustration" and is not necessarily to be construed as preferred or advantageous over other embodiments disclosed herein. Furthermore, references to "implementations" of this disclosure or invention include specific references to one or more embodiments thereof, and vice versa, and are intended to provide illustrative examples rather than to limit the scope of the invention, which is indicated by the appended claims rather than by the following description.

[0295] As used in the specification, unless otherwise implied or expressly understood or stated, words appearing in the singular form encompass their plural equivalents, and words appearing in the plural form encompass their singular equivalents. Therefore, it should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “described” include plural indicators unless the context clearly specifies otherwise. For example, a reference to a singular indicator (e.g., “widget”) includes one, two, or more indicators unless otherwise implied or expressly understood or stated. Similarly, a reference to multiple indicators should be interpreted as including a single indicator and / or multiple indicators unless the content and / or context clearly specify otherwise. For example, a reference to a plural indicator (e.g., “widget”) does not necessarily require multiple such indicators. Conversely, it should be understood that one or more indicators are contemplated herein, independent of the inferred number of indicators, unless otherwise stated.

[0296] As used herein, directional terms (e.g., “top,” “bottom,” “left,” “right,” “up,” “down,” “upper,” “lower,” “proximal,” “farthest,” “adjacent,” etc.) are used only to indicate relative directions and are not otherwise intended to limit the scope of this disclosure and / or the claimed invention.

[0297] Summary of exemplary embodiments: The following is an item-by-item list of some non-limiting exemplary embodiments of this disclosure: 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 a opposite distal end of the electroporation chamber, wherein at least one of the first electrode or the second electrode is movable between a capped position for electroporation and an uncapped position for loading a sample, and / or the electroporation cartridge is configurable between a sealed state and an unsealed state.

[0298] 2. The electroporation cartridge as described in item 1, wherein the elongated body comprises or is made of one or more of non-conductive plastic, glass, or ceramic, and is configured to receive cell-containing fluid to be electroporated within the electroporation chamber defined by the elongated body.

[0299] 3. The electroporation chamber as described in item 2, wherein the electroporation chamber comprises or is made of glass and / or ceramic.

[0300] 4. The electroporation chamber as described in item 2 or 3, wherein the electroporation chamber comprises or is made of polycarbonate or other non-conductive radiation-stabilized plastic.

[0301] 5. The electroporation chamber as described in any one of items 1-4, wherein at least a portion of the electroporation chamber tapers gradually between the first electrode and the second electrode.

[0302] 6. The electroporation chamber as described in item 5, wherein the tapering portion of the electroporation chamber does not substantially interfere with the generation of a uniform electric field between the first electrode and the second electrode.

[0303] 7. The electroporation chamber as described in any one of items 1-6, wherein the electroporation chamber comprises a uniform cross-section along the length of the reaction chamber.

[0304] 8. The electroporation cartridge as described in item 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 deployed between the first electrode and the second electrode.

[0305] 9. The electroporation chamber as described in item 7 or item 8, wherein the electroporation chamber comprises a cylindrical cavity and the uniform cross-section comprises a circle.

[0306] 10. The electroporation chamber as described in any one of items 1-7, further comprising a proximity sidewall defined between the proximity opening of the elongated body and the inflection point of the sidewall defining the electroporation chamber, wherein the proximity sidewall narrows from a first diameter defined by the proximity opening to a second smaller diameter defined at a position distal from the inflection point.

[0307] 11. The electroporation cartridge of any one of items 1-10, wherein the first electrode includes a spherical extension having a substantially flat distal surface.

[0308] 12. The electroporation cartridge of any one of items 1-10, wherein the first electrode includes a spherical extension having a distal surface with a convex or angled profile.

[0309] 13. The electroporation cartridge as described in item 11 or 12, wherein the spherical extension is separated from the base portion of the first electrode by a narrow rod.

[0310] 14. The electroporation cartridge as described in any one of items 11-13, wherein the spherical extension is operable to: displace one or more air bubbles associated with the cell-containing fluid to be electroporated in the electroporation chamber while the first electrode is fixed in the electroporation chamber.

[0311] 15. The electroporation cartridge of any one of items 1-14 further includes a sealing member disposed between the first electrode and the proximal surface of the elongated body, the sealing member being operable to form a fluid-tight connection between the first electrode and the elongated body.

[0312] 16. The electroporation cartridge as described in item 15, wherein the first electrode includes a first electrode flange, and the elongated body includes a proximal body flange oriented in a plane substantially parallel to the first electrode flange, and the sealing member is disposed between the first electrode flange and the proximal body flange to form the fluid-tight connection therebetween.

[0313] 17. The electroporation cartridge as described in any one of items 1-16, wherein the first electrode is operable to: configure the electroporation cartridge between a sealed state and an unsealed state.

[0314] 18. The electroporation cartridge as described in item 17, wherein the first electrode is operable to: configure the electroporation cartridge between a sealed state and an unsealed state without the need for an additional removable capping workpiece.

[0315] 19. The electroporation cartridge as described in item 17 or 18, wherein the first electrode is a removable cap.

[0316] 20. The electroporation cartridge as described in any one of items 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 elongated body.

[0317] 21. The electroporation chamber as described in any one of items 1-20, wherein the diameter of the proximal end of the second electrode is substantially equal to the cross-section of the electroporation chamber.

[0318] 22. The electroporation cartridge of any one of items 1-21, wherein the second electrode includes a protrusion extending from the distal end of the elongated body into the electroporation chamber.

[0319] 23. The electroporation cartridge as described in item 22, wherein the perimeter of the protruding portion includes a complementary shape to the inner surface of the elongated body defining the electroporation chamber.

[0320] 24. The electroporation cartridge as described in item 22 or 23, wherein the second electrode further includes a first sealing member disposed between the second electrode and the distal surface of the elongated body, the first sealing member being operable to: form a fluid-tight connection between the second electrode and the distal surface of the elongated body.

[0321] 25. The electroporation cartridge as described in item 24, wherein the second electrode includes an electrode flange, and the elongated body includes a remote body flange oriented in a plane substantially parallel to the electrode flange, and the sealing member is disposed between the electrode flange and the remote body flange to form the fluid-tight connection therebetween.

[0322] 26. The electroporation chamber of any one of items 22-25, wherein the second electrode further comprises a second sealing member disposed around the protrusion of the second electrode and positioned distal from the proximal surface of the second electrode, the second sealing member being operable to form a fluid-tight connection between the protrusion and the inner surface of the elongated body defining the electroporation chamber.

[0323] 27. The electroporation cartridge as described in any one of items 22-26, wherein the proximal surface of the second electrode comprises a flat and uniform surface.

[0324] 28. The electroporation chamber as described in any one of items 22-27, wherein the proximal surface of the second electrode is orthogonal to the longitudinal axis of the electroporation chamber.

[0325] 29. The electroporation cartridge as described in any one of items 1-28 further includes a retaining pin associated with the second electrode and configured to secure the second electrode to the elongated body.

[0326] 30. The electroporation cartridge as described in item 29, wherein the second electrode defines a channel configured in size and shape to receive the retaining pin, the channel being aligned with and configured to receive the retaining pin by a pair of holes defined by the sidewall of the elongated body, thereby securing the second electrode in a fixed position relative to the elongated body.

[0327] 31. The electroporation cartridge as described in item 30, wherein the channel is formed to pass through the central region of the protruding portion of the second electrode located distal to the first sealing member and / or the second sealing member.

[0328] 32. The electroporation cartridge as described in any one of items 1-31, 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.

[0329] 33. The electroporation cartridge as described in any one of items 1-32 further includes a volume-reducing sleeve configured in terms of size and shape to fit within the electroporation chamber.

[0330] 34. The electroporation cartridge as described in item 33, wherein the volume-reducing sleeve defines a secondary electroporation chamber having a smaller volume than the electroporation chamber.

[0331] 35. The electroporation cartridge as described in item 33 or 34, wherein the volume-reducing sleeve includes a distal opening configured to abut the second electrode when fixed within the electroporation chamber.

[0332] 36. The electroporation cartridge as described in any one of items 33-35, wherein the volume-reducing sleeve includes a vent hole deployed adjacent to the proximal end of the volume-reducing sleeve, the vent hole being configured to allow air to pass through it during the introduction of the volume-reducing sleeve into or out of the electroporation chamber, thereby preventing the formation of a vacuum between the sub-electroporation chamber and the electroporation chamber, thereby allowing cell-containing fluid of electroporation to fill the sub-electroporation chamber upon introduction of the volume-reducing sleeve and to exit the sub-electroporation chamber upon removal of the volume-reducing sleeve.

[0333] 37. The electroporation cartridge as described in any one of items 33-36, wherein the volume-reducing sleeve includes a radial sealing member configured to secure the volume-reducing sleeve within the electroporation chamber.

[0334] 38. The electroporation cartridge as described in item 37, wherein the radial sealing member forms a fluid-tight seal with the sidewall defining the electroporation chamber to prevent leakage of cellular fluid within the secondary electroporation chamber through the distal opening of the volume-reduced sleeve.

[0335] 39. The electroporation cartridge as described in any one of items 33-38, wherein the first electrode is configured to selectively associate with the volume-reducing sleeve and form a fluid-tight seal with the volume-reducing sleeve.

[0336] 40. The electroporation cartridge as described in any one of items 33-39, wherein a space is defined between the outer surface of the volume-reducing sleeve and the inner sidewall of the elongated body to form a fluid overflow space, which is configured to receive the overflow volume displaced by the first electrode when the electroporation chamber is sealed.

[0337] 41. The electroporation cartridge as described in any one of items 1-40, further comprising a fluid overflow space associated with a proximal region of the electroporation chamber and configured to receive an overflow volume displaced by the first electrode when the electroporation chamber is sealed.

[0338] 42. The electroporation cartridge as described in any one of items 1-41 further includes one or more springs longitudinally disposed on the proximal side of the elongated body and configured to position the first electrode in the unsealed position at a distance from the electroporation chamber.

[0339] 43. The electroporation cartridge as described in item 42, wherein the electroporation cartridge in the capped position configures the one or more springs to be compressed, and configures the first electrode to be deployed in an electrode chamber and operable to electroporate a cell-containing fluid deployed therein.

[0340] 44. The electroporation cartridge as described in any one of items 1-43, wherein the electroporation cartridge comprises a flow-through electroporation cartridge.

[0341] 45. The electroporation cartridge as described in item 44, further comprising a port associated with the first electrode, the port defining a lumen within the first electrode such that the lumen is fluidly connected to the electroporation chamber.

[0342] 46. ​​The electroporation chamber as described in item 44 further includes a port associated with the proximal portion of the elongated body, the port being configured to: discharge displaced air from the electroporation chamber when the electroporation chamber is being filled, and / or introduce filtered or purified air into the electroporation chamber when the electroporation chamber is being emptied.

[0343] 47. The electroporation chamber as described in any one of items 44-46, further comprising a chamber inlet and a chamber outlet, each of the chamber inlet and the chamber outlet being fluidly connected to the electroporation chamber.

[0344] 48. The electroporation cartridge as described in item 47, wherein one or more of the chamber inlet or chamber outlet are disposed above the proximal surface of the second electrode.

[0345] 49. The electroporation cartridge as described in item 47 or claim 48, wherein the lumen of the chamber inlet and / or chamber outlet is substantially parallel to the proximal surface of the second electrode.

[0346] 50. The electroporation chamber as described in any one of items 47-49, wherein one or more of the chamber inlet or chamber outlet are associated with a plug and / or a valve to control the inward flow of the cell-containing fluid to be electroporated into the electroporation chamber, and / or to control the outward flow of the cell-containing fluid to be electroporated from the electroporation chamber.

[0347] 51. The electroporation cartridge as described in any one of items 44-50, further comprising a fluid overflow space associated with the first electrode and / or the elongated body, the fluid overflow space being configured to receive overflow volume displaced from the electroporation chamber when the electroporation chamber is filled.

[0348] 52. The electroporation chamber as described in any one of items 44-50 further includes a fluid overflow space associated with a sealing cap, the fluid overflow space being configured to receive overflow volume displaced from the electroporation chamber when the electroporation chamber is sealed by the sealing cap.

[0349] 53. An electroporation system configured to provide flow-through electroporation of a sample, the electroporation system comprising: a modular housing having a plurality of chambers for holding and arranging a plurality of electroporation system assemblies, the electroporation system assemblies comprising: one or more pumps configured to move a sample through the system; an electroporation chamber configured to receive a flow-through electroporation cartridge configured to hold a sub-volume of sample within the electroporation chamber for electroporation of the sub-volume; and a conduit having an inlet end and an outlet end, the conduit being routed through the housing to fluidly connect the plurality of electroporation system assemblies.

[0350] 54. The electroporation system as described in item 53 further includes a bag compartment configured to receive and support an input bag and / or an output bag.

[0351] 55. The electroporation system as described in item 54, wherein the compartment includes an insertion portion slidably connected to the compartment to allow selective extraction from or enclosing within the housing.

[0352] 56. The electroporation system as described in item 55, wherein the compartment includes one or more magnetic latches for holding the compartment in a closed position within the housing.

[0353] 57. The electroporation system of any one of items 53-56 further includes a cooling module that is in thermal contact with the electroporation chamber and is configured to regulate the temperature of the electroporation chamber.

[0354] 58. The electroporation system as described in item 57, wherein the cooling module comprises a ceramic block.

[0355] 59. The electroporation system as described in item 57 or 58, wherein the cooling module is cooled via thermoelectric cooling.

[0356] 60. The electroporation system of any one of items 53-59 further includes a mixer container disposed downstream of the inlet and upstream of the electroporation cartridge, the mixer container including a mixing element configured to provide mixing to a portion of the sample contained within the mixing container.

[0357] 61. The electroporation system as described in item 60, wherein the mixing element comprises a mixing blade.

[0358] 62. The electroporation system as described in item 60 or 61, wherein the mixer container includes a mixer magnet assembly mechanically coupled to the mixing element, the mixer magnet assembly being deployed so as not to contact the portion of the sample contained within the mixer container.

[0359] 63. The electroporation system as described in item 62 further includes 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 with the mixer magnet assembly.

[0360] 64. The electroporation system as described in item 62 or 63, wherein the mixer container includes a cover, and wherein the mixer magnet assembly is deployed at or near the cover.

[0361] 65. The electroporation system of any one of items 60-64 further includes a sample input assembly configured to facilitate the transfer of a sample between the input and the mixer container, the sample input assembly comprising: a main conduit section disposed between the input and the mixer container; and an intermediate conduit section pneumatically coupled to and extending therefrom the main conduit section to a terminal end having an air inlet, the intermediate conduit section thereby allowing air to be delivered to the main conduit section when there is a sufficient pressure drop in the main conduit section.

[0362] 66. The electroporation system as described in item 65, wherein the terminal end of the intermediate conduit section is coupled to an air container having a variable volume, and wherein the sample input assembly is configured to detect a threshold decrease in the variable volume to determine that the sample has moved into the mixer container.

[0363] 67. The electroporation system as described in item 66, wherein the sample input sensor assembly includes 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 the threshold reduction of the variable volume being detected as a result of movement of the plunger.

[0364] 68. The electroporation system of any one of items 53-67 further includes a chamber sealing assembly operatively coupled to the electroporation chamber and configured to regulate the pressure within the electroporation chamber during electroporation and thereby limit bubble formation.

[0365] 69. The electroporation system as described in item 68, wherein the chamber sealing assembly includes one or more linear actuators configured to advance a plunger toward or retract a plunger from the electroporation chamber, thereby regulating the pressure within the electroporation chamber.

[0366] 70. The electroporation system of any one of items 53-69 further includes a precooling assembly disposed upstream of the electroporation chamber and configured to cool the subvolume of the sample prior to electroporation of the subvolume.

[0367] 71. The electroporation system as described in item 70, wherein the precooling assembly includes a cooling block and a pipe section disposed within or adjacent to the cooling block.

[0368] 72. The electroporation system as described in item 70 or 71, wherein the cooling block of the pre-cooled assembly is cooled via thermoelectric cooling.

[0369] 73. The electroporation system of any one of items 70-72, wherein the precooling assembly includes a flexible biasing element that biases the cooling block against the pipe section disposed adjacent to the cooling block.

[0370] 74. The electroporation system of any one of items 53-73 further includes at least one flow sensor, wherein the at least one flow sensor is deployed between the mixer container and the electroporation chamber.

[0371] 75. The electroporation system as described in item 74, wherein the flow sensor is an ultrasonic sensor.

[0372] 76. The electroporation system as described in item 74 or 75, wherein the flow sensor includes an actuable trigger that, when actuated, positions a corresponding pipe section within the flow sensor for detecting flow through the pipe section.

[0373] 77. The electroporation system of any one of items 53-76 further includes one or more flow indicators that route the corresponding pipe section to a location on the outside of the housing so that the flow through the pipe section can be visualized.

[0374] 78. The electroporation system as described in any one of items 53-77, wherein the housing includes one or more handles.

[0375] 79. The electroporation system as described in item 78, wherein the one or more handles include a handle having a latch portion configured to engage with a dashboard to attach the dashboard to the housing.

[0376] 80. The electroporation system of any one of items 53-79 further includes an electroporation cartridge attachment feature coupled to the electroporation cartridge, the attachment feature including a flexible biasing element biasing the electroporation cartridge toward a cooling module integrated within the housing.

[0377] 81. The electroporation system of any one of items 53-80 further includes a capping mechanism configured to engage with the electroporation cartridge, wherein the electroporation cartridge includes a first electrode and a second electrode disposed at opposite ends of the electroporation chamber, wherein at least one of the first electrode or the second electrode is engageable with the capping mechanism and is movable between a capped position for electroporation and an uncovered position for discharge as a result of actuating the capping mechanism.

[0378] 82. The electroporation system as described in item 81, wherein the electroporation cartridge includes a spring mechanism that allows the capping mechanism to overtravel relative to the displacement of the electrode as a result of actuating the capping mechanism.

[0379] 83. The electroporation system of any one of items 53-81, wherein the electroporation chamber includes a chamber inlet and a chamber outlet, wherein the chamber outlet is coupled to an outlet plunger movable between an advance position preventing the subvolume from flowing out of the electroporation chamber and a retracted position allowing the subvolume to flow out of the electroporation chamber.

[0380] 84. The electroporation system of any one of items 53-83, wherein the electroporation cartridge comprises one or more bellows structures configured to enclose a movable component of the electroporation chamber.

[0381] 85. The electroporation system of any one of items 53-84, wherein the electroporation chamber comprises a flow-through electroporation cartridge as claimed in any one of claims 44-52.

[0382] 86. The electroporation system of any one of items 53-85 further includes an electroporation assembly electrically coupled to the electroporation chamber, the electroporation assembly including a conductivity sensor for measuring conductivity across the electroporation chamber, the electroporation assembly being communicatively coupled to a controller having one or more processors and one or more hardware storage devices.

[0383] 87. The electroporation system of claim 86, wherein computer-executable instructions are stored on the one or more hardware storage devices, the computer-executable instructions, when executed by the one or more processors, configuring the controller to perform at least the following operations: determining the conductivity of the sub-volume within the electroporation chamber via the conductivity sensor; determining a voltage drop across the electroporation chamber based on the determined conductivity; and charging a capacitor in the electroporation circuit accompanying the electroporation chamber to a voltage level greater than the determined voltage drop across the electroporation chamber to compensate for other voltage drops between the capacitor and the electroporation chamber.

[0384] 88. An electroporation system as described in Item 86 or 87, wherein computer-executable instructions are stored on the one or more hardware storage devices, which, when executed by the one or more processors, configure the controller to perform at least the following operations: determine the conductivity of the sub-volume within the electroporation chamber via the conductivity sensor; determine a predicted temperature increase of the sub-volume based on the determined conductivity, a predicted pulse voltage, and a predicted pulse duration; and if the predicted temperature increase results in a temperature of the sub-volume greater than a predetermined threshold temperature, perform one or more of the following operations: send an arc risk alarm; and / or withdraw the sample sub-volume to retain the sample sub-volume, and / or adjust cooling accordingly to reduce the temperature of the electroporation chamber.

[0385] 89. The electroporation system as described in item 88, wherein the computer-executable instructions further configure the controller to determine the initial temperature of the sub-volume within the electroporation chamber by correlating the determined conductivity with temperature.

[0386] 90. The electroporation system of any one of items 86-89, wherein computer-executable instructions are stored on the one or more hardware storage devices, which, when executed by the one or more processors, configure the controller to perform at least the following operations: determining the conductivity of the sub-volume in the electroporation chamber by means of the conductivity sensor; and evacuating the sub-volume from the electroporation chamber if the determined conductivity falls below a predetermined threshold indicating the presence of one or more bubbles in the electroporation chamber.

[0387] 91. The electroporation system of any one of items 53-90 further includes a controller having one or more processors and one or more hardware storage devices, wherein computer-executable instructions are stored on the one or more hardware storage devices, which, when executed by the one or more processors, configure the controller to repeatedly tilt the charger voltage based on a previous input voltage applied to the electroporation chamber and a corresponding previously measured actual voltage.

[0388] 92. The electroporation system of any one of items 74-91 further includes a controller having one or more processors and one or more hardware storage devices, wherein computer-executable instructions are stored on the one or more hardware storage devices, the computer-executable instructions, when executed by the one or more processors, configuring the controller to: determine a step volume of movement of the system between each electroporation event, the calibration being performed by performing at least the following: determining a number N of rotations of a drive pump required to move enough to fill the conduit deployed between the flow sensor and the electroporation chamber and to completely fill the sample volume of the electroporation chamber, the number N representing the volume between the flow sensor and the outlet of the electroporation chamber. The process involves: emptying the electroporation chamber; returning the sample to a point upstream of the flow sensor by the drive pump through a fixed number of rotations k, where k represents the volume between the point upstream of the flow sensor and the inlet of the electroporation chamber; determining the number x of rotations x of the drive pump required to move the sample from the point upstream of the flow sensor to the flow sensor, where x represents the volume between the point upstream of the flow sensor and the flow sensor, and the number (kx) represents 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 as N-(kx), and setting this volume as the step volume.

[0389] 93. The electroporation system of any one of items 53-92 further includes a safety gate configured to mechanically open the electroporation circuit to prevent voltage discharge while the safety gate is open.

[0390] 94. The electroporation system of any one of items 53-93, 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.

[0391] 95. The electroporation system of any one of items 74-91 further includes a controller having one or more processors and one or more hardware storage devices, wherein computer-executable instructions are stored on the one or more hardware storage devices, which, 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, wherein calibration is performed by performing at least the following operations: The first filling of the electroporation chamber is performed by monitoring the resistance of the sample fluid in the electroporation chamber during the first filling process from several kiloohms to a stable value in the range of about 600-800 ohms, and stopping the first filling when the stable resistance value is reached. Based on calculated values, such as N from the peristaltic pump. rev The electroporation chamber is coarsely filled for a second filling. After the second filling and the top electrode is capped, the resistance is measured; If the resistance is within a stable value range determined from the first filling, then the second filling is complete, and the sample can continue to be de-perforated. If the resistance is not within the stable value range determined from the first fill, then unseal the top electrode and add an additional n. rev Quantity is used for precise filling; After the fine filling is completed and the top electrode is capped, the resistance is measured. If the resistance is within a stable value range determined from the first filling, then the second filling is complete, and the sample can continue to be de-perforated. If not, repeat the fine filling and measure the resistance step by step until the resistance is within the stable value range determined from the first filling. For the number of pump rotations N rev + n rev x (where x equals the number of attempts at fine filling), complete the subsequent filling (third filling, fourth filling, etc.).

[0392] 96. The system as described in item 95, wherein the stable value of the resistor is approximately 700 ohms.

[0393] 97. The system as described in item 95, wherein the step of coarsely filling the electroporation chamber for a second filling is further based on calculations using one or more parameters, such as the inner diameter of the pipe, the number of rollers in the pump, the diameter of the electroporation chamber, and / or the height of the electroporation chamber.

[0394] in conclusion

[0395] The terminology and expressions used herein are for descriptive purposes and not for limitation, and there is no intention to use these terms and expressions to exclude any equivalents of the features shown and described or any parts thereof, but it is recognized that various modifications are possible within the scope of the invention as enumerated. Therefore, it should be understood that while the invention has been disclosed in part by way of preferred embodiments, exemplary embodiments, and optional features, modifications and variations can be made by those skilled in the art using the concepts disclosed herein, and such modifications and variations are considered to be within the scope of the invention as defined in the appended entries. The specific embodiments provided herein are examples of useful embodiments of the invention and various changes and / or modifications to the inventive features shown herein, and additional applications of the principles shown herein to those skilled in the art and with knowledge of this disclosure may be made with respect to the illustrated embodiments without departing from the spirit and scope of the invention as defined in the entries, and should be considered to be within the scope of this disclosure.

[0396] It should also be understood that systems, devices, products, kits, methods, and / or processes according to specific embodiments of this disclosure may include, incorporate, or otherwise include the properties or features (e.g., components, members, elements, parts, and / or portions) described in other embodiments disclosed and / or described herein. Accordingly, various features of specific embodiments may be compatible with, combined with, included in, and / or incorporated into other embodiments of this disclosure. Therefore, the disclosure of specific features with respect to specific embodiments of this disclosure should not be construed as limiting the application or inclusion of said features to the specific embodiments. Rather, it should be understood that other embodiments may also include said features, members, elements, parts, and / or portions without departing from the scope of this disclosure.

[0397] Furthermore, unless a feature is described as another feature to be combined with, any feature herein may be combined with any other feature of the same or different embodiments disclosed herein. Moreover, to avoid obscuring aspects of the exemplary embodiments, various well-known aspects of the illustrative systems, methods, apparatuses, etc., have not been described in particular detail herein. However, such aspects are contemplated herein.

[0398] All references cited in this application are therefore incorporated in their entirety to the extent that they do not conflict with the disclosures herein. It will be apparent to those skilled in the art that methods, apparatuses, apparatus elements, materials, processes, and techniques other than those specifically described herein can be applied to the practice of the invention as broadly disclosed herein without the need for excessive experimentation. All well-known functional equivalents of the methods, apparatuses, apparatus elements, materials, processes, and techniques specifically described herein are intended to be covered by this disclosure.

[0399] When groups of materials, compositions, ingredients, or compounds are disclosed herein, it should be understood that all individual members of these groups and all their subgroups are disclosed separately. When Markush groups or other groupings are used herein, all individual members of the group, as well as all possible combinations and subcombinations of the group, are intended to be included separately in this disclosure. Unless otherwise stated, every chemical formula and combination of components described or illustrated herein can be used to practice the invention. Whenever a range (e.g., a temperature range, a time range, or a composition range) is given in the specification, all intermediate ranges and subranges, as well as all individual values ​​included in the given range, are intended to be included in this disclosure.

[0400] All changes to the meaning and scope of equivalence derived from an entry should be covered within its scope.

[0401] Example

[0402] The following examples are provided to illustrate implementations of the various exemplary embodiments disclosed herein. While general paradigms or method flows are illustrated in the following examples, those skilled in the art will understand that the disclosed method actions may be modified, excluded, or replaced by alternatives or additional actions known in the art. Accordingly, the following examples are intended to be exemplary in nature and not to unnecessarily limit the scope and / or content of the disclosure provided therein.

[0403] Example 1

[0404] To illustrate aspects of the functionality of the cassettes, systems, and methods of this disclosure for electroporation cells, the following are performed: Figure 28 , Figure 29A and Figure 30A The illustrative embodiments provide exemplary cell preparation and processing methods.

[0405] Reference Figure 28 The diagram illustrates an exemplary method flow for preparing cells for conversion via a batch or flow-through electroporation system and associated cartridges (as described above). As shown, an exemplary pre-electroporation method may include: obtaining cultured cells. This may include, for example: expanding a stock of immortalized cell cultures, and / or isolating primary cells from a patient and culturing them in vitro to stabilize the cells, then electroporating to expand the cells to confluence, and / or expanding the cells through continuous passage. This action can be performed in an incubator with a suitable growth medium, as known in the art.

[0406] Figure 28The method also includes mild enzymatic treatments known in the art to release any cells adhering to the surface of the growth chamber. The method can be performed in a sterile environment (e.g., a biosafety cabinet or other enclosed, ventilated laboratory workspace) to minimize the possibility of contamination.

[0407] Figure 28 The method further provides actions for removing the growth medium and washing the cells with a washing solution (e.g., phosphate-buffered saline (PBS)), followed by resuspension and titration for the desired cell density. For this purpose, these actions can be performed using any materials known in the art and by any method. However, it is worth noting that... Figure 28 A standard method is provided that includes centrifugation and pipette-based washing and resuspension. Cells to be electroporated can be resuspended to a desired concentration by diluting washed cells in a calculated volume of electroporation buffer (e.g., from cultured cells of a known concentration, and / or by using a cytometer or other cell counting apparatus / system). A “payload” or electroporation target, as defined herein, can be added to the full volume of the resuspended cells or to an appropriate volume of the desired aliquot for batch or flow-through processing as described herein.

[0408] exist Figure 28 In the preparation protocols shown, note that for some primary and / or immortalized cell lines, cell viability decreases approximately 15 minutes after cells are removed from the growth medium and / or resuspended in electroporation buffer. Therefore, note that it is preferable to add the washed and / or resuspended cells to the electroporation system and / or cartridge for transformation using the systems and methods disclosed above within 15 minutes of washing and / or resuspending the cells. It should be understood that this time can vary between different cell and buffer types and between treatment conditions.

[0409] Now refer to Figure 29A The above is shown as an example of what is used in... Figure 28 This document outlines an exemplary scheme for the batch processing and transformation of cells prepared using the exemplary methods described herein. Figure 29A As shown in the exemplary electroporation scheme, according to the relevant Figure 28The aliquots or total volumes of cells prepared by the methods outlined and discussed are loaded into electroporation (EP) cartridges along with the desired payload or electroporation target. The EP cartridges are inserted into an electroporation system (e.g., an electroporation system described herein configured for batch processing of samples), electroporation parameters are set and executed on the system, and the EP cartridges are removed from the system. The electroporated cells can then be transferred to complete culture medium or other recovery media and incubated for a period of time (e.g., 24–72 hours). The viability and electroporation efficiency of the electroporated cells can then be inquired using appropriate biochemical, optical, or molecular reader methods (e.g., Western blotting for protein concentration / protein expression, flow cytometry for expression of transformed fluorescent proteins, qPCR for molecular analysis, etc.).

[0410] For example, such as Figure 29B As shown, primary T cells activated using CD3 / CD28 immunomagnetic beads (dynabeads) were cultured for 4 days, followed by... Figure 28 and Figure 29A The procedure disclosed and shown describes the preparation of electroporation cells. Washed primary cells were resuspended in R buffer to 50 × 10⁻⁶. 6 Cells were selected at a concentration of [number] cells / mL and contained a Cas9+ gRNA payload targeting TRAC-1. Electroporation was performed using a batch processing method and associated EP cassettes at 2300V / 3ms for each of the four pulses. After electroporation, cells were transferred to complete culture medium and incubated for 48 hours. Electroporated cells were then stained with TCRα / β antibodies and evaluated for residual expression via flow cytometry compared to non-electroplated control cells (negative control). Figure 29B As shown, the conversion efficiency achieved by the disclosed system is within the expected limits compared to commercial platforms. Cells were also not significantly affected by electroporation, as determined by Sytox viability staining, which was also confirmed by flow cytometry.

[0411] Now refer to Figure 30A The above is shown as an example of what is used in... Figure 28 This describes an exemplary scheme for the flow-through processing and transformation of cells prepared using the exemplary methods outlined herein. Figure 30AAs shown in the exemplary electroporation protocol, resuspended cells, along with the desired payload or electroporation target, are transferred or resuspended in an input bag. The input bag is attached to an input port on a flow-through consumable, as described above. The flow-through consumable cartridge is then loaded into the electroporation system (e.g., those flow-through electroporation systems described above). Electroporation parameters are set and executed (e.g., using an associated computing system and / or user interface on the electroporation system), and an 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 culture medium or other reconstitution medium and incubated for a period of time (e.g., 24–72 hours). The viability and electroporation efficiency of the electroporated cells can then be inquired using appropriate biochemical, optical, or molecular readers (e.g., Western blotting for protein concentration / protein expression, flow cytometry for expression of transformed fluorescent proteins, qPCR for molecular analysis, etc.).

[0412] For example, such as Figure 30B As shown, primary T cells activated using CD3 / CD28 Dynabeads™ were cultured for up to 4 days, followed by... Figure 28 and Figure 30A The procedure disclosed and shown describes the preparation of electroporation cells. Washed primary cells were resuspended in R buffer to 50 × 10⁻⁶. 6 Cells were selected at a concentration of [number] cells / mL and contained a Cas9+ gRNA payload targeting TRAC-1. Electroporation was performed for each of the four pulses using electroporation parameters of 2300V / 3ms, employing a flow-through processing method and associated flow-through consumables. After electroporation of each sample in an automated batch process (e.g., a "flow-through" method), cells were collected to assess inter-sample variability (if any). Each isolated sample was transferred to complete culture medium and incubated for 48 hours. Electroporated cells were then stained with TCRα / β antibodies and evaluated for residual expression via flow-through cytometry compared to non-electroplated control cells (negative control). Figure 30B The results for each sample in the automated batch processing or flow-through method are shown. As the data clearly demonstrate, the conversion efficiency achieved by the disclosed system is within the expected limits compared to commercial platforms. Cells were also not significantly affected by electroporation, as determined by Sytox viability staining, which was also confirmed by flow-through cytometry.

[0413] As mentioned above Figure 28 , Figure 29A and Figure 30A The cell viability and electroporation efficiency of single-batch processed samples of the same cell culture described are compared with those of flow-through or automated batch processing. Figure 31As shown, flow-through or automated batch processing of samples exhibits greater viability and electroporation efficiency than existing technology systems, while single-batch processing of samples demonstrates similar transformation efficiency and cell viability.

[0414] The disclosed electroporation system and method are also shown for use with immortalized cell cultures. Now refer to... Figure 32 The graph shown illustrates the viability and transformation efficiency of the exemplary flow-through system and method using Jurkat cells. Specifically, according to Figure 28 and Figure 30A The protocol outlined herein prepares Jurkat cells that have undergone up to four passages-post thaw cycles for electroporation. Electroporation is performed using exemplary flow-through consumables, and each individual electroporation well is collected to assess variability between samples.

[0415] The washed Jurkat cells were resuspended in R buffer to 50 × 10⁻⁶. 6 Cells were selected at a concentration of 1700V / mL and carried a 4.6kB GFP plasmid payload. Electroporation was performed using a flow-through process and associated flow-through consumables, with each electroporated sample receiving a single pulse at 1700V / 20ms. Cells were collected after electroporation of each sample in the automated batch / flow-through process to assess inter-sample variability (if any). Each isolated sample was transferred to complete culture medium and incubated for 24 hours. Electroporated cells were then assessed for GFP expression via flow-through cytometry compared to a non-electroplated control. Figure 32 and Figure 33 The results for each sample are shown in the automated batch processing or flow-through method. Figure 32 (Negative control data are not shown above). As the data clearly demonstrate, the transformation efficiency achieved by the disclosed system is within the expected limits compared to commercial platforms, and the cells were not significantly affected by electroporation, as determined by sytox viability staining, which was also confirmed by flow cytometry.

[0416] Example 2

[0417] To illustrate aspects of the functionality of the cassettes, instruments, systems, and methods of this disclosure for electroporating cells, exemplary cell preparation and processing methods provided by the illustrative embodiments described herein are performed.

[0418] Cell source and culture conditions: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor leukocytes using the standard Ficoll-Paque method and cryopreserved. Upon thawing, the PBMCs were activated via CD3 / CD28 Dynabeads™, cultured in OpTmizer™ medium containing 2% human serum or 5% immune cell serum substitutes, and maintained at 37°C and 5% CO2. This resulted in the generation of activated primary human T cells.

[0419] Electroporation: After three days of activation, cells were prepared for electroporation by centrifugation and resuspended in standard electroporation buffer or gene editing buffer. Ribonuclear protein (RNP) was formed by combining Invitrogen™ TrueCut™ Cas9 protein v2 and Invitrogen™ TrueGuide™ (from Thermo Fisher Scientific) synthesized gRNA. The prepared cells and RNP combination were incubated for 5 minutes, donor DNA template was added, and then electroporated using the newly developed large-scale electroporation system in single-use or flow-through cassettes as described below. Cells were immediately returned to complete culture medium after electroporation and cultured for 48–72 hours. Locus-specific antibody target analysis was performed using the Invitrogen™ Attune™ NxT flow cytometer (from Thermo Fisher Scientific).

[0420] In one exemplary embodiment, activated primary human T cells are generated and electroporated via Cas9 RNP-targeted delivery transfected with Rab11a and TRAC loci for homology-directed repair. Activated primary human T cells are prepared as described above (under the heading “Cell Source and Culture Conditions”). The activated primary human T cells are then resuspended in gene-editing buffer under the following conditions: cells = 2 x 10⁻⁶. 7c / mL; Cas9 = 80 μg / mL; gRNA = 20 μg / mL; and dsDNA = 80 μg / mL. In this single-use cassette, a newly developed large-scale electroporation system is used to immediately electroporate cells under either cell electroporation condition A (i.e., 1700 V / 10 ms / 1-pulse) or electroporation condition F (i.e., 2300 V / 3 ms / 4-pulse) to deliver a Cas9:gRNA RNP targeting the Rab11a or TRAC locus and a 1.4 kb linear dsDNA template encoding GFP via a 100 bp homologous arm. Knock-in efficiency was assessed via flow cytometry. Figure 34 The KI efficiency and cell viability were analyzed after 48 hours of electroporation. Figure 34 The results are shown in the figure.

[0421] Figure 34 The cell viability, transformation efficiency, and knock-in efficiency of transformed and activated primary human T cells as described above according to one or more embodiments of this disclosure are shown, and the successful use of exemplary single-use consumables and electroporation systems and methods for electroporation is demonstrated.

[0422] In another exemplary embodiment, activated primary human T cells are generated and electroporated via Cas9:gRNARNP-targeted delivery of the TRAC locus to generate CAR-T cells. Activated primary human T cells are prepared as described above (under the heading "Cell Source and Culture Conditions"). The activated primary human T cells are then resuspended in gene editing buffer under the following conditions: cells = 2.5 x 10⁻⁶. 7 c / mL; Cas9 = 100 μg / mL; gRNA = 25 μg / mL; and dsDNA = 80 μg / mL. This disclosure describes a 1 mL single-use electroporation cartridge (in... Figure 35 Described as “small-scale EP” in the text) and the 1 mL flow-through electroporation cartridge of this disclosure (in Figure 35 Described as "large-scale EP," both methods used electroporation conditions F (2300V / 3ms / 4 pulses) to electroporate cells to deliver a Cas9:gRNA RNP targeting the TRAC locus and a linear dsDNA template (2.8kb) encoding an Anti-CD19 CAR via a 100bp homologous arm. Knock-in efficiency was assessed via flow cytometry. Figure 35 The KI efficiency and cell viability were analyzed after 96 hours of electroporation. Figure 35 The results are shown in the figure.

[0423] Figure 35The present invention illustrates transformation efficiency, knock-in efficiency, and cell viability during the generation of CAR-T cells using the flow-through and single-use cartridges and electroporation systems and methods disclosed herein, according to one or more embodiments of the present disclosure.

Claims

1. An electroporation box, comprising: Electroporation chamber, which is defined by an elongated body; The first electrode is deployed at the proximal end of the electroporation chamber; and A second electrode is deployed at the opposite distal end of the electroporation chamber, wherein at least one of the first electrode or the second electrode is movable between a capped position for electroporation and an uncapped position for loading a sample, and / or the electroporation chamber is configurable between a sealed state and an unsealed state.

2. The electroporation box as described in claim 1, wherein, The elongated body comprises one or more of non-conductive plastic, glass, or ceramic, or is made therefrom, and is configured to receive cell-containing fluid to be electroporated within the electroporation chamber defined by the elongated body.

3. The electroporation box tube as described in claim 2, wherein, The electroporation chamber comprises or is made of glass and / or ceramic.

4. The electroporation cartridge as described in claim 2 or claim 3, wherein, The electroporation chamber comprises or is made of polycarbonate or other non-conductive radiation-stabilized plastic.

5. The electroporation cartridge as described in any one of claims 1-4, wherein, At least a portion of the electroporation chamber gradually tapers between the first electrode and the second electrode.

6. The electroporation cartridge as described in claim 5, wherein, The gradually tapering portion of the electroporation chamber does not substantially interfere with the generation of a uniform electric field between the first electrode and the second electrode.

7. The electroporation cartridge as described in any one of claims 1-6, wherein, The electroporation chamber comprises a uniform cross-section along the length of the reaction chamber.

8. The electroporation cartridge as described in claim 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 deployed between the first electrode and the second electrode.

9. The electroporation cartridge as claimed in claim 7 or claim 8, wherein, The electroporation chamber includes a cylindrical cavity, and the uniform cross-section includes a circle.

10. The electroporation chamber as claimed in any one of claims 1-7, further comprising a proximal sidewall defined between the proximal opening of the elongated body and the inflection point of the sidewall defining the electroporation chamber, wherein, The proximal sidewall narrows from a first diameter defined by the proximal opening to a second, smaller diameter defined at a position distal to the inflection point.

11. The electroporation cartridge as described in any one of claims 1-10, wherein, The first electrode includes a spherical extension having a substantially flat distal surface.

12. The electroporation cartridge as described in any one of claims 1-10, wherein, The first electrode includes a spherical extension having a distal surface with a convex or angled profile.

13. The electroporation cartridge as claimed in claim 11 or claim 12, wherein, The spherical extension is separated from the base portion of the first electrode by a narrow rod.

14. The electroporation cartridge as described in any one of claims 11-13, wherein, The spherical extension is operable to: while the first electrode is fixed in the electroporation chamber, displace one or more air bubbles associated with the cell-containing fluid to be electroporated in the electroporation chamber.

15. The electroporation cartridge of any one of claims 1-14, further comprising a sealing member disposed between the first electrode and the proximal surface of the elongated body, the sealing member being operable to form a fluid-tight connection between the first electrode and the elongated body.

16. The electroporation cartridge as described in claim 15, wherein, The first electrode includes a first electrode flange, and the elongated body includes a proximal body flange oriented in a plane substantially parallel to the first electrode flange. The sealing member is disposed between the first electrode flange and the proximal body flange to form the fluid-tight connection therebetween.

17. The electroporation cartridge as described in any one of claims 1-16, wherein, The first electrode is operable to configure the electroporation cartridge between a sealed state and an unsealed state.

18. The electroporation cartridge as claimed in claim 17, wherein, The first electrode is operable to configure the electroporation cartridge between a sealed and unsealed state without the need for an additional removable capping workpiece.

19. The electroporation cartridge as claimed in claim 17 or claim 18, wherein, The first electrode has a removable cap.

20. The electroporation cartridge of any one of claims 1-17, 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 elongated body.

21. The electroporation cartridge as described in any one of claims 1-20, 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 as described in any one of claims 1-21, wherein, The second electrode includes a protruding portion that extends from the distal end of the elongated body into the electroporation chamber.

23. The electroporation cartridge as described in claim 22, wherein, The perimeter of the protruding portion includes the complementary shape of the inner surface of the elongated body that defines the electroporation chamber.

24. The electroporation cartridge as claimed in claim 22 or claim 23, wherein, The second electrode further includes a first sealing member disposed between the second electrode and the distal surface of the elongated body, the first sealing member being operable to form a fluid-tight connection between the second electrode and the distal surface of the elongated body.

25. The electroporation cartridge as described in claim 24, wherein, The second electrode includes an electrode flange, and the elongated body includes a distal body flange oriented in a plane substantially parallel to the electrode flange. The sealing member is disposed between the electrode flange and the distal body flange to form the fluid-tight connection therebetween.

26. The electroporation cartridge as described in any one of claims 22-25, wherein, The second electrode further includes a second sealing member disposed around the protrusion of the second electrode and positioned distal from the proximal surface of the second electrode, the second sealing member being operable to form a fluid-tight connection between the protrusion and the inner surface of the elongated body defining the electroporation chamber.

27. The electroporation cartridge as described in any one of claims 22-26, wherein, The proximal surface of the second electrode comprises a flat and uniform surface.

28. The electroporation cartridge as described in any one of claims 22-27, wherein, The proximal surface of the second electrode is orthogonal to the longitudinal axis of the electroporation chamber.

29. The electroporation cartridge as claimed in any one of claims 1-28, further comprising a retaining pin associated with the second electrode and configured to secure the second electrode to the elongated body.

30. The electroporation cartridge as described in claim 29, wherein, The second electrode defines a channel configured in size and shape to receive the retaining pin. The channel is aligned with a pair of holes defined by the sidewall of the elongated body and configured to receive the retaining pin, thereby fixing the second electrode in a fixed position relative to the elongated body.

31. The electroporation cartridge as described in claim 30, wherein, The channel is formed to pass through the central region of the protruding portion of the second electrode, which is located distal to the first sealing member and / or the second sealing member.

32. The electroporation cartridge as described in any one of claims 1-31, 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 as claimed in any one of claims 1-32, further comprising a volume-reducing sleeve configured in terms of size and shape to fit within the electroporation chamber.

34. The electroporation cartridge as described in claim 33, wherein, The volume-reducing sleeve defines a secondary electroporation chamber having a smaller volume than the electroporation chamber.

35. The electroporation cartridge as claimed in claim 33 or claim 34, wherein, The volume-reducing sleeve includes a distal opening configured to mate with the second electrode when fixed within the electroporation chamber.

36. The electroporation cartridge as described in any one of claims 33-35, wherein, The volume reduction sleeve includes a vent deployed adjacent to the proximal end of the volume reduction sleeve. The vent is configured to allow air to pass through it during the introduction of the volume reduction sleeve into or out of the electroporation chamber, thereby preventing a vacuum from forming between the sub-electroporation chamber and the electroporation chamber. This allows cellular fluid from electroporation to fill the sub-electroporation chamber during the introduction of the volume reduction sleeve and to exit the sub-electroporation chamber during the introduction and removal of the volume reduction sleeve.

37. The electroporation cartridge as described in any one of claims 33-36, wherein, The volume reduction sleeve includes a radial sealing member configured to secure the volume reduction sleeve within the electroporation chamber.

38. The electroporation cartridge as described in claim 37, wherein, The radial sealing member forms a fluid-tight seal with the sidewall defining the electroporation chamber to prevent leakage of cellular fluid within the secondary electroporation chamber through the distal opening of the volume-reduced sleeve.

39. The electroporation cartridge as described in any one of claims 33-38, wherein, The first electrode is configured to selectively associate with the volume-reducing sleeve and form a fluid-tight seal with the volume-reducing sleeve.

40. The electroporation cartridge as described in any one of claims 33-39, wherein, A space is defined between the outer surface of the volume-reducing sleeve and the inner wall of the elongated body to form a fluid overflow space, which is configured to receive the overflow volume displaced by the first electrode when the electroporation chamber is sealed.

41. The electroporation cartridge of any one of claims 1-40, further comprising a fluid overflow space associated with a proximal region of the electroporation chamber and configured to receive an overflow volume displaced by the first electrode when the electroporation chamber is sealed.

42. The electroporation cartridge of any one of claims 1-41, further comprising one or more springs longitudinally disposed on the proximal side of the elongated body and configured to position the first electrode in the unsealed position at a distance from the electroporation chamber.

43. The electroporation cartridge as described in claim 42, wherein, The electroporation cartridge in the capped position configures the one or more springs to be compressed and the first electrode to be deployed in the electrode chamber and operable to electroporate the cell-containing fluid deployed therein.

44. The electroporation cartridge as described in any one of claims 1-43, wherein, The electroporation cartridge includes a flow-through electroporation cartridge.

45. The electroporation cartridge of claim 44, further comprising a port associated with the first electrode, the port defining a lumen within the first electrode such that the lumen is fluidly connected to the electroporation chamber.

46. ​​The electroporation chamber of claim 44, further comprising a port associated with the proximal portion of the elongated body, the port being configured to: discharge displaced air from the electroporation chamber when the electroporation chamber is being filled, and / or introduce filtered or purified air into the electroporation chamber when the electroporation chamber is being emptied.

47. The electroporation chamber of any one of claims 44-46, further comprising a chamber inlet and a chamber outlet, each of the chamber inlet and the chamber outlet being fluidly connected to the electroporation chamber.

48. The electroporation cartridge as described in claim 47, wherein, One or more of the chamber inlet or chamber outlet are deployed above the proximal surface of the second electrode.

49. The electroporation cartridge as claimed in claim 47 or claim 48, wherein, The lumen of the chamber inlet and / or chamber outlet is substantially parallel to the proximal surface of the second electrode.

50. The electroporation cartridge as described in any one of claims 47-49, wherein, One or more of the chamber inlet or chamber outlet are associated with plugs and / or valves to control the inward flow of the cell-containing fluid to be electroporated into the electroporation chamber and / or to control the outward flow of the cell-containing fluid to be electroporated from the electroporation chamber.

51. The electroporation cartridge of any one of claims 44-50, further comprising a fluid overflow space associated with the first electrode and / or the elongated body, the fluid overflow space being configured to receive overflow volume displaced from the electroporation chamber when the electroporation chamber is filled.

52. The electroporation chamber of any one of claims 44-50, further comprising a fluid overflow space associated with a sealing cap, the fluid overflow space being configured to receive overflow volume displaced from the electroporation chamber when the electroporation chamber is sealed by the sealing cap.

53. An electroporation system configured to provide flow-through electroporation of a sample, the electroporation system comprising: A modular housing having multiple compartments for holding and arranging multiple electroporation system components, the electroporation system components including: One or more pumps configured to move the sample through the system; An electroporation chamber configured to receive a flow-through electroporation cartridge, the electroporation cartridge being configured to retain a sub-volume of a sample within the electroporation chamber for electroporation of the sub-volume; and A conduit having an inlet end and an outlet end, the conduit being routed through the housing to allow fluid-mode connection of the plurality of electroporation system components.

54. The electroporation system of claim 53 further includes a bag compartment configured to receive and support an input bag and / or an output bag.

55. The electroporation system of claim 54, wherein, The compartment includes an insertion portion slidably connected to the compartment, allowing it to be selectively withdrawn from or enclosed within the housing.

56. The electroporation system of claim 55, wherein, The compartment includes one or more magnetic latches for holding the compartment in a closed position within the housing.

57. The electroporation system of any one of claims 53-56 further includes a cooling module that is in thermal contact with the electroporation chamber and is configured to regulate the temperature of the electroporation chamber.

58. The electroporation system of claim 57, wherein, The cooling module includes a ceramic block.

59. The electroporation system as claimed in claim 57 or 58, wherein, The cooling module is cooled by thermoelectric cooling.

60. The electroporation system of any one of claims 53-59 further includes a mixer container deployed downstream of the inlet and upstream of the electroporation cartridge, the mixer container including a mixing element configured to provide mixing to a portion of the sample contained within the mixing container.

61. The electroporation system of claim 60, wherein, The mixing element includes mixing blades.

62. The electroporation system of claim 60 or 61, wherein, The mixer container includes a mixer magnet assembly mechanically coupled to the mixing element, the mixer magnet assembly being deployed so as not to contact the portion of the sample contained within the mixer container.

63. The electroporation system of claim 62 further includes 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 with the mixer magnet assembly.

64. The electroporation system as claimed in claim 62 or 63, wherein, The mixer container includes a cover, and the mixer magnet assembly is deployed at or near the cover.

65. The electroporation system of any one of claims 60-64, further comprising a sample input assembly configured to assist in the transfer of a sample between an input and the mixer container, the sample input assembly comprising: The main piping section is positioned between the input and the mixer container; And an intermediate pipe section that is pneumatically coupled to the main pipe section and extends therefrom to a terminal end with an air inlet, wherein the intermediate pipe section thereby allows air to be delivered to the main pipe section when there is a sufficient pressure drop in the main pipe section.

66. The electroporation system of claim 65, wherein, The terminal end of the intermediate pipe section is coupled to an air container with a variable volume, and the sample input assembly is configured to detect a decrease in the threshold of the variable volume to determine that the sample has moved into the mixer container.

67. The electroporation system of claim 66, wherein, The sample input sensor assembly includes a syringe having a barrel and a plunger deployed within the barrel, the variable volume being defined by the position of the plunger within the barrel, and the threshold reduction of the variable volume being detected as a result of the plunger's movement.

68. The electroporation system of any one of claims 53-67 further includes a chamber sealing assembly operably coupled to the electroporation chamber and configured to regulate the pressure within the electroporation chamber during electroporation and thereby limit bubble formation.

69. The electroporation system of claim 68, wherein, The chamber sealing assembly includes one or more linear actuators configured to advance a plunger toward or retract a plunger away from the electroporation chamber, thereby regulating the pressure within the electroporation chamber.

70. The electroporation system of any one of claims 53-69 further includes a pre-cooling assembly disposed upstream of the electroporation chamber and configured to cool the sub-volume prior to electroporation of the sub-volume of the sample.

71. The electroporation system of claim 70, wherein, The precooling assembly includes a cooling block and pipe sections, the pipe sections being deployed within or adjacent to the cooling block.

72. The electroporation system as claimed in claim 70 or 71, wherein, The cooling block of the pre-cooled assembly is cooled by thermoelectric cooling.

73. The electroporation system according to any one of claims 70-72, wherein, The precooling assembly includes a flexible biasing element that biases the cooling block against the pipe section deployed adjacent to the cooling block.

74. The electroporation system according to any one of claims 53-73, further comprising at least one flow sensor, wherein, The at least one flow sensor is deployed between the mixer container and the electroporation chamber.

75. The electroporation system of claim 74, wherein, The flow sensor is an ultrasonic sensor.

76. The electroporation system of claim 74 or 75, wherein the flow sensor includes an actuable trigger that, when actuated, positions a corresponding pipe section within the flow sensor for detecting flow through the pipe section.

77. The electroporation system of any one of claims 53-76 further includes one or more flow indicators that route the corresponding pipe section to a location on the exterior of the housing so that the flow through the pipe section can be visualized.

78. The electroporation system as described in any one of claims 53-77, wherein, The housing includes one or more handles.

79. The electroporation system of claim 78, wherein, The one or more handles include handles having a latch portion configured to engage with the dashboard to attach the dashboard to the housing.

80. The electroporation system of any one of claims 53-79 further includes an electroporation cartridge attachment feature coupled to the electroporation cartridge, the attachment feature including a flexible biasing element biasing the electroporation cartridge toward a cooling module integrated within the housing.

81. The electroporation system of any one of claims 53-80, further comprising a capping mechanism configured to engage with the electroporation cartridge, wherein, The electroporation cartridge includes a first electrode and a second electrode, both disposed at opposite ends of the electroporation chamber, wherein at least one of the first electrode or the second electrode is engageable with the capping mechanism and is movable between a capped position for electroporation and an uncovered position for discharge as a result of actuating the capping mechanism.

82. The electroporation system of claim 81, wherein, The electroporation cartridge includes a spring mechanism that allows the capping mechanism to overtravel relative to the displacement of the electrode as a result of actuating the capping mechanism.

83. The electroporation system as described in any one of claims 53-81, wherein, The electroporation chamber includes a chamber inlet and a chamber outlet, wherein the chamber outlet is coupled to an outlet plunger movable between an advance position that prevents the subvolume from flowing out of the electroporation chamber and a retracted position that allows the subvolume to flow out of the electroporation chamber.

84. The electroporation system as described in any one of claims 53-83, wherein, The electroporation cartridge includes one or more bellows structures, all configured to enclose the movable components of the electroporation chamber.

85. The electroporation system as described in any one of claims 53-84, wherein, The electroporation chamber includes a flow-through electroporation cartridge as described in any one of claims 44-52.

86. The electroporation system of any one of claims 53-85, further comprising an electroporation assembly electrically coupled to the electroporation chamber, the electroporation assembly including a conductivity sensor for measuring conductivity across the electroporation chamber, the electroporation assembly communicatively coupled to a controller having one or more processors and one or more hardware storage devices.

87. The electroporation system of claim 86, wherein, The one or more hardware storage devices store computer-executable instructions that, when executed by the one or more processors, configure the controller to perform at least the following operations: The conductivity of the sub-volume within the electroporation chamber is determined using the conductivity sensor. Based on the determined conductivity, the voltage drop across the electroporation chamber is determined; as well as The capacitor in the electroporation circuit accompanying the electroporation chamber is charged to a voltage level greater than the determined voltage drop across the electroporation chamber to compensate for other voltage drops between the capacitor and the electroporation chamber.

88. The electroporation system as claimed in claim 86 or 87, wherein, The one or more hardware storage devices store computer-executable instructions that, when executed by the one or more processors, configure the controller to perform at least the following operations: The conductivity of the sub-volume within the electroporation chamber is determined using the conductivity sensor. Based on the determined conductivity, expected pulse voltage, and expected pulse duration, the predicted temperature increase of the sub-volume is determined; and If the predicted temperature increase results in a temperature in the sub-volume that exceeds a predetermined threshold temperature, then one or more of the following operations are performed: Send an arc risk alarm. Withdraw the sample sub-volume to retain the sample sub-volume, and / or The cooling is adjusted accordingly to reduce the temperature of the electroporation chamber.

89. The electroporation system of claim 88, wherein, The computer-executable instructions also configure the controller to determine the initial temperature of the sub-volume within the electroporation chamber by correlating the determined conductivity with temperature.

90. The electroporation system as described in any one of claims 86-89, wherein, The one or more hardware storage devices store computer-executable instructions that, when executed by the one or more processors, configure the controller to perform at least the following operations: The conductivity of the sub-volume within the electroporation chamber is determined using the 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 sub-volume is emptied from the electroporation chamber.

91. The electroporation system of any one of claims 53-90, further comprising a controller having one or more processors and one or more hardware storage devices, wherein, The one or more hardware storage devices store computer-executable instructions that, when executed by the one or more processors, configure the controller to repeatedly tilt the charger voltage based on a previous input voltage applied to the electroporation chamber and a corresponding previously measured actual voltage.

92. The electroporation system of any one of claims 74-91, further comprising a controller having one or more processors and one or more hardware storage devices, wherein, The one or more hardware storage devices store computer-executable instructions that, when executed by the one or more processors, configure the controller to: determine the step volume to which the system should move between each electroporation event, wherein calibration is performed by performing at least the following operations: Determine the number N of rotations of the drive pump required to fill the pipe deployed between the flow sensor and the electroporation chamber and to completely fill the sample volume of the electroporation chamber, where N represents the volume between the flow sensor and the outlet of the electroporation chamber; Empty the electroporation chamber; The driving pump returns the sample to a point upstream of the flow sensor by a fixed number of rotations k, where the fixed number k represents the volume between the point upstream of the flow sensor and the inlet of the electroporation chamber; Determine the number x of rotations of the drive pump required to move the sample from the point upstream of the flow sensor to the flow sensor, where the number x represents the volume between the point upstream of the flow sensor and the flow sensor, and the number (kx) represents the volume between the flow sensor and the inlet of the electroporation chamber; as well as The volume between the inlet and outlet of the electroporation chamber is determined to be N-(kx), and this volume is set as the step volume.

93. The electroporation system of any one of claims 53-92 further includes a safety gate configured to mechanically open the electroporation circuit to prevent voltage discharge while the safety gate is open.

94. The electroporation system according to any one of claims 53-93, 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. The electroporation system of any one of claims 74-91, further comprising a controller having one or more processors and one or more hardware storage devices, wherein, The one or more hardware storage devices store computer-executable instructions that, when executed by the one or more processors, configure the controller to: determine the step volume to which the system should move between each electroporation event, wherein calibration is performed by performing at least the following operations: The first filling of the electroporation chamber is performed by monitoring the resistance of the sample fluid in the electroporation chamber during the first filling process from several kiloohms to a stable value in the range of about 600-800 ohms, and stopping the first filling when the stable resistance value is reached. Based on calculated values, such as N from the peristaltic pump. rev The electroporation chamber is coarsely filled for a second filling. After the second filling and the top electrode is capped, the resistance is measured; If the resistance is within a stable value range determined from the first filling, then the second filling is complete, and the sample can continue to be de-perforated. If the resistance is not within the stable value range determined from the first fill, then unseal the top electrode and add an additional n. rev Quantity is used for precise filling; After the fine filling is completed and the top electrode is capped, the resistance is measured. If the resistance is within a stable value range determined from the first filling, then the second filling is complete, and the sample can continue to be de-perforated. If not, repeat the fine filling and measure the resistance step by step until the resistance is within the stable value range determined from the first filling. For the number of pump rotations N rev + n rev x (where x equals the number of attempts at fine filling), complete the subsequent filling (third filling, fourth filling, etc.).

96. The system of claim 95, wherein, The stable value of the resistor is approximately 700 ohms.

97. The system of claim 95, wherein, The step of coarsely filling the electroporation chamber for the second filling is also based on calculations using one or more parameters, such as the inner diameter of the pipe, the number of rollers in the pump, the diameter of the electroporation chamber, and / or the height of the electroporation chamber.

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