Systems, devices, and methods for electroporation within a cell processing system

Abstract

This disclosure relates to systems, devices, and methods for electroporation. In one embodiment, the disclosure relates to an electroporation system comprising: at least one electroporation chamber, each electroporation chamber including a first electrode and a second electrode; a plurality of ports, including an inlet port in a first part of each electroporation chamber and an outlet port in a second part of each electroporation chamber; a fluid conduit disposed between the first electrode and the second electrode, the fluid conduit being in fluid communication with the inlet port and the outlet port; and an automated system for electroporation operably connected to the first electrode and the second electrode of the at least one electroporation chamber.

Description

Technical Field

[0001] (Field of the Invention) The present disclosure relates to electroporation, for example, systems, devices, and methods for electroporation within a cell processing system.

[0002] (Cross - Reference to Related Applications) This application claims priority to U.S. Provisional Application No. 63 / 453,730, filed on March 21, 2023. All applications are hereby incorporated by reference in their entirety.

Background Art

[0003] Electroporation is a process that applies an electric field to cells to create nano- to micron-scale pores in the cell membrane, thereby increasing the permeability of the cell membrane and allowing chemicals, drugs, and / or biological substances (e.g., DNA) to enter the cells. The electroporation process, in a process referred to as reversible electroporation, may include generating pores, stabilizing pores, and sealing pores. Current electroporation devices are configured to apply electroporation to a specific volume of cell solution. Proper application of electroporation energy requires adjusting electrical parameters based on the volume of the solution. Some electroporation devices may not be able to accurately measure the volume of cell solution before applying electrical energy. Some devices may not be able to remove a specific amount of cell solution prior to applying electrical energy, as is necessary in case of overfilling the device. Some electroporation devices may not be able to provide sufficient electrical energy levels to accommodate large volumes of cell solution. Some electroporation devices may not be able to apply electrical energy to substantially all of the cells contained within the device. Additionally, electroporation devices used by those skilled in the art may only be able to process a single container of cell solution at a time. Therefore, some chambers may not be able to support multiple devices, each containing a cell solution, which increases processing time and reduces clinical efficiency.

[0004] Therefore, further methods for electroporation are desired. [Overview of the Initiative]

[0005] This disclosure generally relates to systems, devices, and methods for electroporation in automated cell processing systems. Generally, the electroporation devices disclosed herein may include an electroporation chamber. In some modifications, the electroporation chamber may include a first electrode and a second electrode, a plurality of ports having an inlet port in the first part of the electroporation chamber and an outlet port in the second part of the electroporation chamber, a fluid conduit positioned between the first electrode and the second electrode and having fluid communication with the inlet port and the outlet port, and at least one window disposed on the wall of the electroporation chamber. The distance between the first electrode and the second electrode may be any preferred distance, and in some modifications, the distance may be about 0.5 mm to about 5 mm. The electroporation chamber may further include a first housing and a second housing, and the first electrode, the fluid conduit, and the second electrode may be positioned between the first housing and the second housing. In some modifications, segments of the fluid conduit may be close to the first part of the electroporation chamber. Additionally or alternatively, segments of the fluid conduit may be adjacent to a second portion of the electroporation chamber. In some modifications, at least one window may include a first window and a second window, the first window having a view to a first segment of the fluid conduit and the second window having a view to a second segment of the fluid conduit. Furthermore, in some modifications, each of the multiple ports may extend through the first electrode and / or the second electrode.

[0006] In some modifications, the multiple ports are in fluid communication with a fluid conduit and may further include a vent port located in a first part of the electroporation chamber. The fluid conduit may be in fluid communication with each of the multiple ports via auxiliary channels extending therefrom. Each of the auxiliary channels may be fluid-coupled to the fluid conduit and to one of the multiple ports. In some modifications, the auxiliary channel of the auxiliary channel that fluid-couples the fluid conduit to the outlet port may be curved. Furthermore, in some modifications, at least one window may include a first window and a second window, the first window having a view to a first segment of the fluid conduit and the second window having a view to a second segment of the fluid conduit, and the second window may be close to an auxiliary channel that is in fluid communication with the outlet port. In addition, the auxiliary channel that is in fluid communication with the outlet port may be curved, and the second window may be close to a curved auxiliary channel. In some modifications, the second window may be close to the base of the curved auxiliary channel. In some modifications, the first window may be located near an auxiliary channel that is in fluid communication with the vent port. Furthermore, the first window may be located near the base of the auxiliary channel that is in fluid communication with the vent port. Furthermore, in some modifications, the inlet port located in the first part of the electroporation chamber may be further positioned toward the first side wall of the electroporation chamber, and the vent port located in the first part of the electroporation chamber may be further positioned toward the second side wall of the electroporation chamber.

[0007] In some modifications, the fluid conduit may have a volume defined by the distance between the first electrode and the second electrode. The volume of the fluid conduit may range from approximately 0.5 μL to approximately 2.5 mL. Furthermore, in some modifications, the fluid conduit may have a triangular cross-section along an axis defined from the first part of the electroporation chamber to the second part of the electroporation chamber.

[0008] Furthermore, this specification also describes systems for electroporation. In some modifications, the system may include an output stage and a multiplexer configured to direct at least a portion of an electrical load to at least one of a first electroporation chamber and a second electroporation chamber. In addition, in some modifications, excess charge in the capacitance bank may be discharged through a charge-discharge circuit.

[0009] In some variations, the system may further include a charge / discharge circuit and a capacitance bank that can be charged via a high-voltage DC-DC converter. The capacitance bank may store approximately 4.5 mF.

[0010] In some variations, the system may further include an integrated pulse module that opens and closes the supply path of electrical load to the output stage. The integrated pulse module may support pulse amperages up to approximately 800A for a pulse length of approximately 1 ms.

[0011] In other variations, the electroporation system described herein may include at least one electroporation chamber, each electroporation chamber comprising a first electrode and a second electrode; a plurality of ports, each electroporation chamber comprising an inlet port in the first part and an outlet port in the second part; a fluid conduit disposed between the first electrode and the second electrode, the fluid conduit being in fluid communication with the inlet port and the outlet port; and an automated system for electroporation operably connected to the first electrode and the second electrode of the at least one electroporation chamber. In some variations, the output from the system may generate an electric field of about 1.0 kV / cm to about 3 kV / cm in the fluid conduit of at least one of the first and second electroporation chambers.

[0012] In some modifications, the system may further include at least one window disposed on the wall of each electroporation chamber, providing a view to the corresponding segment of the fluid conduit in the electroporation chamber. The at least one window may include a first window and a second window, the first window having a view to a first segment of the fluid conduit, and the second window having a view to a second segment of the fluid conduit. In some modifications, the system may further include at least one sensor configured to view at least one window. Each of the multiple ports may extend through the first electrode and / or the second electrode.

[0013] Furthermore, in some modifications, the system may further include a processing circuit configured to generate a first signal for each of at least one electroporation chambers to cause cell solution to flow into a fluid conduit via an inlet port, a second signal to stop the flow of cell solution into the fluid conduit via the inlet port based on data received from at least one sensor, a third signal to apply an electric field into the fluid conduit for a predetermined duration via an automated system for electroporation, and a fourth signal to cause cell solution to drain from the fluid conduit via an outlet port. The multiple ports may further include vent ports that are in fluid communication with the fluid conduit and are located in the first part of each electroporation chamber. In some modifications, the fourth signal generated by the processing circuit may be configured to cause a fluid pump to drain cell solution from the fluid conduit by purging the fluid conduit with air via a vent port. Further comprising a fluid pump, the first, second, and fourth signals are transmitted to the fluid pump.

[0014] In some modifications, the system may include an output stage for switching the polarity of an electrical load, and a multiplexer configured to direct at least a portion of the electrical load to at least one of a first electroporation chamber and a second electroporation chamber. Additionally, the automated system for electroporation may further include a charge / discharge circuit and a capacitance bank rechargeable via a high-voltage DC-DC converter. Furthermore, the system may further include an integrated pulse module capable of opening and closing the supply path of the electrical load to the output stage. In some modifications, the integrated pulse module may support pulse amperages up to approximately 800A for a pulse length of approximately 1 ms.

[0015] Methods for electroporating cells are also described herein. In some variations, the method may include: introducing a cell solution into a fluid conduit of an electroporation chamber through an inlet port of the electroporation chamber; detecting the presence of the cell solution in a second portion of the electroporation chamber through at least one window in the wall of the electroporation chamber; stopping the inflow of the cell solution into the fluid conduit; applying an electric field to the cell solution in the fluid conduit for a predetermined duration through a first electrode and a second electrode of the electroporation chamber; and draining the cell solution from the fluid conduit through an outlet port.

[0016] In some modifications, at least one window may be a second window located adjacent to the outlet port. In some modifications, the method may further include detecting the presence of cell solution in a first portion of the electroporation chamber after detecting the presence of cell solution in a second portion of the electroporation chamber through a first window and through a second window, and an electric field may be applied after detecting the presence of cell solution in the first portion of the electroporation chamber. Detecting the presence of cell solution in the first portion of the electroporation chamber may include detecting the presence of cell solution adjacent to a vent port located in the first portion of the electroporation chamber, which is in fluid communication with a fluid conduit. Detecting the presence of cell solution at the second end of the electroporation chamber may include detecting the presence of cell solution adjacent to an auxiliary channel extending from a fluid conduit, which is in fluid communication with the fluid conduit and the outlet port. Furthermore, in some modifications, draining cell solution from the fluid conduit via the outlet port may include purging the fluid conduit with air via a vent port located in the first portion of the electroporation chamber, which is in fluid communication with the fluid conduit.

[0017] In other variations, the method for electroporating cells as described herein may include: positioning a cell-containing cartridge within an electroporation apparatus of a working cell by a robot; allowing a cell solution to flow into an electroporation chamber of the cartridge, the electroporation chamber being located within an electroporation module that can be coupled to an electroporation apparatus; detecting the presence of the cell solution in a portion of the electroporation chamber by the electroporation apparatus through at least one window provided on the electroporation chamber; stopping the flow of the cell solution into the electroporation chamber; applying an electric field to the cell solution via a first electrode and a second electrode of the electroporation chamber; and draining the cell solution from the electroporation chamber.

[0018] Further embodiments, features, and advantages of the present invention will become apparent from the following detailed description and through the implementation of the invention. [Brief explanation of the drawing]

[0019] [Figure 1A] This is a block diagram illustrating an illustrative variation of a cell processing system. [Figure 1B] This is a block diagram illustrating an example of a modified cartridge. [Figure 2A] This is a block diagram illustrating an illustrative variation of a cell processing system. [Figure 2B] This is a perspective view of an illustrative modification of a working cell in a cell processing system. [Figure 2C] This is a perspective view of an illustrative modification of a working cell and cartridge in a cell processing system. [Figure 2D] This is a block diagram illustrating an illustrative variation of a cell processing system. [Figure 3A] This is a rendering of an exploded view of an exemplary electroporation chamber in a cartridge. [Figure 3B]Schematic diagram of an exemplary electroporation chamber of a cartridge. [Figure 3C] Rendering of a perspective view of an exemplary electroporation chamber of a cartridge. [Figure 3D] Rendering of an aspect of an exemplary electroporation chamber of a cartridge. [Figure 3E] Rendering of an aspect of an exemplary electroporation chamber of a cartridge. [Figure 3F] Rendering of an aspect of an exemplary electroporation chamber of a cartridge. [Figure 4A] Schematic diagram of an exemplary variation of an electroporation system showing an electroporation device in a work cell and a corresponding electroporation module in a cartridge. [Figure 4B] Schematic diagram of an exemplary electroporation system when an electroporation module is coupled to an electroporation device. [Figure 5] Schematic diagram of an aspect of an electroporation device of an electroporation system. [Figure 6] Flowchart of an exemplary variation of a method for cell treatment. [Figure 7A] Flowchart of an exemplary variation of an electroporation method. [Figure 7B] Flowchart of an exemplary variation of an electroporation method. [Figure 7C] Flowchart of an exemplary variation of an electroporation method. [Figure 8A] Flowchart of an exemplary variation of an electroporation method. [Figure 8B] Flowchart of an exemplary variation of an electroporation method.

Modes for Carrying Out the Invention

[0020] Electroporation typically involves applying an electric field to cells to induce the opening of nanoscale and / or microscale pores on the cell membrane in order to increase the permeability of the cell membrane. These pores then allow chemicals, drugs, and / or DNA to be introduced into the cells. An exemplary process of electroporation includes generating pores, stabilizing the pores, and sealing the pores.

[0021] During the electroporation process, host cells and selected molecules may be suspended in a conductive solution, and an electrical circuit is closed around the mixture. Electrical pulses are discharged through the cell suspension. This disrupts the phospholipid bilayer of the membrane, resulting in the formation of transient pores. The potential across the cell membrane simultaneously increases, allowing charged molecules such as DNA and RNA to be driven across the membrane through the pores in a manner similar to electrophoresis.

[0022] More specifically, when an electric field is applied across a cell, the induced membrane potential accumulates across the cell membrane, creating pores on both sides of the cell along the direction of the electric field. Due to the difference in conductivity and dielectric constant between the inside and outside of the cell, there is no electric field inside the cell. Therefore, DNA, RNA, and / or proteins adhere to the pores in the cell membrane and are gradually absorbed by the cell during sealing, becoming integrated into the cell.

[0023] However, current electroporation devices have limitations. For example, many current electroporation devices have fixed cathodes and anodes, resulting in suboptimal cell permeabilization. Furthermore, many current electroporation devices can only perform electroporation within a small range of cell solution volumes. Moreover, certain current electroporation devices cannot accurately understand the cell solution volume and / or fluid position within the electroporation chamber to perform electroporation, thus leaving the possibility of overfilling the device and resulting in suboptimal electroporation results. Therefore, there is a need to provide electroporation systems and methods that enable greater flexibility, precision, and effectiveness when processing cells.

[0024] This specification discloses devices, systems, and methods for the electroporation of cells. The disclosed devices, systems, and methods can be used with a wide range of cell volumes, and in some modifications, the devices, systems, and methods disclosed herein utilize multiple electroporation chambers and / or sensors to assist in accuracy and effectiveness.

[0025] As described throughout, cell processing methods, devices, and systems may involve moving a cartridge containing cell products between multiple instruments within a working cell. One or more instruments may be configured to interface with the cartridge to perform cell processing steps. In some modifications, multiple cell processing steps may be performed within a single cartridge. For example, a robotic arm may be configured to move the cartridge between instruments, each instrument configured to perform a different cell processing step when coupled to a corresponding module within the cartridge. The cartridge may comprise any number of modules, such as a bioreactor module, a counterflow centrifugal elutriation (CCE) module, a magnetic cell sorter module, an electroporation module, a sorting module (e.g., a fluorescence-activated cell sorting (FACS) module), a spinoculation module, an acoustic flow cell module, a microfluidic concentration module, and / or a combination thereof. In some embodiments, a working cell may process two or more cartridges in parallel. For example, a working cell may comprise multiple bays, each configured to interface with a cartridge, so that multiple bays within the working cell can be used at any given time. The cell processing systems described herein may reduce operator intervention and increase throughput by automating the movement of cartridges between instruments using a robot. However, in some embodiments, cartridges may be moved manually between instruments. Furthermore, automated cell processing systems may facilitate the transfer of sterile fluid between cartridges and instruments or other components of the system, such as fluid connectors (e.g., sterile fluid transfer ports), reagent storage units, second cartridges, sampling containers, sterile fluid transfer devices, or combinations thereof.

[0026] I. Cell Processing Systems Figure 1A shows an illustrative cell processing system for use with electroporation devices, systems, and methods. A block diagram of the cell processing system 100, including a working cell 110 and a controller 120, is shown. The working cell 110 may comprise one or more of the following: equipment 112, robot 116 (e.g., a robotic arm), reagent storage unit 118, fluid connector 132, sterilizer source 129, fluid source 136, pump 138, and sensor 140. A cartridge 114 and sterilizer transfer device 142, which are part of the cell processing system 100, may be used within the working cell 110. The controller 120 may comprise one or more of the following: processor 122, memory 124, communication device 126, input device 128, and display 130.

[0027] The working cell 110 may comprise a fully or at least partially sealed housing in which one or more cell processing steps are performed in a fully or at least partially automated process. In some modifications, the working cell may be an open system without a housing, which may be configured for use in a cleanroom, biosafety cabinet, or other sterile location. The cartridge 114 may be moved using a robot 116 to reduce manual labor in the cell processing steps, and the transfer of sterile fluids in and out of the cartridge may also be performed in a fully or partially automated process. For example, one or more fluids may be stored in a sterile fluid transfer device 142. In some modifications, the sterile fluid transfer device is a portable consumable that may be moved within the system 100. The sterile fluid transfer devices and fluid connectors described herein may help enable the transfer of fluids in an automated, metered manner of sterilization for automating cell therapy production.

[0028] In some variations, the robot 116 is configured to move the cartridges 114 between different instruments to perform a predetermined sequence of cell processing steps. In this way, different steps of the cell processing sequence can be performed simultaneously on different cartridges, so that multiple cartridges 114 can be processed in parallel.

[0029] The fluid connector 132 can be coupled between two or more cartridges 114 to transfer cell products and / or fluids between them. Furthermore, the fluid connector 132 can be coupled between any set of fluid transport components of the system 100 (e.g., cartridges 114, reagent storage unit 118, fluid source 136, sterile fluid transfer device 142, fluid conduits, containers, vessels, etc.). For example, a first fluid connector may be coupled between a first cartridge and a sterile fluid transfer device, and a second fluid connector may be coupled between a sterile fluid transfer device and a second cartridge.

[0030] As illustrated in Figure 1B, the cartridge 114 may comprise one or more of the following, as will be described in more detail herein: a bioreactor 150, a cell separation system 152, an electroporation module 160, a fluid transfer bus 162, a sensor 164, and a fluid connector 166. The cell separation system 152 may comprise one or more of the following: a rotor 154, a flow cell 156, and a magnet 158. In some embodiments, the magnet 158 ​​may comprise one or more magnets and / or a magnet array. For example, the cell separation system 152 may comprise a first magnet configured to magnetically rotate the rotor 154 and a second magnet (e.g., a magnet array) configured to magnetically separate cells in the flow cell 156.

[0031] Any suitable cell processing may be performed using the systems and devices described herein and may include steps such as growth, concentration, selection, sorting, proliferation, activation, transduction, electroporation, and washing. In some variations, a method for processing a solution containing cell products includes the steps of digesting tissue using an enzymatic reagent to release a selected cell population into a solution, enriching cells using a CCE instrument, washing cells using a CCE instrument, selecting cells in a solution using a selection instrument, sorting cells in a solution using a sorting instrument, differentiating or growing cells in a bioreactor, activating cells using an activation reagent, electroporating cells, transduction of cells using a vector, and finishing the cell products.

[0032] Figure 2A shows an illustrative cell processing system for use with the devices, systems, and methods described herein. A working cell 203 is shown. The working cell may be divided into an internal zone 204 with feedthrough access 206 and quality control (QC) equipment 212. An air filtration inlet (not shown) may provide high-efficiency particulate air (HEPA) filtration to provide air quality of ISO 7 or higher within the internal zone 204. This air filtration may maintain sterile cell processing in an ISO 8 or ISO 9 manufacturing environment. The working cell 203 may also have an air filter at the air outlet to maintain the ISO rating of the room. Similar to the working cell described above with reference to Figure 1A, the working cell 203 may further comprise, within the internal zone 104, a bioreactor instrument 214, a cell selection instrument 216 (e.g., a magnetic separation instrument), an electroporation instrument 220, a counterflow centrifugation elutriation (CCE) instrument 222, a sterile fluid transfer instrument 224 (e.g., a fluid connector), a reagent storage unit 226, and a sterilization system 260. The reagent storage unit 226 may be accessible to the user through a sample pickup port 228. A robot 230 (e.g., a support arm, a robotic arm) may be configured to move one or more cartridges 250 (e.g., consumables) from any instrument to any other instrument, and / or move one or more cartridges 250 to and from the reagent storage unit. In some embodiments, the working cell 203 may comprise one or more movable barriers 213 (e.g., access doors) configured to facilitate access to one or more instruments within the working cell 203. Figure 2B is a perspective view of the working cell 205 of the cell processing system. Figure 2C is a perspective view of the cell processing system depicting the cartridge 250 introduced into the working cell 205. Multiple cartridges can be inserted into the working cell 205 to perform one or more cell processing operations in parallel.

[0033] Figure 2D is a schematic diagram illustrating one embodiment of the work cell 200. The work cell 200 may comprise a housing 202 having four walls, a base, and a roof. The work cell may be divided into an internal zone 204 with feedthrough access 206, a biosafety cabinet (BSC) 208, a computing server rack 210 (e.g., controller 120), and quality control (QC) equipment 212. An air filtration inlet (not shown) may provide high-efficiency particulate air (HEPA) filtration to provide air quality of ISO 7 or higher within the internal zone 204. The work cell may also have an air filter at the air outlet to maintain the ISO rating of the room. Similar to the working cell described above, the working cell 200 may further include, within its internal zone 204, an instrument 211 (e.g., located in a general-purpose instrument bay), a bioreactor instrument 214, a cell selection instrument 216 (e.g., a magnetic separation instrument, a cell selection system), a cell sorting instrument 218 (e.g., a FACS), an electroporation instrument 220, a countercurrent centrifugation elutriation (CCE) instrument 222, a sterile fluid transfer instrument 224 (e.g., a fluid connector), a reagent storage unit 226, and a sterilization system 260 including one or more of a sterilizer source, a fluid source, and a pump. The reagent storage unit 226 may be accessible to the user through a sample pickup port 228. A robot 230 (e.g., a support arm, a robotic arm) may be configured to move one or more cartridges 250 from any instrument to any other instrument or reagent storage unit.

[0034] In some embodiments, a human operator may load one or more cartridges 250 into the feedthrough 206. The cartridges 250 may be pre-sterilized, or the feedthrough 206 may sterilize the cartridges 250 using ultraviolet (UV) light or chemical sterilizers provided as spray or wash. The feedthrough 206 chamber may optionally be configured to automatically spray, wash, irradiate, or otherwise treat the cartridges (e.g., with ethanol and / or isopropyl alcohol solution) to maintain the sterility of the internal zone 204 (e.g., ISO 7 or higher) or the biosafety cabinet 208 (e.g., ISO 5 or higher). The cartridges 250 may be passed to the biosafety cabinet 206, where input cell products are provided and loaded into the cartridges using a sterile liquid transfer device 224 (e.g., a fluid connector). The user may then return the cartridges 250 to the feedthrough 206 and initiate automated processing using a computer processor (e.g., a controller 120) in a computer server rack 210. The robot 230 may be configured to move the cartridge 250 to multiple devices and stations in a predefined sequence, and the components of the work cell 200 are controlled by a computer processor in a computer server rack 210.

[0035] Other preferred cell processing systems and embodiments thereof are provided, for example, in U.S. Patent Application No. 17 / 198,134, published as U.S. Patent Application Publication No. 2021 / 0283565, which is incorporated herein by reference.

[0036] A. Working cell i. Robot In general, the robot in a work cell may be equipped with any mechanical device capable of moving a cartridge from one location to another within the work cell. For example, the robot may be equipped with a mechanical manipulator (e.g., an arm) that is in a fixed position or mounted on a linear rail, or with a two-dimensional or three-dimensional rail system. In some of the figures, the robot is shown to be fixed in a fixed position or fixed to a rail system, but it does not have to be so. For example, in some modifications, the robot may be equipped with a wheeled device. Any number of robots may be used within the work cell described herein. For example, in some embodiments, the work cell may be equipped with two or more robots of the same or different types (e.g., two robotic arms, each independently configured to move a cartridge between devices). The robot may also be equipped with end effectors for precise handling of different cartridges or for barcode scanning or radio-frequency identification tag (RFID) reading.

[0037] A robot for use with the cell processing system described herein can move the cartridge between slots or bays within the working cell so that the modules within the cartridge can be coupled to the corresponding equipment within the working cell to perform different cell processing steps.

[0038] ii. Controller In an embodiment, the cell processing system 100 may include a controller 120 (e.g., a computing device) which includes one or more processors 122, memory 124, a communication device 126, an input device 128, and a display 130. The controller 120 may be configured to control (e.g., operate) the working cell 110. The controller 120 may include multiple devices. For example, the working cell 110 may surround one or more components of the controller 120 (e.g., the processor 122, memory 124, and communication device 126), and one or more components of the controller 120 (e.g., the input device 128 and display 130) may be located away from the working cell 110.

[0039] iii. Processor A processor as described herein (e.g., processor 122) may process data and / or other signals and control one or more components of a system. A processor may be configured to receive, process, compile, compute, store, access, read, write, and / or transmit data and / or other signals. Additionally or alternatively, a processor may be configured to control one or more components of a device (e.g., a console, a touchscreen, a personal computer, a laptop, a tablet, a server).

[0040] In some embodiments, the processor may be configured to access one or more of the working cell 110, the server, the controller 120, and storage media (e.g., memory, flash drive, memory card, database) or to receive data and / or other signals from them. In some embodiments, the processor may be any suitable processing device configured to operate and / or execute a set of instructions or code, and may include one or more data processors, image processors, graphics processing units (GPUs), physical processing units, digital signal processors (DSPs), analog signal processors, mixed signal processors, machine learning processors, deep learning processors, finite state machines (FSMs), compression processors (e.g., data compression to reduce data rate and / or memory requirements), cryptography processors (e.g., for secure wireless data transfer), and / or central processing units (CPUs). The processor may be, for example, a general-purpose processor, a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), a processor board, and / or similar. The processor may be configured to run and / or execute application processes and / or other modules, system-associated processes and / or functions.The underlying device technologies can be provided by various component types, such as metal-oxide semiconductor field-effect transistor (MOSFET) technologies like complementary metal-oxide semiconductor (CMOS), bipolar technologies like emitter-coupled logic (ECL), polymer technologies (e.g., silicone-conjugated polymers, and metal-conjugated polymer-metal structures), and analog and digital hybrid technologies.

[0041] The systems, devices, and / or methods described herein may be implemented by software (running on hardware), hardware, or a combination thereof. Hardware modules may include, for example, general-purpose processors (or microprocessors or microcontrollers), field-programmable gate arrays (FPGAs), and / or application-specific integrated circuits (ASICs). Software modules (running on hardware) may be expressed in various software languages ​​(e.g., computer code), including structured text, TypeScript, C, C++, C#, Java®, Python, Ruby, Visual Basic®, and / or other object-oriented, procedural, or other programming languages ​​and development tools. Examples of computer code include, but are not limited to, microcode or microinstructions, machine instructions such as those generated by a compiler, code used to generate web services, and files containing high-level instructions executed by a computer using an interpreter. Additional examples of computer code include, but are not limited to, control signals, encryption code, and compression code.

[0042] iv. Memory The cell processing systems and devices described herein may include a memory (e.g., memory 124) configured to store data and / or information. In some embodiments, the memory may include one or more of the following: random access memory (RAM), static RAM (SRAM), dynamic RAM (DRAM), memory buffer, erasable programmable read-only memory (EPROM), electrically erasable read-only memory (EEPROM), read-only memory (ROM), flash memory, volatile memory, non-volatile memory, or combinations thereof. In some embodiments, the memory may store instructions for causing a processor to execute device-related modules, processes, and / or functions, such as image processing, image display, sensor data, data and / or signal transmission, data and / or signal reception, and / or communication. Some embodiments described herein may relate to computer storage products with a non-temporary computer-readable medium (which may also be referred to as a non-temporary processor-readable medium) having instructions or computer code for performing various computer implementation operations. Computer-readable media (or processor-readable media) are non-transient in the sense that they do not contain transient propagating signals themselves (e.g., propagating electromagnetic waves that carry information on a transmission medium such as space or a cable). Computer code (which may also be called code or algorithms) may be designed and constructed for one or more specific purposes. In some embodiments, memory may be configured to store received data, as well as data generated by the controller and / or working cells. In some embodiments, memory may be configured to store data temporarily or permanently.

[0043] v. Input devices In some embodiments, the input device 128 may include, for example, a display or be coupled thereto. The input device may be any suitable device capable of receiving input from a user, such as a keyboard, buttons, or a touchscreen. The input device may include at least one switch configured to generate user input. For example, the input device may include a touch surface for the user to provide input corresponding to user input (e.g., finger contact to the touch surface). An input device including a touch surface may be configured to detect contact and movement on the touch surface using any of a plurality of touch sensitivity techniques, including capacitive, resistive, infrared, optical imaging, dispersed signaling, acoustic pulse recognition, and surface acoustic wave techniques. In embodiments of an input device including at least one switch, the switch may have at least one of the following: a button (e.g., a hard key, a soft key), a touch surface, a keyboard, an analog stick (e.g., a joystick), a directional pad, a mouse, a trackball, a jog dial, a step switch, a rocker switch, a pointer device (e.g., a stylus), a motion sensor, an image sensor, and a microphone. The motion sensor may receive user movement data from an optical sensor and classify the user's gestures as user input. The microphone can receive acoustic data and recognize the user's voice as user input.

[0044] In some embodiments, the cell processing system may optionally include one or more output devices in addition to a display, such as an audio device and a haptic device. The audio device may audibly output any system data, alarms, and / or notifications. For example, the audio device may output an audible alarm when a malfunction is detected. In some embodiments, the audio device may include at least one of a speaker, a piezoelectric audio device, a magnetostrictive speaker, and / or a digital speaker. In some embodiments, a user may communicate with other users using the audio device and communication channels. For example, a user may form a voice communication channel (e.g., a VoIP call).

[0045] Additionally or alternatively, the system may include a haptic device configured to provide the user with additional sensory output (e.g., force feedback). For example, the haptic device may generate a tactile response (e.g., vibration) to confirm user input to an input device (e.g., a touch surface). As another example, haptic feedback may indicate that user input is being invalidated by the processor.

[0046] vi. Communication devices In some embodiments, the controller may include a communication device (e.g., communication device 126) configured to communicate with another controller and one or more databases. The communication device may be configured to connect the controller to another system (e.g., the Internet, a remote server, a database, a work cell) by a wired or wireless connection. In some embodiments, the system may communicate with other devices via one or more wired and / or wireless networks. In some embodiments, the communication device may include a radio frequency receiver, transmitter, and / or optical (e.g., infrared) receiver and transmitter configured to communicate with one or more devices and / or networks. The communication device may communicate by wire and / or wireless.

[0047] vii. Display Image data can be output onto a display of the cell processing system (e.g., display 130). In some embodiments, the display may include at least one of the following: light-emitting diodes (LEDs), liquid crystal displays (LCDs), electroluminescent displays (ELDs), plasma display panels (PDPs), thin-film transistors (TFTs), organic light-emitting diodes (OLEDs), electronic paper / electronic ink displays, laser displays, and / or holographic displays.

[0048] viii. Graphical User Interface In some embodiments, as shown above, the GUI may be configured to design processes and monitor products. For example, the GUI may be a process design homepage. The GUI may indicate that no processes are selected or loaded. A create icon (e.g., “Create Process”) may be selectable by the user to initiate the process design process. In some embodiments, one or more of the GUIs described herein may include a search bar.

[0049] B. Cartridge The cell processing systems described herein may comprise one or more cartridges having one or more modules configured to interface with one or more instruments in a working cell.

[0050] Various materials may be used to construct the cartridge and cartridge housing, including metal, plastic, rubber, and / or glass, or combinations thereof. The cartridge, its components, and its housing may be molded, machined, extruded, 3D printed, or any combination thereof. The cartridge may house commercially available components (e.g., pipes, valves, fittings), which may be mounted on or integrated with custom components or devices. The cartridge housing may constitute an additional sealing layer to further protect the sterility of the cell products.

[0051] In some embodiments, modules may be integrated in a fixed configuration within a cartridge. Additionally or alternatively, modules may be configurable or movable within a cartridge, allowing for the assembly of various types of cartridges. For example, a cartridge may be a single enclosed unit with fixed components for each module, or a cartridge may house configurable modules coupled by configurable fluid, mechanical, optical, and electrical connections. In some modifications, one or more subcartridges, each housing a set of modules, may be used to perform various cell processing workflows. Modules may be provided in separate housings or integrated with other modules within a cartridge or subcartridge. While this disclosure generally presents modules as separate groups of components for simplicity, it should be noted that these modules may be arranged in any preferred configuration. For example, components for different modules may be scattered amongst themselves, such that each module is defined by a set of connected components that collectively perform a predetermined function. However, the components of each module may or may not be physically grouped within a cartridge. In some embodiments, a single cell product can be processed using multiple cartridges by transferring the cell product from one cartridge to another cartridge of the same or different type, and / or by dividing the cell product into more cartridges, and / or by pooling multiple cell products into fewer cartridges.

[0052] Generally, each instrument within a working cell interfaces with its respective module(s) on a cartridge. For example, if a cartridge has an electroporation module, it is moved by a robot to the electroporation instrument within the working cell to perform electroporation on the cells in the cartridge. One advantage of such a segmented module / instrument design is that expensive components (e.g., motors, sensors, heaters, lasers, etc.) can be kept within the system's instruments, while less expensive components reside in cartridges, which are typically disposable and configured for single use. The use of disposable cartridges can eliminate the need to sterilize the cartridge between uses. Furthermore, having multiple instruments within a working cell is even more helpful in enabling parallel use of these instruments when multiple cartridges are used within the working cell. In contrast, most conventional semi-automated instruments have instrument components that are idle and cannot be used simultaneously.

[0053] Additional preferred embodiments of the cartridge are provided, for example, in U.S. Patent Application No. 17 / 198,134, published as U.S. Patent Application Publication No. 2021 / 0283565, which is incorporated herein by reference.

[0054] C. Electroporation System As described above, one of the cell processing steps that can be performed is electroporation. In the modified form in which electroporation is performed, the cartridge comprises an electroporation module, and the working cell comprises a corresponding electroporation apparatus. The electroporation module in the cartridge may comprise one or more electroporation chambers, and the electroporation apparatus may be configured to apply electroporation energy to one or more electroporation chambers to electroporate the cells therein.

[0055] Energy can be applied to the electroporation chamber simultaneously or continuously. For this purpose, the electroporation apparatus can be configured to deliver electrical energy to the electroporation chamber. The amount of energy supplied to the electroporation chamber can be predetermined or based on the volume of cell solution in the electroporation chamber.

[0056] Figures 3A to 3F provide schematic and illustrative diagrams of the electroporation chamber of this disclosure.

[0057] Figure 3A provides an exploded view of an exemplary electroporation chamber 306. The electroporation chamber 306 may comprise a first housing 305A, a first electrode 308A, an electroporation cell 315, a second electrode 308B, and a second housing 305B. The first housing 305A and the second housing 305B may sandwich, surround, enclose, or otherwise house the first electrode 308A, the electroporation cell 315, and the second electrode 308B. As illustrated in Figure 3A, the first electrode 308A may form a layer between the first housing 305A and the electroporation cell 315. The second electrode 308B may form a layer between the second housing 305B and the electroporation cell 315. The first electrode 308A may be bonded to a first surface of the electroporation cell 315. The second electrode 308B may be coupled to the second surface of the electroporation cell 315. The distance between the first surface and the second surface of the electroporation cell 315, together with the opening 315' of the electroporation cell 315, defines the fluid conduit of the electroporation chamber 306. The fluid conduit will be described in more detail below. The first electrode 308A and the second electrode 308B may have at least one alignment feature corresponding to at least one alignment feature of the electroporation cell 315. For example, the at least one alignment feature may be an orifice, a hole, a projection, a recess, or any other surface feature. The first electrode 308A and the second electrode 308B may be coupled to the electroporation cell 315 in any preferred manner, such as by glue, screw, adhesive, friction, or other similar fastening devices or mechanisms.

[0058] The first electrode 308A, the second electrode 308B, and the electroporation cell 315 may be aligned relative to each other using at least one alignment feature and subsequently compressed to form a watertight seal around the combined assembly. The coupling of the first housing 305A and the second housing 305B around the first electrode 308A and the second electrode 308B and the electroporation cell 315 may be fluidically sealed. The fluid seal may be achieved via adhesives, polymers, welding procedures, coupling tabs, interlocking fingers, or any other similar mechanism. The fluid seal may prevent substantially any cell solution from leaking through the layers into the external environment. The fluid seal may prevent substantially any substance in the external environment from penetrating into the electroporation chamber and / or electroporation system.

[0059] Figure 3B is a schematic diagram of a system including one or more electroporation chambers, such as electroporation chamber A 307 and electroporation chamber B 309. For simplicity, Figures 3B to 3F are described with reference to a single electroporation chamber (e.g., chamber A 307) where necessary. Chamber A 307 may comprise at least one electrode 308. For example, referring to Figure 3A, chamber A 307 may comprise a first electrode 308A and a second electrode 308B, where these electrodes are conductive and may be configured to generate an electric field between them. The electric field may be generated to induce membrane permeabilization of cells within the fluid conduit 314 of chamber A 307, as described below.

[0060] The electric field can be determined by the applied voltage (V) and the distance (d) between electrodes 308A and 308B. For example, the electric field (E) can be calculated by the formula E = V / d. In some modifications, the distance between the first electrode 308A and the second electrode 308B may be about 0.5 mm to about 5 mm. For example, the distance between the first electrode 308A and the second electrode 308B may be at least about 0.5 mm, at least about 1 mm, at least about 1.5 mm, at least about 2 mm, at least about 2.5 mm, at least about 3 mm, at least about 3.5 mm, at least about 4 mm, and / or at least about 4.5 mm. In some embodiments, the distance between electrodes 308A and 308B may be fixed at, for example, d = 2 mm. The applied voltage may be determined by the type of cells being treated. For example, the pores of T cells may be opened with an effective electric field of about 1 to 3 kV / cm. If an effective electric field of 1 kV / cm is desired, the required voltage may be 200 V. If an effective electric field of 3 kV / cm is desired, the required voltage may be 600 V. In general, the effective electric field between at least two electrodes in at least one electroporation chamber may be at least 100 V / cm, at least 200 V / cm, at least 300 V / cm, at least 400 V / cm, at least 500 V / cm, at least 600 V / cm, at least 700 V / cm, at least 800 V / cm, at least 900 V / cm, at least 1 kV / cm, at least 2 kV / cm, at least 3 kV / cm, at least 4 kV / cm, and at least 5 kV / cm.

[0061] Chamber A 307 may further comprise a fluid conduit 314. During the electroporation process, the fluid conduit 314 may be configured to receive a volume of cell solution of 2.5 mL or less. For example, the fluid conduit 314 can accommodate volumes of at least 0.5 mL, at least 1 mL, at least 1.5 mL, at least 2 mL, at least 3 mL, at least 3.5 mL, at least 4 mL, at least 4.5 mL, and at least 5 mL. In some modifications, the fluid conduit 314 may be configured to accommodate about 1.5 mL to about 2.0 mL of fluid. For this purpose, the volume of the fluid conduit 314 is determined based on the shape and depth of the opening (opening "O" in Figure 3A) in the electroporation cell 315, where the depth defines the distance separating the first electrode 308A from the second electrode 308B when the electroporation chamber is assembled. The fluid conduit 314 may be configured to accommodate a mixture of gas and liquid.

[0062] Chamber A 307 may further include an inlet port 312, an outlet port 316, and a vent port 310. At least one of the inlet port 312, the outlet port 316, and the vent port 310 may be configured to control and / or allow a fluid (e.g., a gas, liquid, or a mixture thereof) to flow into and / or out of the fluid conduit 314. For example, each port may have a closed configuration and an open configuration. In a closed configuration, each of the multiple ports may prevent the flow of fluid through it. In an open configuration, each of the multiple ports may allow the flow of fluid through it. In some embodiments, each of the multiple ports may have a partially open configuration. For example, a partially open configuration may allow the flow of fluid at a reduced flow rate compared to an open configuration. Each of the multiple ports may cause a pressure drop in the fluid flowing through it. The pressure drop associated with one port may be different from the pressure drop associated with any other port. Also note that ports do not need to be configured to have the same configuration at the same time. Some ports can be opened, while others are closed or partially open.

[0063] Figure 3C provides an illustrative depiction of an electroporation chamber that may be used with the systems, devices, and methods described herein. The electroporation chamber may be electroporation chamber A 307 and / or electroporation chamber B 309. Electroporation chambers 307, 309 may include a first portion 320 and a second portion 330. The first portion 320 may include an outlet port 316 and a first window 318A. The second portion may include an inlet port 312, a vent port 310, and a second window 318B.

[0064] In some embodiments, there may be a first window through the side wall providing a view to a first portion of the electroporation chambers 307, 309. There may also be a second window providing a view to a second portion of the electroporation chambers 307, 309. The first and second windows may, advantageously, provide visibility to each portion of the fluid conduit 314. In some embodiments, there may be three or more windows. For example, a third window may be located close to the inlet port 312. In some embodiments, windows may be located close to any of a plurality of ports. In some embodiments, windows 318A, 318B may comprise a transparent material such as glass, polymer, or a combination thereof. The window material may comprise a flexible, durable material that can withstand one or more of vibration, deflection, and torsion without cracking or shattering.

[0065] In some embodiments, the electroporation apparatus may include at least one sensor corresponding to one of the windows 318A, 318B. For example, a first sensor may be located close to the first window 318A, and a second sensor may be located close to the second window 318B. The sensors may be outside the electroporation chamber and may be located within 1, 2, 3, 4, 5, or 6 inches of the window. In some embodiments, the sensors have a direct line of sight to the fluid conduit 314 of the electroporation chambers 307, 309. At least one sensor may be configured to detect the presence or absence of fluid in the fluid conduit 314. The at least one sensor may be any suitable sensor, such as an optical sensor, a pressure sensor, an infrared sensor, a capacitive sensor, or any combination thereof.

[0066] In some embodiments, at least one sensor may be located near at least one of the outlet port 316, the inlet port 312, and the vent port 310. At least one sensor may be located near other components in the electroporation system, such as a fluid pump and fluid pipes. At least one sensor may be configured to detect the filling state of the fluid conduit 314. For example, at least one sensor may detect whether the fluid conduit 314 is substantially empty or whether the fluid conduit 314 contains a target volume of fluid. In some embodiments, at least one sensor may detect whether the fluid conduit 314 contains a volume of fluid exceeding the target volume. At least one sensor may also detect whether fluid is flowing into or out of the fluid conduit 314 through at least one of the inlet port 312, the outlet port 316, and the vent port 310. At least one sensor may detect whether the flow rate into or out of the fluid conduit 314 is substantially zero.

[0067] The inlet port 312, outlet port 316, and vent port 310 may extend from or protrude from the electroporation cell 315. In some embodiments, the inlet port 312, outlet port 316, and vent port 310 extend from or protrude from the electroporation cell 315 through at least one of the first electrode 308A and the first housing 305A and / or the second electrode 308B and the second housing 305B. This allows the windows 318A, 318B to be suitably positioned at any location around the electroporation chamber. In some embodiments, any of the multiple ports may protrude through any other wall or surface of the electroporation chamber. For example, at least one of the multiple ports may extend from the electroporation cell 315 through the side wall of the electroporation chamber and not through any one of the electrodes. In some embodiments, the positioning of the multiple ports may help minimize the dimensions of at least one of the electroporation chambers.

[0068] Figures 3D to 3F provide additional illustrative diagrams of electroporation chambers 307 and 309. Electroporation chambers 307 and 309 may include a first portion 320. The first portion 320 may comprise at least one auxiliary channel, such as a first auxiliary channel 325A. The first auxiliary channel 325A may fluidly connect the outlet port 316 to a fluid conduit 314. The first portion 320 may further comprise a first window 318A, which may fluidly connect to at least one of the auxiliary channel 325A and the fluid conduit 314. The first window 318A may be located on the side wall of the electroporation chamber 307 or 309, adjacent to the first auxiliary channel 325A connected to the outlet port 316. The fluid conduit 314 may comprise a cavity configured to receive a fluid, such as a cell solution. The fluid conduit 314 may comprise a first segment 335 and a second segment 345. In some embodiments, the first segment 335 of the fluid conduit 314 may be adjacent to a first portion 320 of the electroporation chamber 307. In some embodiments, the second segment 345 of the fluid conduit 314 may be adjacent to a second portion 330 of the electroporation chamber 307.

[0069] The second portion 330 may comprise at least one auxiliary channel. The second portion 330 may comprise a second auxiliary channel 325B and a third auxiliary channel 325C. The second auxiliary channel 325B may fluidly connect the inlet port 312 to the fluid conduit 314. The third auxiliary channel 325C may fluidly connect the vent port 310 to the fluid conduit 314. Similar to the first auxiliary channel 325A, the second auxiliary channel 325B and the third auxiliary channel 325C may each have a window adjacent to it. The second viewing window 318B may be positioned adjacent to the second auxiliary channel 325B coupled to the vent port 310. In some embodiments, the third window may be positioned adjacent to the third auxiliary channel 325C coupled to the inlet port 312.

[0070] In some embodiments, the fluid conduit 314 may be in fluid communication with each of the multiple ports 310, 312, and 316 via auxiliary channels 325A, 325B, and 325C extending therefrom. The auxiliary channels 325A, 325B, and 325C may have any preferred cross-sectional shape, such as circular, square, rectangular, triangular, or a combination thereof. In some embodiments, the fluid conduit 314 may have a triangular cross-section along an axis defined from the first portion 320 of the electroporation chambers 307, 309 to the second portion 330 of the electroporation chambers 307, 309. In some embodiments, the inner surfaces of the auxiliary channels 325A, 325B, and 325C may have surface features. The auxiliary channel 325A that is in fluid communication with the outlet port 316 may be curved. In some embodiments, the first window 318A is adjacent to at least one curved auxiliary channel. In some embodiments, the first window 318A may be located near the base of a curved auxiliary channel. In some embodiments, the second window 318B may be located near an auxiliary channel 525B that is in fluid communication with the vent port 310. In some embodiments, the second window 318B may be located near the base of the auxiliary channel 525B that is in fluid communication with the vent port 310. The inlet port 312, outlet port 316, and vent port 310 may each be configured to direct the fluid flow. For example, the inlet port 312 may be configured to receive flow from an external source and direct the fluid into the fluid conduit 314 via the auxiliary channel 325C. At least one sensor may be located near at least one of the auxiliary channels 325A, 325B, and 325C.

[0071] In some embodiments, the inlet port 312 may receive fluid from the cartridge's fluid transfer bus. The outlet port 316 may be configured to receive fluid from the fluid conduit 314 and direct the fluid to an external component. In some embodiments, the outlet port 316 may be configured to direct the fluid to a fluid pump device or fluid transfer bus. The vent port 310 may be configured to discharge fluid from the fluid conduit 314 and / or purge the fluid. For example, the fluid discharged and / or purged through the vent port 310 may be a gas or a liquid. The fluid in the fluid conduit 314 may be air, a byproduct of the electroporation process of the cell solution, or any other gas.

[0072] Referring here to Figure 4A, an illustrative schematic diagram of an electroporation system 400 is provided, comprising at least one electroporation module 405 in at least one cartridge 401 and at least one electroporation device 413 in a working cell 411. The electroporation system 400 has at least one electroporation chamber (e.g., 407, 409) of the electroporation module 405 in cartridge 401. The electroporation module 405 is configured to be coupled to the electroporation device 413 in the working cell 411 so that electroporation can be performed. The electroporation module 405 can be operably coupled to the module or otherwise interfaced with the module so that electrical energy can be delivered to at least one electroporation chamber.

[0073] At least one electroporation chamber of the electroporation module 405 may comprise a first electroporation chamber 307 ("EP Chamber A") and optionally a second electroporation chamber 409 ("EP Chamber B"). The electroporation chambers 407 and 409 may, but do not have to be, located adjacent to each other within the cartridge 401. In some embodiments, the electroporation chambers 407 and 409 may be separated from each other within the cartridge 401.

[0074] Cartridge 401 may include a fluid transfer bus 403 that fluid-couples the electroporation module 405 to other modules of cartridge 401. The fluid transfer bus 403 may be configured to transport at least one type of fluid between modules of cartridge 401 via tubes or pipes. The fluid may be a liquid, a gas, or a mixture thereof. The fluid transported within the fluid transfer bus 403 may be a reagent or a cell solution containing any type of cell, including patient-derived cells or allogeneic cells.

[0075] The electroporation apparatus 413 may include a docking station 417 configured to receive a cartridge 401. The docking station 417 may include a cavity having structural features corresponding to features on the cartridge 401. For example, the docking station may have at least one track configured to receive at least one rail, wheel, projection, indentation, or similar feature on the cartridge 401. The cartridge 401 may be configured to slidably fit within the docking station 417 of the electroporation apparatus 413. For example, the cavity may be slightly larger in size than the shape and size of the cartridge 401. The cartridge may rely on frictional force to hold it within the docking station 417. In some embodiments, the cartridge may be held in place via at least one of magnets, latches, push rods, or similar features. The docking station may further include at least one electroporation feature (e.g., a high-voltage connector) on the surface of the cavity to interface with the electrodes of the electroporation module 305 of the cartridge 401 when the cartridge 401 is inside the docking station 417.

[0076] The electroporation apparatus 413 may include a high-voltage connector 423 and a programmable logic controller (PLC) control system 327. The PLC control system 327 may be electrically connected to the high-voltage connector 423. The PLC control system 327 may transmit at least one of digital and analog signals to the high-voltage connector 423. In some embodiments, the PLC control system 327 may transmit electrical signals to the high-voltage connector 423.

[0077] The high-voltage connector 423 may be electrically connected to the docking station 317. The high-voltage connector 423 may be connected to at least one surface feature of the docking station 317. In some embodiments, the high-voltage connector 423 may be electrically coupled to at least one electrical lead or electrode on the docking station. In some embodiments, the high-voltage connector 423 may contact a feature of the electroporation module 305 of the cartridge 401 via an electroporation feature on the surface of the cavity when the cartridge 401 is inside the docking station 317.

[0078] In some embodiments, the high-voltage connector 423 further comprises a multiplexer (MUX) 425. The MUX 425 may be a mechanical multiplexer. For example, the MUX 425 may include a mechanical switch that moves physically in response to at least one input signal. The MUX 425 may be configured to adjust the output of the high-voltage connector 423. For example, the MUX 425 may isolate the output of the high-voltage connector 423 to a selected electroporation chamber, such as EP chamber A 407 or EP chamber B 409. In some embodiments, the MUX 425 may rapidly switch the output between the electroporation chambers. In some embodiments, the MUX 425 may select both electroporation chambers simultaneously and thus provide an electrical output to both EP chamber A 407 and EP chamber B 409 at the same time.

[0079] The electroporation device 413 may further comprise an electroporation device controller 419 and a fluid transfer bus controller 421. The electroporation device controller 419 may control at least one function of the electroporation device 413. For example, the electroporation device controller 419 may control at least one of the fluid transfer bus controller 421, at least one sensor 420, or a programmable logic controller (PLC) control system 427 via at least one electrical signal. In some modifications, the electroporation device controller 419 transmits a signal to the fluid transfer bus controller 421 to start or stop the fluid flow from the fluid transfer bus to EP chambers A and B (407 and 409). In some modifications, the electroporation device controller 319 receives a signal from the fluid transfer bus controller 421 indicating the status of the fluid flow through the fluid transfer bus 403 or the absence thereof. The electroporation device controller 419 may transmit an electrical signal to at least one sensor 420. For example, the electroporation equipment controller 419 can switch the state of at least one sensor 420 from on to off, or vice versa. In some modifications, the electroporation equipment controller 419 receives a signal from at least one sensor 420. For example, at least one sensor 420 may provide the electroporation equipment controller 419 with a continuous or discontinuous output signal. In some embodiments, the output signal from at least one sensor 420 may be one or more of a video stream, a picture, a digital or analog electrical signal, a pressure measurement, a capacitance measurement, or a combination thereof.

[0080] The electroporation equipment controller 419 may also transmit electrical signals to the PLC control system 427. For example, the electroporation equipment controller 419 may transmit a power input to the PLC control system 427 that can switch the state of the PLC control system 427 from on to off or vice versa. In some embodiments, the power signal transmitted from the electroporation equipment controller 419 to the PLC control system 427 includes a desired output voltage to be supplied to the high-voltage connector 423, and therefore to the electroporation module 405 of the cartridge 401. The desired output voltage may be preset, predetermined, or depend on one or more factors and may be determined in real time using one or more sensors. For example, the output voltage may be determined at least in part on the type of cells in the cell solution being electroporated. The electroporation equipment controller 419 may receive signals from the PLC control system 427. For example, the PLC control system may transmit one or more of the following to the electroporation equipment controller 419: resistance value, voltage value, and on / off state. The electroporation controller 419, the PLC control system 427, the fluid transfer bus controller 421, and the sensor 420 can all be electrically connected. It should be understood that data received from any one of the fluid transfer bus controller 421, at least one sensor 420, and the PLC control system 427 may influence subsequent commands generated by the electroporation equipment controller. For example, data received from at least one sensor 420 may cause the electroporation equipment controller 419 to send a command to the fluid transfer bus controller 421 to stop the fluid flow. In another example, data received from at least one sensor 420 may cause the electroporation equipment controller 419 to send a command to the PLC control system 427 to start electroporation.

[0081] In some variations, the electroporation equipment controller 419 sends a signal to the fluid transfer bus controller 421, which in turn sends a signal to the docking station 417. For example, the fluid transfer bus controller 421 may send at least one signal to at least one feature of the docking station 417 configured to engage with the cartridge 401. For example, the fluid transfer bus controller 421 may turn on or off a locking mechanism in the docking station configured to hold the cartridge 401 within the docking station. The fluid transfer bus controller 421 can indicate the status of the docking station 417 and the cartridge 401. For example, the status may be an indicator that the cartridge 401 is successfully engaged with the docking station 417. In another example, the status may be an indicator that there is a fluid sealing between the fluid paths between system components.

[0082] The fluid transfer bus control 421 can control at least one function of at least one fluid transfer bus 403 within the cartridge 401. For example, as described above, the fluid transfer bus control 421 can send signals to start or stop the flow in the fluid transfer bus 403. In embodiments, the fluid bus control 421 can send signals to multiple components on the cartridge 401. For example, the fluid transfer bus controller 421 can send a signal to a fluid pump fluid-coupled to the fluid transfer bus 403 of the cartridge 401 to start the fluid flow, and can also send a signal to an inlet port to open the inlet port. In embodiments, the fluid bus control 421 can send multiple signals to multiple components simultaneously.

[0083] Figure 4B is a schematic diagram of an electroporation system, in which the electroporation module of a cartridge is coupled to the electroporation equipment 413 of a working cell. In this modified example, the electroporation module includes electroporation chambers 407, 409. The electroporation equipment 413 may include an electroporation equipment controller 419 that can be electrically connected to a PLC control system 427, at least one sensor 420, and a fluid transfer bus controller 421. In this embodiment, the PLC control system 427, at least one sensor 420, and the fluid transfer bus controller 421 can be electrically connected to components housed in the electroporation chambers 407, 409. In this way, the electroporation equipment controller 419 can indirectly control at least one component within the electroporation chambers 407, 409 when the electroporation chambers 407, 409 are docked into the docking station 417 of the electroporation equipment 413. For example, the electroporation equipment controller 419 may transmit a signal to the fluid transfer bus controller 421, which then transmits a signal to the fluid transfer bus 403 and / or controls the fluid transfer bus to cause fluid flow in and between the inlet port 412, vent port 410, and outlet port 416. For example, the fluid transfer bus controller 421 may transmit a signal to open or close each of the inlet port 412, vent port 410, and outlet port 416. In an embodiment, the electroporation equipment controller 419 may receive signals from one or more of the fluid transfer bus 403, inlet port 412, vent port 410, and outlet port 416. For example, one or more of the inlet port 412, vent port 410, and outlet port 416 may transmit a signal indicating their open, closed, or partially closed configuration.

[0084] In another example, the electroporation equipment controller 419 may transmit a signal to at least one sensor 420 that transmits or receives signals through at least one window 418. For example, the at least one sensor 420 may include an optical sensor positioned near at least one window 418 such that the optical sensor can measure the presence or absence of fluid inside the window 418 based on reflected light.

[0085] In one embodiment, the electroporation equipment controller 419 may transmit a signal to and / or receive a signal from the PLC control system 427, and the PLC control system 210 may transmit a signal to and / or receive a signal from the high-voltage connector 423. For example, the high-voltage connector 423 may transmit an electrical signal to at least one electrode 408 when the cartridge and at least one electroporation chamber therein are docked in the docking station. In another example, the high-voltage connector 423 may receive a signal, such as a resistance value or a voltage value, from at least one electrode 408 of at least one electroporation chamber 407, 409.

[0086] The PLC control system 427 can supply continuous or discontinuous electrical signals to at least one electrode 408. In some embodiments, the PLC control system 427 can supply pulsed electrical signals to at least one electrode 408. In some embodiments, the pulsed electrical signals can have a variety of electrical characteristics. In some embodiments, the electrical signals may include about 1 pulse to about 50 pulses, at least about 5 pulses, at least about 10 pulses, at least about 15 pulses, at least about 20 pulses, at least about 25 pulses, at least about 30 pulses, at least about 35 pulses, at least about 40 pulses, and at least about 45 pulses.

[0087] The electrical signal may include any suitable voltage, e.g., approximately 0V to approximately 700V, at least approximately 50V, at least approximately 100V, at least approximately 150V, at least approximately 200V, at least approximately 250V, at least approximately 300V, at least approximately 350V, at least approximately 400V, at least approximately 450V, at least approximately 500V, at least approximately 550V, at least approximately 600V, and at least approximately 650V. The above range of voltages applied to at least one electrode 408 is not limiting, and it should be understood that in many cases the voltage applied to at least one electrode 408 is based on various factors, including the cell solution being electroporated (e.g., the volume of the cell solution, the type of cells in the cell solution, etc.).

[0088] In some embodiments, the electrical signal includes pulse widths of approximately 100 μs to approximately 1 ms, at least 100 μs, at least 200 μs, at least 300 μs, at least 400 μs, at least 500 μs, at least 600 μs, at least 700 μs, at least 800 μs, and at least 900 μs. In some embodiments, the electrical signal includes pulse widths of approximately 1 ns to approximately 100 μs, at least 1 ns, at least 100 ns, at least 250 ns, at least 500 ns, at least 750 ns, at least 1 μs, at least 25 μs, at least 50 μs, and at least 75 μs. The electrical signal may include a series of electrical pulses (constituting a predetermined duration) with pulse intervals of approximately 1 second to approximately 30 seconds, at least approximately 5 seconds, at least approximately 10 seconds, at least approximately 15 seconds, at least approximately 20 seconds, and at least approximately 25 seconds.

[0089] The electroporation system may further comprise a processing circuit as part of (or separately from) the electroporation instrument controller 419. The processing circuit may be configured to perform any number of functions. For example, it may be configured to generate a first signal for inflowing cell solution into the fluid conduit 414 via the inlet port 412, a second signal (for example, based on data received from at least one sensor 420) for stopping the inflow of cell solution into the fluid conduit 414 via the inlet port 412, a third signal for applying an electric field into the fluid conduit 414 for a predetermined duration, and a fourth signal for outflowing cell solution from the fluid conduit 414 via the outlet port 416. In some modifications, the electroporation chambers described herein are in fluid communication with the fluid conduit 414 and comprise vent ports 410 located in the first part of each electroporation chamber 407, 409. The electroporation chambers described herein may further comprise a fluid pump. In some variations, the fourth signal generated by the processing circuit may be configured to cause the cell solution to drain from the fluid conduit 414 by purging the fluid conduit 414 with air through the vent port 410.

[0090] Figure 5 is a schematic diagram of an exemplary embodiment of the electroporation system 500. The electroporation system 500 generally supplies electrical energy to at least one other component within the electroporation system. The electroporation system 500 may include a PLC control system 502. The PLC control system 502 may include a programmable logic controller ("PLC") 501. The PLC 501 may transmit at least one signal to at least one high-voltage DC-DC converter ("high-voltage DC-DC, HVDC-DC") 503. For example, the PLC 501 may transmit a signal 523 to the HVDC-DC 503. The signal 523 may include an electrical signal of 0 to 24V configured to power the HVDC-DC. For example, the HVDC-DC 503 may convert a 24V input from the PLC 501 to a 100 to 700V output. The output from the 100-700V HVDC-DC503 can charge other system components such as a capacitance bank. In one embodiment, signal 523 may include electrical signals greater than 24V, for example, at least about 25V, at least about 30V, at least about 40V, at least about 50V, at least about 60V, at least about 70V, at least about 80V, at least about 90V, and at least about 100V. Signal 423 may also include amperes from 0 to 6.5A. In some embodiments, signal 523 may include electrical signals greater than 6.5A, for example, at least about 7A, at least about 8A, at least about 9A, and at least about 10A. Signal 523 may be configured to turn the HVDC-DC503 on or off. The PLC may send a separate signal 525 to the HVDC-DC403. Signal 525 may include electrical signals from 0 to 4.75V. In embodiments, signal 525 may include electrical signals greater than 4.75V, such as at least about 5V, at least about 6V, at least about 7V, at least about 8V, at least about 9V, and at least about 10V. Signal 525 may be a variable voltage signal configured to set the voltage of the electrical signal output from the HVDC-DC503. For example, signal 525 may be a specific voltage corresponding to a specific voltage output from the HVDC-DC503.

[0091] The electroporation system 500 may also include a charging / discharging circuit (CDC) 505. The CDC 505 may include a switching relay. The switching relay may include a high-wattage, low-resistance damping resistor. In some embodiments, the switching relay may include a low-wattage, high-resistance damping resistor. The CDC 505 may be electrically connected to an HVDC-DC 503. The CDC 505 may be configured to receive an output of 100-700V from the HVDC-DC 503. The CDC 505 may be further configured to receive a signal 527 from a PLC 501. The signal 527 may include an electrical signal of 0-24V. In some embodiments, the signal 527 may enable or disable the CDC 505. The CDC 505 may be configured to output an electrical signal to at least one other component. The CDC 505 may further include a connection to earth.

[0092] The electroporation system 500 may also include a capacitance ("cap") bank 507. The cap bank 507 may be electrically connected to the CDC 505. The cap bank 507 may be configured to receive at least one electrical signal from the CDC 505. The cap bank may include at least one capacitor configured to be charged by the electrical signal provided by the CDC 505. The at least one capacitor may have a capacitance of at least 4.5 mF. In some embodiments, at least one capacitor may have capacitances of at least about 0.5mF, at least about 1mF, at least about 1.5mF, at least about 2mF, at least about 2.5mF, at least about 3mF, at least about 3.5mF, at least about 4mF, at least about 5.5mF, at least about 6mF, at least about 6.5mF, at least about 7mF, at least about 7.5mF, at least about 8mF, at least about 8.5mF, at least about 9mF, at least about 9.5mF, and at least about 10mF. In some embodiments, the cap bank 407 may comprise a plurality of capacitors, each having a capacitance of at least 4.5mF. When the cap bank 507 reaches its maximum capacitance level, the CDC 505 may be configured to discharge or shunt the energy stored in the cap bank 507 to ground.

[0093] Cap bank 507 may also be electrically connected to PLC 501. Cap bank 507 may be configured to transmit signal 529 to PLC 401. Signal 529 may include an electrical signal of approximately 1V to 100V. In some embodiments, signal 529 may be used to read the voltage of cap bank 507. Cap bank 507 may further include a connection to ground.

[0094] The electroporation system 500 enables the switching of the polarity of the electrical load applied to the electroporation chamber to improve the efficiency of the electroporation process. For this purpose, the electroporation system 500 comprises an integrated pulse module 509 that opens and closes a load path, and an output stage 511 which is an electrical circuit that switches the polarity of the voltage output from the integrated pulse module 509. In some embodiments, the output stage 511 sends the output voltage to a positive high-voltage lead 513 and a negative high-voltage lead 515. A cap bank 507 may be electrically connected to the integrated pulse module 509. The integrated pulse module 509 may include a switch array. The switch array may be configured to turn on the release path to the output stage 511. For example, the switch array may have an initial configuration that prevents the charge output by the integrated pulse module from reaching the output stage 511. The switch array may have an additional configuration that allows the charge output by the integrated pulse module to reach the output stage 511. The output stage 511 may include an H-bridge configured to enable switching of the output polarity. For example, the polarity of the output signal transmitted to one of the electrodes (e.g., the first electrode 308A and the second electrode 308B in Figure 3A) can be switched from positive to negative, or from negative to positive. In other words, in some modifications, the first electrode 308A may be the anode and the second electrode 308B may be the cathode. In the first example, a positive electrical signal may be transmitted from the output stage 511 to the cathode 308B, and a negative electrical signal may be transmitted to the anode 308A. Switching the polarity of the electrical signal transmitted by the output stage 511 may result in the anode 308A receiving a positive electrical signal and the cathode 308B receiving a negative electrical signal in the second example.

[0095] The output polarity can be switched during the application of electrical energy to the cell solution. For example, the cell solution may first be treated with electrical energy to electroporate cells from a first direction. Then, the polarity of the electrical energy may be switched to electroporate cells from a second direction. The polarity of the electrical energy may be switched to improve the efficiency of the electroporation process.

[0096] The output stage 511 may be configured to support output signals up to 880 A. In some embodiments, the output stage 511 may output signals of at least about 100 A, at least about 200 A, at least about 300 A, at least about 400 A, at least about 500 A, at least about 600 A, at least about 700 A, at least about 800 A, at least about 900 A, and at least about 1000 A. The output signal from the output stage 511 may also include at least one pulse. At least one pulse may have a pulse width of 1 ms or less. In some embodiments, the pulse width of the output signal from the output stage 511 may be at least 100 μs, at least 200 μs, at least 300 μs, at least 400 μs, at least 500 μs, at least 600 μs, at least 700 μs, at least 800 μs, and at least 900 μs. In some embodiments, the pulse width of at least one pulse may be between 100 μs and 5 ms.

[0097] At least one of the integrated pulse module 509 and the output stage 511 may be configured to receive at least one input signal from the PLC 501. For example, one of the integrated pulse module 509 and the output stage 511 may receive at least one of signals 531 and 533. In some embodiments, signal 531 may be a negative power signal. In some embodiments, signal 533 may be a positive power signal. One of the integrated pulse module 509 and the output stage 511 may also be configured to transmit at least one signal to the PLC 501. For example, the integrated pulse module 509 may transmit at least one of signals 535, 537, and 539 to the PLC 501. In some embodiments, signal 535 may be a positive high-voltage readout signal. In some embodiments, signal 537 may be a negative high-voltage readout signal. In some embodiments, signal 539 may include a shunt voltage signal.

[0098] The electroporation system 500 may also include at least one electrical output lead. At least one electrical output lead may include a positive high-voltage lead 513 and a negative high-voltage lead 515. Naturally, the polarity of the leads can be switched, with high-voltage lead 513 being the negative lead and high-voltage lead 515 being the positive lead, so the terms used for high-voltage leads 513 and 515 are representative. Leads 513 and 515 may be electrically connected to an output stage 511. Leads 513 and 515 may be further electrically connected to a high-voltage connector 523. The high-voltage connector 523 may include a multiplexer 517. The multiplexer 517 may be electrically connected to at least one electroporation flow chamber. For example, the multiplexer 517 may be electrically connected to electroporation chamber A 519 and electroporation chamber B 521.

[0099] II. Methods for electroporating cells In general, the systems and devices described herein can perform one or more cell processing steps to produce cell products. Figure 6 is a flowchart of an illustrative method of cell processing 600 that can be performed via the systems and devices described above. Method 600 may include, in step 602, concentrating a selected population of cells in a solution (e.g., a fluid). For example, the solution may be transported to the CCE module of the cartridge via a fluid transfer bus. A robot may be operated to move the cartridge to the CCE instrument so that the CCE module interfaces with the CCE instrument. The CCE instrument may be operated to cause the CCE module to concentrate the selected cell population. Additionally or alternatively, cell products may be introduced into and out of the cartridge (either manually or automatically) via a sterile fluid transfer port for any of the steps described herein. In some embodiments, the cartridge may be sterilized (either manually or automatically) within a feedthrough port.

[0100] In some embodiments, a selected population of cells in the solution may be washed (604). For example, the solution may be transported to the cartridge's CCE module via a fluid transfer bus. A robot may be operated to move the cartridge to the CCE instrument so that the CCE module interfaces with the CCE instrument. The CCE instrument may be operated to cause the CCE module to remove culture medium from the solution, introduce culture medium into the solution, and / or replace the culture medium in the solution.

[0101] In some embodiments, a population of cells in a solution may be selected in step 606. For example, the solution may be transported to a selection module in a cartridge via a fluid transfer bus. A robot may be operated to move the cartridge to a selection device so that the selection module interfaces with the selection device. The selection device may be operated to cause the selection module to select the selected population of cells.

[0102] In some embodiments, a population of cells in a solution may be sorted in step 608. For example, the solution may be transported to a sorting module in a cartridge via a fluid transfer bus. A robot may be operated to move the cartridge to a sorting device so that the sorting module interfaces with the sorting device. The sorting device may be operated to cause the sorting module to sort the cell population.

[0103] In some embodiments, the solution may be transported to the bioreactor module of the cartridge via a fluid transfer bus in step 610 and allowed to settle. For example, a robot may be operated to move the cartridge to the bioreactor instrument so that the bioreactor module interfaces with the bioreactor instrument. The bioreactor instrument may be operated to cause the bioreactor module to maintain cells under a set of predetermined conditions.

[0104] In some embodiments, in step 612, cells can be grown in solution. For example, the solution may be delivered to the bioreactor module of the cartridge via a fluid transfer bus. A robot may be operated to move the cartridge to the bioreactor instrument so that the bioreactor module interfaces with the bioreactor instrument. The bioreactor instrument may be operated to grow cells in the bioreactor module by cell replication.

[0105] In some embodiments, the tissue may be digested in step 614 by transporting an enzyme reagent via a fluid transfer bus to a module containing a solution containing the tissue, such that the tissue releases a selected cell population into the solution.

[0106] In some embodiments, a selected population of cells in solution is activated in step 616 by transporting an activation reagent via a fluid transfer bus to a module that may contain a solution containing cell products.

[0107] In some embodiments, the solution is transported in step 618 via a fluid transfer bus to the electroporation module of the cartridge, which can receive an electroporation signal and electroporate the cells in the solution. For example, a robot may be operated to move the cartridge to the electroporation instrument so that the electroporation module interfaces with the electroporation instrument. The electroporation instrument may be operated to cause the electroporation module to electroporate a selected population of cells in the presence of genetic material.

[0108] In some embodiments, an effective amount of vector is transported via a fluid transfer bus to a module containing a solution containing cell products, thereby enabling transduction of a selected cell population in the solution in step 620.

[0109] In some embodiments, in step 622, the formulation solution may be transported via a fluid transfer bus to a module containing the cell product to produce the finished cell product. For example, the finished cell product may be transported to one or more product collection bags. In some embodiments, finishing the cell product may include one or more steps of washing the cells, concentrating the cells, exchanging the cell buffer with the formulation buffer, and administering the cells in the formulation buffer in predetermined amounts to one or more product collection bags and / or containers.

[0110] In some embodiments, the cell product may be removed from the cartridge, either manually or automatically, in step 624, and the cells may be collected.

[0111] In some embodiments, the cell product may include one or more of the following: immune cells, genetically engineered chimeric antigen receptor T cells, genetically engineered T cell receptor (TCR) cells, hematopoietic stem cells (HSCs), and tumor infiltrating lymphocytes (TILs). In some embodiments, the immune cells may include natural killer (NK) cells.

[0112] Any of the electroporation systems and devices described above may be used when performing an electroporation method. For example, referring to the electroporation of a cell solution described in step 618 above, a method for electroporating cells may include: introducing the cell solution into a fluid conduit of an electroporation chamber through an inlet port; detecting the presence of the cell solution in a first portion of the electroporation chamber through at least one window; stopping the inflow of the cell solution into the fluid conduit; applying an electric field to the cell solution in the fluid conduit for a predetermined duration through first and second electrodes; and draining the cell solution from the fluid conduit through an outlet port. In some embodiments, the at least one window may be a first window located adjacent to the outlet port.

[0113] The method may further include detecting the presence of a cell solution in a second portion of the electroporation chamber after detecting the presence of a cell solution in a first portion of the electroporation chamber through a second window and through a first window, and the electric field is applied after detecting the presence of a cell solution in the second portion of the electroporation chamber. In some embodiments, detecting the presence of a cell solution in the second portion of the electroporation chamber includes detecting the presence of a cell solution adjacent to a vent port located in the second portion of the electroporation chamber, which is in fluid communication with a fluid conduit. In some embodiments, detecting the presence of a cell solution at the first end of the electroporation chamber includes detecting the presence of a cell solution adjacent to an auxiliary channel extending from a fluid conduit, which is in fluid communication with the fluid conduit and the outlet port. In some embodiments, draining the cell solution from the fluid conduit through the outlet port includes purging the fluid conduit with air via a vent port located in the second portion of the electroporation chamber, which is in fluid communication with the fluid conduit.

[0114] Figure 7A provides a flow chart illustrating an exemplary method 701 of cell electroporation. Method 701 can be controlled by an electroporation instrument controller (see 419 in Figure 4A). In step 705 of Method 701, a signal may be generated to cause a fluid (e.g., cell solution) to flow into the fluid conduit of the electroporation chamber via an inlet port located in a second part of the electroporation chamber. For example, the fluid may flow through the inlet port, through an auxiliary channel, and into the fluid conduit. In step 710, the amount of fluid in the fluid conduit may be determined. For example, a sensor near the window may determine the presence or absence of fluid in the fluid conduit and / or auxiliary channel. If the sensor near the window detects the presence of fluid, the volume of fluid to be contained in the fluid conduit of the electroporation chamber may be estimated based on the known dimensions of the fluid conduit, auxiliary channel, and multiple ports, as well as the position of the sensor relative to those dimensions. The volume of fluid in the fluid conduit can then be useful in determining how much voltage and / or current to apply to the cell solution to achieve electroporation.

[0115] In step 715, a signal may be generated to stop the flow of fluid into the fluid conduit. For example, the fluid may be stopped by stopping the fluid pump used to pump the fluid to the inlet port. In some embodiments, the fluid flow may be stopped by closing the inlet port in response to the signal. The outlet port may also be closed. The vent port may be open, closed, or partially open or closed. Then, in step 720, a signal may be generated to apply electrical energy to the fluid contained in the fluid conduit in order to electroporate cells. In some embodiments, the electrical energy may also electroporate cells contained in at least one auxiliary channel. In step 725, a signal may be generated to cause the fluid in the fluid conduit to flow out of the fluid conduit through the outlet port. The fluid may begin to flow out of the fluid conduit by opening the outlet port. The fluid may flow out of the fluid conduit to the outlet port through an auxiliary channel fluidically connected to the fluid outlet.

[0116] Figure 7B provides a flow diagram illustrating another preferred method 702 of cell electroporation in which the amount of fluid in the fluid conduit is determined using multiple sensors. Method 702 can be controlled by an electroporation instrument controller (see 419 in Figure 4A). In step 705, a signal may be generated to cause fluid to flow into the fluid conduit of the electroporation chamber via an inlet port located in a second part of the electroporation chamber. For example, the fluid may flow through the inlet port, through an auxiliary channel, and into the fluid conduit. In step 706, the presence or absence of fluid may be detected by at least one sensor adjacent to the second part of the electroporation chamber. For example, at least one sensor adjacent to the second part may determine the presence or absence of fluid in the fluid conduit and / or auxiliary channel. In step 707, the presence or absence of fluid may be detected by another sensor adjacent to the first part of the electroporation chamber. In this way, if the measurements from both sensors indicate the presence of fluid in both the first and second portions of the fluid conduit, the volume of fluid in the fluid conduit can be estimated based on the known dimensions of the fluid conduit, auxiliary channels, and multiple ports, as well as the relative positions of each of the at least two sensors to those dimensions.

[0117] Next, the estimated volume of fluid in the fluid conduit can be used to determine the electrical properties of electrical energy, such as voltage and current, to be used to electroporate the cell solution contained therein. Then, in step 715, a signal may be generated to stop the flow of fluid into the fluid conduit. For example, the fluid may be stopped by stopping the fluid pump used to pump the fluid to the inlet port. In embodiments, the fluid flow may be stopped by closing the inlet port in response to the signal. The outlet port may also be closed. The vent port may be opened or closed. Then, in step 720, a signal may be generated to apply electrical energy to the fluid contained in the fluid conduit in order to electroporate cells. In some embodiments, the electrical energy may also electroporate the fluid contained in at least one auxiliary channel. In step 725, a signal may be generated to cause the fluid in the fluid conduit to flow out of the fluid conduit. The fluid may begin to flow out of the fluid conduit by opening the outlet port. The fluid may flow out of the fluid conduit to the outlet port via an auxiliary channel fluidically connected to the outlet port.

[0118] Figure 7C provides a flow chart illustrating another preferred method 703 of cell electroporation. Similar to the above, method 703 can be controlled by an electroporation instrument controller (see 419 in Figure 4A). However, unlike the previous method, method 703 integrates the use of a timer. In the initial configuration, the inlet port of the electroporation chamber is closed, and the outlet port and vent port of the electroporation chamber are open. In step 704, the target volume of fluid to be electroporated in the fluid conduit can be obtained or determined. Based on the target volume, the time required for the fluid flow to fill the fluid conduit to the target volume can be estimated. For example, the estimated filling time can be based on parameters such as (1) the size (e.g., diameter) of the fluid piping connecting the fluid transfer bus to the inlet port, (2) fluid properties such as density, temperature, and pressure, and (3) the velocity of the fluid in the fluid piping connecting the fluid transfer bus to the inlet port. By knowing these parameters, it is possible to estimate the estimated flow rate (e.g., μl / second) of the fluid flow through the inlet port, through the auxiliary channel, and into the fluid conduit of the electroporation chamber. By comparing this flow rate with the target volume, the estimated filling time can be obtained.

[0119] In some variations, the initial volume contained within the electroporation chamber can also be determined. For example, at least one sensor can determine the presence or absence of fluid in any portion of the fluid conduit, auxiliary channels, and multiple ports. If the first sensor detects the presence of fluid, the known dimensions of the fluid conduit, auxiliary channels, and multiple ports can be used to estimate the volume of fluid contained therein, based on the relative position of the sensor to those dimensions. Based on this initial determination, the target volume may represent the difference between the initial volume and the target volume.

[0120] Next, in step 705, a signal may be generated to open the inlet port, thereby allowing fluid to flow into the fluid conduit through the inlet port and through an auxiliary channel fluidically connected to the inlet port, and the timer is started. The timer is started simultaneously with the opening of the inlet port. Once the fluid has flowed into the fluid conduit, in step 706, a sensor proximal to the second part of the electroporation chamber (e.g., an optical sensor proximal to the first window) may detect the presence of fluid in it. Simultaneously with the detection of the presence of fluid by the sensor proximal to the second part, an electrical signal is sent to the outlet port to close the outlet port. In step 708, the elapsed time is compared with the estimated filling time. When the elapsed time equals the estimated filling time, the fluid flow through the inlet port may be stopped in step 716 in response to the generated signal. For example, the fluid may be stopped by stopping the fluid pump used to pump the fluid into the inlet port and / or by closing the inlet port in response to the signal sent thereto. The outlet port may also be closed. The vent port may be open, closed, or partially open. In step 720, a signal may be generated to apply an electric field to the fluid contained in the fluid conduit via electrodes in order to electroporate the cells. In embodiments, the electric field may also electroporate the cells contained in at least one auxiliary channel. After electroporation, in step 725, a signal may be generated to cause the fluid in the fluid conduit to flow out of the fluid conduit. The fluid may begin to flow out of the fluid conduit by opening the outlet port. The fluid may flow out of the fluid conduit to the outlet port via an auxiliary channel fluidically connected to the fluid outlet.

[0121] Figure 8A provides a flow diagram of yet another preferred method 801 of cell electroporation, in which the presence of fluid is determined by sensors located near the inlet and vent ports, and excess fluid can be drained prior to the application of electroporation energy to the cell solution. As described above, method 801 can be controlled by an electroporation instrument controller (see 419 in Figure 4A). In the initial configuration, the inlet and outlet ports of the electroporation chamber are closed, and the vent port of the electroporation chamber is open. Then, in step 805, the presence or absence of fluid may be detected by a first sensor located near the inlet port. Then, in step 810, the inlet port may be opened, allowing the fluid to begin flowing through the inlet port and through an auxiliary channel fluid-connected thereto into the fluid conduit. While the fluid is flowing into the fluid conduit, the presence of fluid may or may not be detected by a second sensor in step 815. The second sensor may be located near the vent port. In particular, in step 815, the detection of fluid by the second sensor indicates the volume of fluid contained in the electroporation chamber that exceeds the maximum volume of the fluid conduit. The excess fluid may flow out of the fluid conduit in step 820. For example, the excess fluid may flow through the auxiliary channel and through the inlet port. In some embodiments, the excess fluid may flow through the inlet port by negative pressure applied to the fluid conduit and / or by air purging applied to the fluid conduit via the vent port. Subsequently, the second sensor may detect the absence of fluid in step 825. As long as the second sensor detects the presence of fluid, the excess fluid may continue to flow through the inlet port. When the fluid is no longer detected by the second sensor, the flow of fluid flowing out of the fluid conduit through the inlet port may be stopped in step 830.

[0122] In step 815, if no fluid is detected by the second sensor, in step 830, the flow of fluid into the fluid conduit may be stopped. For example, a fluid pump in a fluid transfer bus configured to pump fluid into an inlet port may be turned off. In another example, an inlet port may be closed to prevent the flow of fluid through it.

[0123] The presence or absence of fluid detected by at least one sensor can be used to determine the volume of fluid in the fluid conduit. For example, if the fluid flow into the fluid conduit is stopped before a sensor near the vent port detects the presence of fluid, the known volumes of the fluid conduit, auxiliary channels, and multiple ports can be used to estimate the volume of fluid contained therein, similar to embodiments of the method in Figures 7A and 7C. In another example, if in step 815 fluid is detected by a sensor near the vent port, and in step 820 excess fluid must be removed, then in step 825 the change in sensor output from detecting the presence of fluid to detecting the absence of fluid can help determine the volume of fluid contained in the fluid conduit.

[0124] Next, the volume of fluid in the fluid conduit can be used to determine the electrical properties of electrical energy, such as voltage and current, used to electroporate the cell solution contained therein. In step 835, electrical energy can be applied to the fluid contained in the fluid conduit to electroporate the cells therein. Electrical energy can also electroporate cells contained in at least one auxiliary channel. Next, in step 840, the fluid in the fluid conduit may flow out of the fluid conduit through the outlet port. The fluid may begin to flow out of the fluid conduit by opening the outlet port. The fluid may flow out of the fluid conduit to the outlet port through the fluid outlet and the auxiliary channel fluidically connected to the fluid conduit.

[0125] Figure 8B provides a flow diagram illustrating another preferred method 802 of cell electroporation described herein, in which the presence of fluid is determined by sensors in the first and second windows of the electroporation chamber, and excess fluid can be drained before the application of electroporation energy to the cell solution. As described above, method 802 can be controlled by an electroporation instrument controller (see 419 in Figure 4A). In the initial configuration, the inlet and outlet ports of the electroporation chamber are closed, and the vent port of the electroporation chamber is open. The fluid can then begin to flow out in step 845 through the inlet port of the electroporation chamber, through an auxiliary channel fluid-coupled to the inlet port, and into the fluid conduit of the electroporation chamber.

[0126] Next, in step 850, the presence or absence of fluid may be detected by a first sensor in the first part. For example, a first sensor located near a first window in the first part of the electroporation chamber may determine the presence of fluid in the fluid conduit and / or auxiliary channel. In step 855, the presence of fluid may or may not be detected by a second sensor in step 855. The second sensor may be located near a second window in the second part of the electroporation chamber. If fluid is detected by the second sensor (step 855), the volume of fluid contained in the electroporation chamber exceeds the maximum volume of the fluid conduit. Therefore, the excess fluid may flow out of the fluid conduit in step 860. For example, the excess fluid may flow out of the fluid conduit through the inlet port. Subsequently, the second sensor may detect the absence of fluid in step 865. If the second sensor continues to detect the presence of fluid, the excess fluid may continue to flow out of the fluid conduit and flow out through the inlet port. When the second sensor detects the absence of fluid, the excess fluid flow from the fluid source can be stopped.

[0127] If no fluid is detected by the second sensor in step 855, the flow of fluid into the fluid conduit may be stopped in step 870. The fluid flow may be stopped by closing the inlet port. In one embodiment, the fluid flow may be stopped by turning off a fluid pump in a fluid transfer bus configured to pump fluid into the inlet port. The presence or absence of fluid detected by at least one sensor may help determine the volume of fluid in the fluid conduit. For example, if the flow of fluid into the fluid conduit is stopped before the second sensor adjacent to the second window detects the presence of fluid, the known volumes of the fluid conduit, auxiliary channels, and multiple ports may be used to estimate the volume of fluid contained therein, similar to embodiments of the method in Figures 7A and 7C. In another example, if fluid is detected by the second sensor adjacent to the second window in step 855, and the excess fluid must be removed in step 860, the change in sensor output from the detection of the presence of fluid to the detection of the absence of fluid in step 855 may help determine the volume of fluid contained in the fluid conduit.

[0128] Next, the volume of fluid in the fluid conduit, auxiliary channels, and multiple ports can be used to determine the electrical properties of electrical energy, such as voltage and current, which will be used to electroporate the cell solution contained therein. In step 875, electrical energy can be applied to the fluid contained in the fluid conduit to electroporate the cells therein. Electrical energy can also electroporate cells contained in at least one auxiliary channel. Next, in step 880, the fluid in the fluid conduit may flow out of the fluid conduit through the outlet port. The fluid may flow out of the fluid conduit through the fluid outlet and auxiliary channels fluid-connected to the fluid conduit.

[0129] While described above as including specific steps, please understand that a cell processing method may include any subset of the cell processing steps in any preferred order.

[0130] All cited references are incorporated herein by reference in their entirety.

[0131] Throughout this application, the term “approximately” is used to indicate that a value includes inherent variations in error in the device or method used to determine that value, or variations present between the samples being measured. Unless otherwise stated or evident from the context, “approximately” means within plus or minus 10 percent of the reported number (except where such a number exceeds 100% of the possible value or falls below 0%). When used in conjunction with a range or set of values, the term “approximately” applies to each of the endpoints of the range or each of the range values ​​listed in a series, unless otherwise indicated. As used herein, the terms “approximately” and “about” are synonymous.

[0132] While embodiments of the present invention have been shown and described herein, those skilled in the art will understand that such embodiments are provided only as examples. Those skilled in the art will recall numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in carrying out the invention. The following claims define the scope of the present invention, and methods and structures within the scope of these claims, as well as their equivalents, are intended to be covered thereby.

Claims

1. Electroporation chamber, A first electrode and a second electrode, Multiple ports, The inlet port in the first part of the electroporation chamber, Multiple ports, including an outlet port in the second part of the electroporation chamber, A fluid conduit disposed between the first electrode and the second electrode, wherein the fluid conduit is in fluid communication with the inlet port and the outlet port, An electroporation chamber comprising: at least one window disposed on the wall of the electroporation chamber, providing a view to the segments of the fluid conduit.

2. The aforementioned multiple ports are, The electroporation chamber according to claim 1, further comprising a vent port that is in fluid communication with the fluid conduit and is located in the first portion of the electroporation chamber.

3. The electroporation chamber according to claim 1, wherein the fluid conduit has a volume defined by the distance between the first electrode and the second electrode.

4. The electroporation chamber according to claim 3, wherein the volume of the fluid conduit is approximately 0.5 μL to approximately 2.5 mL.

5. The electroporation chamber according to claim 1, wherein the distance between the first electrode and the second electrode is approximately 0.5 mm to approximately 5 mm.

6. The electroporation chamber according to claim 1, further comprising a first housing and a second housing, wherein the first electrode, the fluid conduit, and the second electrode are positioned between the first housing and the second housing.

7. The electroporation chamber according to claim 1, wherein the segment of the fluid conduit is adjacent to the first portion of the electroporation chamber.

8. The electroporation chamber according to claim 1, wherein the segment of the fluid conduit is adjacent to the second portion of the electroporation chamber.

9. The electroporation chamber according to claim 1, wherein the at least one window includes a first window and a second window, the first window having a view to a first segment of the fluid conduit and the second window having a view to a second segment of the fluid conduit.

10. The electroporation chamber according to claim 2, wherein the fluid conduit is in fluid communication with each of the plurality of ports and with auxiliary channels extending therefrom.

11. The electroporation chamber according to claim 10, wherein each of the auxiliary channels is fluidly coupled to one of the fluid conduits and one of the plurality of ports.

12. The electroporation chamber according to claim 11, wherein one of the auxiliary channels that fluidly couples the fluid conduit to the outlet port is curved.

13. The electroporation chamber according to claim 11, wherein the at least one window includes a first window and a second window, the first window having a view to a first segment of the fluid conduit, the second window having a view to a second segment of the fluid conduit, and the second window being close to the auxiliary channel having fluid communication with the outlet port.

14. The electroporation chamber according to claim 13, wherein the auxiliary channel communicating with the outlet port is curved, and the second window is adjacent to the curved auxiliary channel.

15. The electroporation chamber according to claim 14, wherein the second window is adjacent to the base of the curved auxiliary channel.

16. The electroporation chamber according to claim 11, wherein the first window is adjacent to the auxiliary channel which is in fluid communication with the vent port.

17. The electroporation chamber according to claim 16, wherein the first window is adjacent to the base of the auxiliary channel which is in fluid communication with the vent port.

18. The electroporation chamber according to claim 2, wherein the inlet port located in the first portion of the electroporation chamber is further positioned toward the first side wall of the electroporation chamber, and the vent port located in the first portion of the electroporation chamber is further positioned toward the second side wall of the electroporation chamber.

19. The electroporation chamber according to claim 4, wherein the fluid conduit has a triangular cross-section along an axis defined from the first portion of the electroporation chamber to the second portion of the electroporation chamber.

20. The electroporation chamber according to claim 1, wherein each of the plurality of ports extends through the first electrode and / or the second electrode.

21. An automated system for electroporation, An output stage that switches the polarity of the electrical load, An automated system comprising: a multiplexer configured to direct at least a portion of the electrical load to at least one of a first electroporation chamber and a second electroporation chamber.

22. Charging and discharging circuits, The automation system according to claim 21, further comprising a capacitance bank that can be charged via a high-voltage DC-DC converter.

23. The automation system according to claim 22, wherein the capacitance bank can store approximately 4.5 mF.

24. The automation system according to claim 21, further comprising an integrated pulse module for opening and closing the supply path of the electrical load to the output stage.

25. The automated system according to claim 24, wherein the integrated pulse module can support pulse amperages up to approximately 800 A for a pulse length of approximately 1 ms.

26. The automation system according to claim 22, wherein excess charge in the capacitance bank can be discharged via the charge / discharge circuit.

27. It is an electroporation system, At least one electroporation chamber, each electroporation chamber is A first electrode and a second electrode, Multiple ports, An inlet port located in the first part of each electroporation chamber, Multiple ports, each having an outlet port in a second part of the electroporation chamber, An electroporation chamber comprising at least one fluid conduit disposed between the first electrode and the second electrode, the fluid conduit being in fluid communication with the inlet port and the outlet port, An electroporation system comprising an automated system for electroporation, operably connected to the first electrode and the second electrode of the at least one electroporation chamber.

28. The electroporation system according to claim 27, wherein each electroporation chamber is disposed on the wall of each electroporation chamber and further includes at least one window providing a view to the corresponding segment of the fluid conduit of the electroporation chamber.

29. The electroporation system according to claim 28, wherein the at least one window includes a first window and a second window, the first window having a view to a first segment of the fluid conduit and the second window having a view to a second segment of the fluid conduit.

30. The automated system for electroporation is, An output stage that switches the polarity of the electrical load, The electroporation system according to claim 27, comprising a multiplexer configured to direct at least a portion of the electrical load to each of the at least one electroporation chambers.

31. The automated system for electroporation is, Charging and discharging circuits, The electroporation system according to claim 30, further comprising a capacitance bank that can be charged via a high-voltage DC-DC converter.

32. The electroporation system according to claim 30, further comprising an integrated pulse module for opening and closing the supply path of the electrical load to the output stage.

33. The electroporation system according to claim 32, wherein the integrated pulse module can support pulse amperages up to approximately 800 A for a pulse length of approximately 1 ms.

34. The electroporation system according to claim 27, wherein the output from the automated system for electroporation generates an electric field of about 1.0 kV / cm to about 3 kV / cm in the fluid conduit of at least one of the first electroporation chamber and the second electroporation chamber.

35. The electroporation system according to claim 28, further comprising at least one sensor configured to view the at least one window.

36. For each of the at least one electroporation chambers, A first signal is generated to cause the cell solution to flow into the fluid conduit via the inlet port. Based on the data received from at least one of the sensors, a second signal is generated to stop the inflow of the cell solution into the fluid conduit via the inlet port. A third signal is generated via the automated system for electroporation to apply an electric field into the fluid conduit for a predetermined duration. The electroporation system according to claim 35, further comprising a processing circuit configured to generate a fourth signal for discharging the cell solution from the fluid conduit via the outlet port.

37. The electroporation system according to claim 36, wherein the plurality of ports are in fluid communication with the fluid conduit and further comprises vent ports located in the first portion of each electroporation chamber.

38. The electroporation chamber according to claim 37, wherein the fourth signal generated by the processing circuit is configured to cause a fluid pump to discharge the cell solution from the fluid conduit by purging the fluid conduit with air through the vent port.

39. The electroporation system according to claim 36, further comprising a fluid pump, wherein the first signal, the second signal, and the fourth signal are transmitted to the fluid pump.

40. The electroporation system according to claim 27, wherein each of the plurality of ports extends through the first electrode and / or the second electrode.

41. A method of electroporating cells, The cell solution is introduced into the fluid conduit of the electroporation chamber described in claim 1 via the inlet port, The presence of the cell solution in the second portion of the electroporation chamber is detected through the at least one window, To stop the inflow of the cell solution into the fluid conduit, Applying an electric field to the cell solution in the fluid conduit for a predetermined duration via the first electrode and the second electrode, A method comprising draining the cell solution from the fluid conduit through the outlet port.

42. The method according to claim 41, wherein the at least one window is a second window positioned adjacent to the outlet port.

43. The method further includes detecting the presence of the cell solution in the first portion of the electroporation chamber after detecting the presence of the cell solution in the second portion of the electroporation chamber through the first window and the second window, The method according to claim 42, wherein the electric field is applied after detecting the presence of the cell solution in the first portion of the electroporation chamber.

44. Detecting the presence of the cell solution in the first portion of the electroporation chamber is: The method according to claim 43, comprising detecting the presence of the cell solution in close proximity to a vent port located in the first portion of the electroporation chamber, which is in fluid communication with the fluid conduit.

45. Detecting the presence of the cell solution at the second end of the electroporation chamber is, The method according to claim 44, comprising detecting the presence of the cell solution adjacent to an auxiliary channel extending from the fluid conduit, wherein the auxiliary channel is in fluid communication with the fluid conduit and the outlet port.

46. Discharging the cell solution from the fluid conduit through the outlet port is, The method according to claim 42, comprising purging the fluid conduit with air through a vent port that is in fluid communication with the fluid conduit and is located in the first portion of the electroporation chamber.

47. A method of electroporating cells, The robot positions the cartridge containing the cells within the electroporation device of the working cell, The process involves introducing the cell solution into the electroporation chamber of the cartridge, wherein the electroporation chamber is located within an electroporation module that can be coupled to the electroporation device. The electroporation apparatus detects the presence of the cell solution in a portion of the electroporation chamber through at least one window disposed on the electroporation chamber, To stop the flow of the cell solution into the electroporation chamber, Applying an electric field to the cell solution via the first electrode and the second electrode of the electroporation chamber, A method comprising draining the cell solution from the electroporation chamber.

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