Systems, devices, and methods for cell processing

Abstract

Disclosed herein are cell processing and manufacturing systems, devices, and methods thereof. A system for cell processing may include a plurality of docking station modules, each configured to cooperate with a corresponding plurality of cartridge modules to perform one or more cell processing operations on cells in the cartridges.

Description

[Technical Field]

[0001] Incorporation by Reference This application claims priority to U.S. Provisional Patent Application No. 63 / 427,720, filed November 23, 2022, the entire contents of which are incorporated herein by reference.

[0002] The devices, systems, and methods herein relate to the production of cellular products for biomedical applications using automated systems. [Background technology]

[0003] Generally, cell therapies based on hematopoietic stem cells (HSCs), chimeric antigen receptor (CAR) T cells, NK cells, tumor-infiltrating lymphocytes (TILs), T-cell receptors (TCRs), regulatory T cells (Tregs), gamma delta (γδ) T cells, and the like, rely on the manufacturing of cell products. Cell product manufacturing typically involves multiple cell processing steps. Conventional solutions for cell product manufacturing may rely on cumbersome manual operations performed in costly biosafety cabinets and / or clean rooms. However, skilled laboratory technicians, appropriate sterile enclosures such as clean room facilities, and associated protocols and procedures for regulated (GMP) manufacturing can be expensive to design and use. Many current manufacturing processes may also employ numerous manual reagent preparation and instrument manipulation steps during the manufacturing protocol, and the process may require days or even weeks. However, even platforms described for automating cell processing in closed systems may generally rely on pre-configured instrumentation and piping sets that limit operational flexibility and do not reliably prevent process failures due to accidental operator / human error. Furthermore, most efforts to automate the manufacturing of cellular products have been directed toward automating individual processing steps in the cell therapy manufacturing workflow, and even systems that automate several steps may lack end-to-end process flexibility, process robustness, and process scalability. These and other limitations of previous attempts at automating cell processing are addressed in various embodiments disclosed herein. Summary of the Invention

[0004] According to one embodiment, the present disclosure generally relates to methods and systems for processing cellular products.

[0005] In some variations, the present disclosure provides a system including a cartridge comprising a plurality of cartridge modules, including a bioreactor module, a counterflow centrifugal elutriation module, and at least one of an electroporation module, a magnetic-activated cell selection module, a fluorescence-activated cell selection module, or a spinoculation module, at least one sterile liquid transfer port, and a liquid transfer bus fluidly coupled to each cartridge module; and a docking station comprising a plurality of docking station modules corresponding to the plurality of cartridge modules, each of the plurality of docking station modules independently configured to cooperate with a respective cartridge module to perform one or more cell processing operations on the cartridge, the docking station being sized and shaped to receive a single cartridge, the liquid transfer bus being fluidly coupled to each cartridge module by a fluid conduit disposed between a port on the liquid transfer bus and a respective port on each cartridge module, the fluid conduit comprising tubing or channeling, the system further comprises a sterile liquid transfer device configured to facilitate transfer of liquid between the cartridge and the closed volume fluidic device, the closed volume fluidic device being a fluid container, the sterile liquid transfer device comprising at least one actuator, the actuator actuating one of the at least one sterile liquid transfer port of the cartridge and a corresponding sterile liquid transfer port of the closed volume fluidic device, the system further comprises a fluid reservoir disposed within a housing of the system, the fluid reservoir being accessible by a user to insert and / or remove the one or more closed volume fluidic devices, each of the one or more cell processing operations performed on the cartridge is performed on cells within the cartridge, the plurality of cartridge modules comprising electroporation modules, the plurality of cartridge modules comprising magnetic-activated cell selection modules, the plurality of cartridge modules comprising fluorescence-activated cell selection modules, the plurality of cartridge modules comprising spinoculation modules, and the plurality of docking station modules comprising corresponding bioreactor modules;a docking station, wherein a plurality of docking station modules comprise corresponding magnetic-activated cell selection modules, a plurality of docking station modules comprise corresponding fluorescence-activated cell selection modules, a plurality of docking station modules comprise corresponding electroporation modules, a plurality of docking station modules comprise corresponding counterflow centrifugal elutriation modules, a plurality of docking station modules comprise corresponding spinoculation modules, a cartridge comprises a pump fluidly coupled to a liquid transfer bus, the system further comprises a pump actuator configured to interface with the pump, the system further comprises a waste reservoir for capturing waste generated by the cartridge, and / or the transfer of liquid between the cartridge and the closed volume fluidic device includes extraction of at least a portion of the cell processing sample from the cartridge;

[0006] In some variations, the present disclosure further provides an automated cell processing method, comprising performing at least two cell processing operations in a cartridge positioned in a docking station, the cartridge comprising a plurality of cartridge modules, at least one sterile liquid transfer port, and a liquid transfer bus fluidly coupled to each cartridge module; the docking station comprising a plurality of docking station modules corresponding to the plurality of cartridge modules and sized and shaped to receive a single cartridge; the plurality of cartridge modules comprising at least one of a bioreactor module, a counterflow centrifugation elutriation module, and an electroporation module, a magnetic-activated cell selection module, a fluorescence-activated cell selection module, or a spinoculation module; the cell processing operations are automatic upon execution of the received set of instructions; and the method further comprising activating one of the at least one sterile liquid transfer port of the cartridge and a corresponding sterile liquid transfer port of the closed volume fluidic device to facilitate transfer of liquid therebetween. facilitating the transfer of liquid between the cartridge and the closed volume fluidic device comprises activating one of the at least one sterile liquid transfer port and a corresponding sterile liquid transfer port, wherein the transfer of liquid between the cartridge and the closed volume fluidic device comprises extracting at least a portion of the cell processing sample from the cartridge, wherein the plurality of cartridge modules comprise electroporation modules, the plurality of cartridge modules comprise magnetic-activated cell selection modules, the plurality of cartridge modules comprise fluorescence-activated cell selection modules, the plurality of cartridge modules comprise spinoculation modules, the plurality of docking station modules comprise corresponding bioreactor modules, the plurality of docking station modules comprise corresponding counterflow centrifugation elutriation modules, the plurality of docking station modules comprise corresponding electroporation modules, the plurality of docking station modules comprise corresponding magnetic-activated cell selection modules, the plurality of docking station modules comprise corresponding fluorescence-activated cell selection modules;The present invention relates to an automated cell processing method, in which a plurality of docking station modules are equipped with corresponding spinoculation modules, and two or more cell processing steps performed in the cartridge are performed on cells in the cartridge.

[0007] Further modifications, features, and advantages of the present invention will become apparent from the following detailed description, as well as through practice of the invention. [Brief explanation of the drawings]

[0008] The patent or patent application file contains at least one drawing executed in color. Copies of this patent and patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1A] FIG. 1 is a block diagram of an illustrative variation of a cell processing and manufacturing system. [Figure 1B] FIG. 10 is a block diagram of an illustrative variation of the cartridge. [Figure 2A] FIG. 1 is a block diagram of an illustrative variation of a cell processing and manufacturing system. [Figure 2B] FIG. 1 is a perspective view of an illustrative variation of a cell processing station of a cell processing and manufacturing system. [Figure 2C] FIG. 10 is a side view of an illustrative variation of a cell processing station and cartridge of a cell processing and manufacturing system. [Figure 2D] FIG. 1 is a perspective view of an illustrative variation of a cell processing and manufacturing system. [Figure 2E] FIG. 1 is an internal view of an illustrative variation of a cell processing and manufacturing system. [Figure 2F] FIG. 1 is a block diagram of an illustrative variation of a cell processing and manufacturing system. [Figure 2G] FIG. 10 is a block diagram of another illustrative variation of a cell processing and manufacturing system. [Figure 3] FIG. 10 is a block diagram of another illustrative variation of a cell processing and manufacturing system. [Figure 4A]FIG. 10 is a perspective view of another illustrative variation of a docking station and cartridge of a cell processing and manufacturing system. [Figure 4B] FIG. 10 is another perspective view of another illustrative variation of the docking station and cartridge of the cell processing and manufacturing system. [Figure 4C] FIG. 10 is another perspective view of another illustrative variation of the docking station and cartridge of the cell processing and manufacturing system. [Figure 5A] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5B] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5C] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5D] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5E] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5F] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5G] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5H]10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5I] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5J] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5K] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5L] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5M] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5N] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5O] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5P] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5Q]10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5R] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5S] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5T] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5U] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 5V] 10 provides an additional perspective view of another illustrative variation embodiment of at least one of the docking station, cartridge, and sterile liquid transfer device of the cell processing and manufacturing system. [Figure 6] 10A-10C are schematic diagrams of illustrative variations of cartridges. [Figure 7] FIG. 10 is a schematic diagram of another illustrative variation of the cartridge. [Figure 8A] FIG. 10 is a side view of an illustrative variation of the cartridge. [Figure 8B] FIG. 10 is a top view of an illustrative variation of the cartridge. [Figure 8C] FIG. 10 is a side view of an illustrative variation of the cartridge. [Figure 8D] 10A and 10B are perspective views of illustrative variations of cartridges. [Figure 9] 10 shows a cross-sectional side view of an illustrative variation of the cartridge. [Figure 10A] 1 shows illustrative variations of rotary valves and actuators. [Figure 10B] 10 shows an illustrative variation of a rotary valve docked with an actuator. [Figure 11A] 10 is a perspective view of an illustrative variation of a cartridge with a CCE module in an extended configuration. [Figure 11B] 10 is a cross-sectional side view of an illustrative variation of a CCE module in a retracted configuration. FIG. [Figure 11C] 10A and 10B are cross-sectional side views of illustrative variations of CCE modules in an extended configuration. [Figure 12A] FIG. 10 is a perspective view of an illustrative variation of a docking station magnetic-activated cell sorting (MACS) module with magnets in an ON configuration. [Figure 12B] 13 is a perspective view of an illustrative variation of a docking station MACS module with the magnet in an OFF configuration. FIG. [Figure 13] FIG. 10 is a perspective view of an illustrative variation of a cartridge and docking station bioreactor module. [Figure 14] FIG. 10 is a perspective view of an illustrative variation of a cartridge coupled to a docking station bioreactor module. [Figure 15] FIG. 10 is a block diagram of an illustrative variation of a fluid connector system. [Figure 16A] 10A-10C are schematic diagrams of illustrative variations of fluid connectors. [Figure 16B] FIG. 16B is a detailed schematic diagram of the fluid connector depicted in FIG. 16A. [Figure 16C] FIG. 16B is a schematic diagram of the fluid connector depicted in FIG. 16A in a coupled configuration. [Figure 16D] FIG. 16B is a schematic diagram of the fluid connector depicted in FIG. 16A in an open port configuration. [Figure 16E] FIG. 16B is a schematic diagram of the fluid connector depicted in FIG. 16A receiving a gas. [Figure 16F] FIG. 16B is a schematic diagram of the fluid connector depicted in FIG. 16A receiving a sterilant. [Figure 16G]FIG. 16B is a schematic diagram of the fluid connector depicted in FIG. 16A in an open valve configuration. [Figure 16H] FIG. 16B is a schematic diagram of the fluidic connector depicted in FIG. 16A transferring fluid between fluidic devices coupled to the fluidic connector. [Figure 16I] FIG. 16B is a schematic diagram of the fluid connector depicted in FIG. 16A in a closed valve configuration. [Figure 16J] FIG. 16B is a schematic diagram of the fluid connector depicted in FIG. 16A in a closed port configuration. [Figure 16K] FIG. 16B is a schematic diagram of the fluid connector depicted in FIG. 16A in an uncoupled configuration. [Figure 16L] FIG. 16B is a schematic diagram of the fluid connector depicted in FIG. 16A uncoupled from the sterilant source. [Figure 17A] FIG. 16 is a front perspective view of a fluid connector in a closed port configuration. [Figure 17B] FIG. 17B is a rear perspective view of the fluid connector depicted in FIG. 17A in a closed port configuration. [Figure 17C] FIG. 17C is a rear view of the fluid connector depicted in FIG. 17B in a closed port configuration. [Figure 17D] FIG. 16 is a front perspective view of a fluid connector in an open port configuration. [Figure 17E] FIG. 17E is a rear perspective view of the fluid connector depicted in FIG. 17D in an open port configuration. [Figure 17F] FIG. 17F is a rear view of the fluid connector depicted in FIG. 17E in an open port configuration. [Figure 18A] FIG. 1 is a side view of a fluid connector in an unmated configuration. [Figure 18B] FIG. 1 is a cross-sectional side view of a fluid connector in an unmated configuration. [Figure 18C] FIG. 1 is a side view of a fluid connector in a coupled configuration. [Figure 18D] FIG. 1 is a cross-sectional side view of a fluid connector in a coupled configuration. [Figure 18E] FIG. 12 is a side view of a fluid connector in an open port configuration. [Figure 18F]FIG. 1 is a cross-sectional side view of a fluid connector in an open port configuration. [Figure 18G] FIG. 10 is a side view of a fluid connector in an open valve configuration. [Figure 18H] FIG. 10 is a cross-sectional side view of a fluid connector in an open valve configuration. [Figure 19] 1 is a schematic diagram of an illustrative variation of a fluid connector system. [Figure 20A] 1 is a schematic diagram of an illustrative variation of a fluid connector system. [Figure 20B] 10A-10C are schematic diagrams of illustrative variations of the fluid connector connection process. [Figure 20C] 10A-10C are schematic diagrams of illustrative variations of the fluid connector connection process. [Figure 21] FIG. 10 is a block diagram of an illustrative variation of a fluid connector system. [Figure 22] FIG. 10 is a block diagram of an illustrative variation of a fluid connector system. [Figure 23] FIG. 10 is a block diagram of an illustrative variation of a fluid connector system. [Figure 24A] FIG. 10 is a block diagram of an illustrative variation of a fluid connector system. [Figure 24B] 10A-10C are schematic diagrams of illustrative variations of the fluid connector connection process. [Figure 24C] 1 is a schematic diagram of an illustrative variation of a valve. [Figure 25A] FIG. 10 is a block diagram of an illustrative variation of a fluid connector system. [Figure 25B] 10A-10C are schematic diagrams of illustrative variations of the fluid connector connection process. [Figure 25C] 1 is a schematic diagram of an illustrative variation of a valve. [Figure 26A] FIG. 10 is a side view of an illustrative variation of a pump actuator and pump. [Figure 26B] FIG. 10 is a side view of an illustrative variation of a pump actuator coupled to a pump. [Figure 27] 10 is a flowchart of an illustrative variation of a method for transferring fluid using a fluid connector. [Figure 28] 1 is a flow chart of an illustrative variation of a method for cell processing. [Figure 29-1] 1 is a flow chart of an illustrative variation of a method for cell processing. [Figure 29-2] 1 is a flow chart of an illustrative variation of a method for cell processing. [Figure 29-3] 1 is a flow chart of an illustrative variation of a method for cell processing. [Figure 30A] 1 is a flow chart of an illustrative variation of the method of cell treatment for autologous CAR T cells or engineered TCR cells. [Figure 30B] 1 is a flow chart of an illustrative variation of the method of cell processing for allogeneic CAR T cells or engineered TCR cells. [Figure 31] 1 is a flow chart of an illustrative variation of the method of cell treatment for HSC cells. [Figure 32] 1 is a flow chart of an illustrative variation of the method of cell treatment for TIL cells. [Figure 33] 1 is a flowchart of an illustrative variation of the method of cell treatment for NK-CAR cells. [Figure 34A] 1 is a flow chart of an illustrative variation of the method of cell treatment for Treg cells. [Figure 34B] 1 is a flow chart of an illustrative variation of the method of cell treatment for Treg cells. [Figure 34C] 1 is a flow chart of an illustrative variation of the method of cell treatment for Treg cells. [Figure 35] 1 is a flow chart of an illustrative variation of a method for cell processing. [Figure 36] 10 is a flowchart of an illustrative variation of a method for implementing a transformation model. [Figure 37] 1 is an illustrative variation of a graphical user interface associated with an initial process design interface. [Figure 38] 10 is an illustrative variation of a graphical user interface associated with creating a process. [Figure 39] 10 is an illustrative variation of a graphical user interface associated with an empty process. [Figure 40] 10 is an illustrative variation of a graphical user interface associated with adding reagent and consumable containers. [Figure 41] 10 is an illustrative variation of a graphical user interface related to process parameters. [Figure 42] 10 is an illustrative variation of a graphical user interface associated with a patient weight process parameter. [Figure 43] 10 is an illustrative variation of a graphical user interface associated with a pre-processing analysis. [Figure 44] 10 is an illustrative variation of a graphical user interface for a white blood cell count pre-processing analysis. [Figure 45] 10 is an illustrative variation of a graphical user interface associated with process parameter calculations. [Figure 46] 10 is an illustrative variation of a graphical user interface associated with a completed process setup. [Figure 47] 10 is an illustrative variation of a graphical user interface associated with process operation activation settings. [Figure 48] 10 is an illustrative variation of a graphical user interface associated with a completed process action activation setting. [Figure 49] 10 is an illustrative variation of a graphical user interface associated with an initial process operation. [Figure 50] 10 is an illustrative variation of a graphical user interface relating to dragging in a process action. [Figure 51] 10 is another illustrative variation of a graphical user interface relating to dragging in a process action. [Figure 52] 10 is an illustrative variation of a graphical user interface associated with a completed process operation. [Figure 53] 10 is an illustrative variation of a graphical user interface associated with product monitoring. [Figure 54] 10 is another illustrative variation of a graphical user interface for product monitoring. [Figure 55] FIG. 1 is a block diagram of an illustrative variation of a cell processing and manufacturing system. [Figure 56] FIG. 1 is a cross-sectional side view of an illustrative variation of a counterflow centrifugal elutriation (CCE) module. [Figure 57] FIG. 10 is a cross-sectional side view of an illustrative variation of a magnetically activated cell selection (MACS) module. [Figure 58] 1 is a perspective view of an illustrative variation of a CCE system. [Figure 59A] 1 is a perspective view of an illustrative variation of a CCE system. [Figure 59B] 1 is a perspective view of an illustrative variation of a CCE system. [Figure 59C] 1 is a side cross-sectional view of an illustrative variation of a CCE system. [Figure 59D] 10A and 10B are side cross-sectional views of illustrative variations of rotors of CCE modules. [Figure 59E] 10A and 10B are side cross-sectional views of illustrative variations of rotors of CCE modules. [Figure 59F] 10A and 10B are side cross-sectional views of illustrative variations of rotors of CCE modules. [Figure 60A] 10A and 10B are plan views of illustrative variations of the rotor of the CCE module. [Figure 60B] 10A and 10B are perspective views of illustrative variations of the rotor of the CCE module. [Figure 60C] 10A and 10B are perspective views of illustrative variations of the rotor of the CCE module. [Figure 60D] 10A and 10B are side views of illustrative variations of the rotor of the CCE module. [Figure 60E] 10 is a perspective view of an illustrative variation of a rotor in a housing. FIG. [Figure 60F]10 is a plan schematic view of an illustrative variation of a rotor of a CCE module. FIG. [Figure 60G] 10 is a plan schematic view of an illustrative variation of a rotor of a CCE module. FIG. [Figure 60H] 10A and 10B are side views of illustrative variations of the rotor of the CCE module. [Figure 60I] FIG. 10 is a perspective view of another illustrative variation of a rotor of a CCE module. [Figure 60J] FIG. 10 is a perspective view of yet another illustrative variation of a rotor of a CCE module. [Figure 60K] FIG. 10 is a schematic plan view of another illustrative variation of rotor dimensions of a CCE module. [Figure 60L] 10 is an image of a set of illustrative variations of the rotor of a CCE module. [Figure 61A] FIG. 1 is a schematic diagram of an illustrative variation of a cell separation process. [Figure 61B] FIG. 1 is a schematic diagram of an illustrative variation of a cell separation process. [Figure 61C] FIG. 1 is a schematic diagram of an illustrative variation of a cell separation process. [Figure 62A] FIG. 1 is a perspective view of an illustrative variation of a MACS system in a first configuration. [Figure 62B] FIG. 10 is a perspective view of an illustrative variation of a MACS system in a second configuration. [Figure 62C] 1 is a cross-sectional side view of an illustrative variation of the MACS system. [Figure 62D] FIG. 10 is a perspective view of an illustrative variation of a MACS system in a second configuration. [Figure 62E] 1 is a plan view of an illustrative variation of the flow cell and magnet array of a MACS system. [Figure 62F] FIG. 10 is a plan view of an illustrative variation of a flow cell of the MACS system. [Figure 62G] 1 is a schematic diagram of an illustrative variation of a flow cell and magnet array. [Figure 63A] 10A and 10B are perspective views of illustrative variations of magnet arrays. [Figure 63B]10A and 10B are perspective views of illustrative variations of magnet arrays. [Figure 63C] 10A and 10B are perspective views of illustrative variations of magnet arrays. [Figure 63D] 10A and 10B are perspective views of illustrative variations of magnet arrays. [Figure 63E] 10A and 10B are perspective views of illustrative variations of magnet arrays. [Figure 64A] FIG. 10 is a perspective view of an illustrative variation of a flow cell. [Figure 64B] 1 shows a cross-sectional side view of an illustrative variation of a flow cell. [Figure 64C] FIG. 1 is a schematic diagram of an illustrative variation of the MACS system. [Figure 65A] FIG. 1 is a schematic diagram of an illustrative variation of a flow cell. [Figure 65B] FIG. 1 is a schematic diagram of an illustrative variation of a flow cell. [Figure 65C] FIG. 1 is a schematic diagram of an illustrative variation of a flow cell. [Figure 66A] FIG. 1 is a schematic diagram of an illustrative variation of a cell separation process. [Figure 66B] FIG. 1 is a schematic diagram of an illustrative variation of a cell separation process. [Figure 66C] FIG. 1 is a schematic diagram of an illustrative variation of a cell separation process. [Figure 67A] FIG. 1 is a schematic diagram of an illustrative variation of a cell processing and manufacturing system. [Figure 67B] FIG. 1 is a schematic diagram of an illustrative variation of a cell processing and manufacturing system. [Figure 68A] FIG. 1 is a cross-sectional perspective view of an illustrative variation of a bioreactor. [Figure 68B] FIG. 1 is a cross-sectional side view of an illustrative variation of a bioreactor. [Figure 68C] FIG. 10 is a perspective view of an illustrative variation of a bioreactor enclosure. [Figure 68D] FIG. 10 is a plan view of an illustrative variation of a bioreactor enclosure. [Figure 68E] FIG. 10 is a perspective view of an illustrative variation of a membrane of a bioreactor. [Figure 68F]FIG. 10 is a side view of an illustrative variation of a membrane of a bioreactor. [Figure 68G] FIG. 10 is a perspective view of an illustrative variation of a membrane of a bioreactor. [Figure 68H] FIG. 10 is a bottom view of an illustrative variation of the membrane of the bioreactor. [Figure 69A] FIG. 10 is a cross-sectional side view of an illustrative variation of a bioreactor enclosure. [Figure 69B] FIG. 10 is a cross-sectional perspective view of an illustrative variation of a bioreactor enclosure. [Figure 70] FIG. 1 is an exploded perspective view of an illustrative variation of a bioreactor. [Figure 71A] FIG. 1 is a plan view of an illustrative variation of a bioreactor. [Figure 71B] FIG. 1 is a cross-sectional side view of an illustrative variation of a bioreactor. [Figure 72] FIG. 1 is a schematic diagram of an illustrative variation of an electroporation system. [Figure 73] FIG. 10 is an exploded perspective view of an illustrative variation of an electroporation module. [Figure 74A] FIG. 1 is a schematic diagram of an illustrative variation of the electroporation process. [Figure 74B] FIG. 1 is a schematic diagram of an illustrative variation of the electroporation process. [Figure 75] FIG. 1 is a circuit diagram of an illustrative variation of an electroporation process. [Figure 76A] 1 is a plot of an illustrative variation of the electroporation process. [Figure 76B] 1 is a plot of an illustrative variation of the electroporation process. [Figure 76C] 1 is a plot of an illustrative variation of the electroporation process. [Figure 76D] 1 is a plot of an illustrative variation of the electroporation process. [Figure 77A] 1 is a flowchart of an illustrative variation of a method for separating cells. [Figure 77B]10 is a flowchart of an illustrative variation of a method for enriching cells. [Figure 77C] 10 is a flow chart of an illustrative variation of a method for buffer exchange. [Figure 78] 10 is a flowchart of another illustrative variation of a method for separating cells. [Figure 79A] 7 is a flowchart of an illustrative variation of a closed-loop method 7900 for separating cells. [Figure 79B] 7 is a flowchart of an illustrative variation of a closed-loop method 7910 for elutriating cells. [Figure 79C] 7 is a flowchart of an illustrative variation of a closed-loop method 7920 for harvesting cells. [Figure 80A] 1 is a flowchart of an illustrative variation of a method for separating cells. [Figure 80B] 10 is a flowchart of an illustrative variation of a method for selecting cells. [Figure 81] 10 is a flowchart of another illustrative variation of a method for separating cells. [Figure 82A] 1 is a flow chart of an illustrative variation of a method for preparing a bioreactor. [Figure 82B] 10 is a flow chart of an illustrative variation of a method for loading a bioreactor. [Figure 82C] 1 is a flow chart of an illustrative variation of a method for preparing a bioreactor. [Figure 82D] 10 is a flow chart of an illustrative variation of a method of calibration for a bioreactor. [Figure 82E] 10 is a flow chart of an illustrative variation of a method for mixing reagents. [Figure 82F] 10 is a flow chart of an illustrative variation of a method for mixing reagents. [Figure 82G] 1 is a flowchart of an illustrative variation of a method for culturing cells. [Figure 82H] 1 is a flow chart of an illustrative variation of a method for freezing cells. [Figure 82I]10 is a flow chart of an illustrative variation of a method for collecting a sample. [Figure 82J] 1 is a flowchart of an illustrative variation of a method for culturing cells. [Figure 82K] 10 is a flow chart of an illustrative variation of a method for medium exchange. [Figure 82L] 10 is a flowchart of an illustrative variation of a method for controlling gas. [Figure 82M] 10 is a flow chart of an illustrative variation of a method for controlling pH. [Figure 83] 1 is a flow chart of an illustrative variation of a method for electroporating cells. [Figure 84] 10 is a flowchart of another illustrative variation of a method for electroporating cells. [Figure 85] 10A-10C are schematic diagrams of illustrative variations of fluid connectors. [Figure 86] 10A-10C are schematic diagrams of illustrative variations of fluid connector ports. [Figure 87] 10A-10C are schematic diagrams of illustrative variations of the fluid connector connection process. [Figure 88] 10A-10C are schematic diagrams of illustrative variations of the fluid connector connection process. [Figure 89] 10A-10C are schematic diagrams of illustrative variations of fluid connectors. [Figure 90A] 10A and 10B are side views of illustrative variations of fluid connectors. [Figure 90B] FIG. 90B is a perspective view of the fluid connector depicted in FIG. 90A. [Figure 90C] FIG. 90B is a cross-sectional side view of the fluid connector depicted in FIG. 90A. [Figure 91A] 10A and 10B are side views of illustrative variations of fluid connectors. [Figure 91B] FIG. 91B is a perspective view of the fluid connector depicted in FIG. 91A. [Figure 91C] FIG. 91B is a cross-sectional side view of the fluid connector depicted in FIG. 91A. [Figure 91D] 10A and 10B are side views of illustrative variations of fluid connectors. [Figure 91E]FIG. 91D is a perspective view of the fluid connector depicted in FIG. 91D. [Figure 91F] FIG. 91D is a cross-sectional side view of the fluid connector depicted in FIG. 91D. [Figure 92A] 10A and 10B are side views of illustrative variations of fluid connectors. [Figure 92B] FIG. 92B is a transparent side view of the fluid connector depicted in FIG. 92A. [Figure 92C] FIG. 92B is a perspective view of the fluid connector depicted in FIG. 92A. [Figure 92D] FIG. 92B is a cross-sectional side view of the fluid connector depicted in FIG. 92A. [Figure 93A] 10A and 10B are perspective views of illustrative variations of fluid connectors. [Figure 93B] FIG. 93B is a transparent perspective view of the fluid connector depicted in FIG. 93A. [Figure 94A] 10A and 10B are perspective views of illustrative variations of fluid connectors. [Figure 94B] FIG. 94B is a transparent perspective view of the fluid connector depicted in FIG. 94A. [Figure 95A] 10A and 10B are perspective views of illustrative variations of fluid connectors. [Figure 95B] FIG. 95B is a transparent perspective view of the fluid connector depicted in FIG. 95A. [Figure 95C] FIG. 10 is a detailed side view of the port in an open port configuration. [Figure 95D] FIG. 10 is a detailed side view of the port in a closed port configuration. [Figure 96A] FIG. 10 is a plan view of an illustrative variation of a fluidic device. [Figure 96B] 10A and 10B are side views of illustrative variations of fluidic devices coupled to a robot. [Figure 96C] 10A and 10B are perspective views of illustrative variations of fluidic devices held by a robot. [Figure 97A] 10 is a perspective view of an illustrative variation of a MACS module. FIG. [Figure 97B] 10 is a cross-sectional perspective view of an illustrative variation of a MACS module. FIG. [Figure 97C]10 is a cross-sectional side view of an illustrative variation of a MACS module. [Figure 98] 1 is a flow chart of an illustrative variation of a method for cell processing. [Figure 99] 1 is a flow chart of an illustrative variation of a method for cell processing. [Figure 100] 1 is a flow chart of an illustrative variation of a method for cell processing. [Figure 101] 1 is a flow chart of an illustrative variation of a method for cell processing. [Figure 102] FIG. 1 is a schematic diagram of an illustrative variation of a cell processing and manufacturing system. [Figure 103A] 10A and 10B are perspective views of illustrative variations of sterile fluid transfer devices. [Figure 103B] 10A and 10B are perspective views of illustrative variations of sterile fluid transfer devices. DETAILED DESCRIPTION OF THE INVENTION

[0009] definition The term "a" or "an" may refer to one or more of that entity, i.e., to a plurality of referents. Similarly, the terms "a," "an," "one or more," and "at least one" are used interchangeably herein. In addition, reference to an "element" by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be only one element.

[0010] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method employed to determine the value or the variation that exists between samples measured. Unless otherwise stated or apparent from the context, the term "about" means within 10% above or below the reported numerical value (except where such numerical value would exceed 100% or be less than 0% of the possible values). When used in conjunction with a range or series of values, the term "about" applies to the endpoints of the range or to each of the values ​​recited in the series of ranges, unless otherwise indicated. As used herein, the terms "about" and "approximately" are used interchangeably.

[0011] As used herein, the term "sterilization" is used as a non-limiting description of several variations and optional features that provide advantages in the operation of certain systems and methods of the present disclosure. Maintaining sterility is typically desirable for cell processing, but may be achieved in a variety of ways, including, but not limited to, providing sterile reagents, media, cells, and other solutions; sterilizing the cartridge and / or cartridge components after loading (protecting the cellular product from destruction); and / or operating the system in a sterile enclosure, environment, building, room, etc. Such user- or system-implemented sterilization steps may sterilize the cartridge or cartridge components and / or maintain the sterility of the cartridge or cartridge components.

[0012] As used herein, the term "and / and" is used interchangeably with "or / or" unless expressly stated otherwise.

[0013] Unless the context clearly requires otherwise, throughout the specification and claims, words like "comprise," "comprising," and the like are to be construed in the inclusive sense, i.e., "including but not limited to," as opposed to the exclusive or exhaustive sense. Words using the singular or plural also include the plural and singular, respectively. Furthermore, the words "herein," "above," "below," and words of similar import, when used in this application, refer to this application as a whole and not to any particular portions of this application.

[0014] Cell Processing and Manufacturing Systems Systems and methods for processing and manufacturing cellular products for biomedical applications are described herein. In variations, these systems may include a cell processing and manufacturing system (CPMS) including a cartridge containing a cellular product and a cell processing station with at least a controller and at least one novel docking station configured to receive the cartridge. In some variations, the CPMS may perform automated manufacturing of the cellular product. Briefly, the cell processing station may also include a pump, a reagent reservoir, a fluid source, a fluid connector, at least one sensor, and a sterile liquid transfer device. The docking station of the cell processing station may include one or more docking station modules (e.g., at least one docking station module, or multiple docking station modules) and may be configured to receive the cartridge. The controller of the cell processing station may include a processor, memory, a display, input devices, and communication devices for performing the methods described herein. The cartridge of the CPMS may include a fluid transfer bus, a sensor (e.g., at least one sensor), a fluid connector, and one or more modules (e.g., at least one cartridge module, or multiple cartridge modules). The one or more cartridge modules may include a cell separation module, which may be a counterflow centrifugation elutriation module, a bioreactor, an electroporation module, a spinoculation module, a fluorescence-activated cell selection module, and a magnetic-activated cell selection module. The docking station may be configured to receive a single cartridge, and the cartridge may be stationary within the docking station. Further, the one or more docking station modules may be configured to engage and / or interface with the cartridge and / or corresponding modules of the cartridge to perform cell processing steps on the cell product, such that the CPMS performs the cell processing steps on the cell product. In some variations, multiple cell processing steps may be performed within a single cartridge.

[0015] Additionally, methods for processing and manufacturing cell products for biomedical applications can include methods for processing a solution containing the cell product. For example, the method can include cell processing steps of digesting tissue using an enzymatic reagent to release a selected cell population into a solution, concentrating the cells using a docking station CCE module, washing the cells using a docking station CCE module, selecting the cells in the solution using a docking station selection module, sorting the cells in the solution using a docking station sorting module, differentiating or expanding the cells in a cartridge bioreactor module, activating the cells using an activation reagent, electroporating the cells, transducing the cells using a vector, and finishing the cell product.

[0016] Advantageously, in some variations, multiple cell processing steps can be performed on a cartridge positioned within the docking station of the cell processing and manufacturing system without the need to move or transport the cartridge from the docking station.

[0017] Cell Processing Station (CPS) The CPMS introduced above may generally include a cell processing station having a docking station including multiple docking station modules, each independently configured to perform one or more cell processing operations on a cartridge in the docking station. The docking station modules may include one or more of a bioreactor module, a cell selection module (e.g., a docking station magnetic-activated cell selection module), a sorting module (e.g., a fluorescence activated cell sorting (FACS) module), an electroporation module, a counterflow centrifugal elutriation (CCE) module, a reagent reservoir, etc. In an alternative, a docking station module of the CPS may be configured to interface with a corresponding cartridge module of a cartridge to perform cell processing on a cellular product in the cartridge.

[0018] The CPS may advantageously include a docking station configured to integrate the functionality of various cell processing tasks into a single structure, thereby allowing cell products to be produced in an end-to-end automated manner without user intervention for changing settings, transferring cell products, and the like. In particular, the single structure may be a cartridge configured to undergo complete end-to-end automation of multiple processes (e.g., washing, concentration, elutriation, magnetic cell separation, sorting, activation, genetic modification, electroporation, spinoculation, formulation, expansion, harvest / formulation, and cryopreservation) while docked to the docking station to produce a cell product. In some variations, the cartridge may include two or more bioreactor modules for producing two or more cell products via a splitting process. In combination with the cartridge of the present disclosure, cell processing tasks may be performed on cells within the cartridge in coordination with corresponding modules of the cartridge according to a predetermined processing schedule to produce a processed cell product.

[0019] In some variations, the cell processing station of the present disclosure may be a stand-alone unit featuring standard power options that can be easily moved (e.g., on rollers or casters) throughout a laboratory space as needed. In particular, the cell processing and manufacturing system of the present disclosure, which may include a mobile cell processing station with a docking station and cartridges housed therein, is compact and can be easily implemented within any laboratory space because no expensive installation is required. Thus, the cell processing and manufacturing system of the present disclosure may enable users to conduct and scale-up process development studies before making a larger investment in production. Furthermore, the stand-alone unit may reduce cell processing design and development risk because cartridges may not need to be moved between modules and / or instruments during cell processing.

[0020] cartridge A cartridge may include multiple cartridge modules (also referred to herein as cartridges, consumable cartridges, consumables, and cell processing devices), such as a bioreactor, a countercurrent centrifugal elutriation (CCE) module, a magnetic cell sorter (e.g., a magnetic-activated cell selection module (MACS)), an electroporation device (e.g., an electroporation module (EP)), a sorting module (e.g., a fluorescence-activated cell sorting (FACS) module), an acoustic flow cell module, a centrifugation module, a microfluidic enrichment module, or combinations thereof. The cell processing and manufacturing systems described herein may reduce operator intervention and increase throughput by automating cartridge (and cell product) processing. For example, an automated cell processing and manufacturing system may facilitate sterile liquid transfer between one or more cartridges and a cell processing station (e.g., one or more docking station modules). Additionally or alternatively, the automated cell processing and manufacturing system may facilitate sterile fluid transfer between one or more cartridges and other components of the system, such as fluid connectors (e.g., sterile fluid transfer ports), reagent reservoirs, sampling containers (e.g., sterile fluid transfer devices as described in detail herein, and combinations thereof).

[0021] The cartridges may be portable and configured to facilitate automated, sterile cell processing, for example, the cartridges may be configured to be moved by a user from a location outside the CPS to a receiving location in a docking station within the cell processing station.

[0022] In some variations, the cartridge may further include one or more of a sterile liquid transfer port, a liquid transfer bus (also referred to herein as a fluid bus) fluidly coupled to each module, and a pump fluidly coupled to the liquid transfer bus.

[0023] In some variations, a cartridge may provide a self-contained device capable of performing one or more cell processing steps, optionally via one or more modules. The modules may be integrated into a fixed configuration within the cartridge. Additionally or alternatively, cartridge modules may be configurable or movable within the cartridge, allowing various cartridges to be assembled from shared modules. Similarly, a cartridge may be a single closed unit with fixed components for each cartridge module, or the cartridge may contain configurable modules coupled by configurable fluidic, mechanical, optical, and electrical connections. In some variations, one or more sub-cartridges, each containing a set of modules, may be configured to be assembled to perform various cell processing workflows. Furthermore, cartridge modules may each be provided within a separate housing or may be integrated into a cartridge or sub-cartridge with other modules. Although modules may be generally described herein as separate groups of components for simplicity, the modules (and their respective components) may be arranged in any suitable configuration. For example, components of different modules may be interspersed with one another within the CPS, with each module defined by a set of connected components that collectively perform a predetermined function. However, the components of each cartridge module may or may not be physically grouped within a cartridge. In some variations, multiple cartridges may be used to process a single cell product through the transfer of the cell product from one cartridge to another cartridge of the same or different type, and / or by splitting the cell product among more cartridges, and / or by pooling multiple cell products into fewer cartridges.

[0024] Generally, each of the docking station modules of the system may interface with its respective module(s) on the cartridge. For example, the electroporation module on the cartridge (if present) may interface with the docking station electroporation module to perform the electroporation step on the cellular product and may also interface with common components such as fluid bus line components (e.g., pumps, valves, sensors, etc.). An advantage of such a split-module design is that expensive components (e.g., motors, sensors, heaters, lasers, etc.) can be retained within the docking station module of the system. The use of disposable cartridges, in such variations, may eliminate the need to sterilize the cartridge between uses.

[0025] A variety of materials can 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 can be molded, machined, extruded, 3D printed, or any combination thereof. The cartridge can contain commercially available components (e.g., tubing, valves, fittings). These components can be attached to or integrated with custom components or devices. The cartridge housing can constitute an additional enclosure layer that further protects the sterility of the cell product. In some variations, the cartridge can be designed to be single-use or disposable.

[0026] Generally, an operator may load or unload a cartridge in a Class ISO-5 or better environment, utilizing aseptic technique to ensure that the sterility of the cartridge contents is maintained when the cartridge is opened. In some variations, an operator may perform the loading or unloading of a cartridge using manual sterile connections (e.g., sterile tubing welds).

[0027] Reagent storage section The system generally may include one or more reagent reservoirs in which reagents are stored. Non-limiting examples of the one or more reagents may include cell culture media, buffers, cytokines, proteins, enzymes, polynucleotides, transfection reagents, non-viral vectors, viral vectors, antibiotics, nutrients, cryoprotectants, solvents, cellular materials, and pharmaceutically acceptable excipients. Additionally or alternatively, waste products may be stored in the reagent reservoir. In some variations, in-process samples extracted from one or more cartridges may be stored in the reagent reservoir. The reagent reservoir may include one or more controlled temperature compartments (e.g., a freezer, cooler, water bath, warming chamber, or other, e.g., about -80°C, about -20°C, about 4°C, about 25°C, about 30°C, about 37°C, and about 42°C). The temperature within these compartments may be varied during the cell manufacturing process to heat or cool the reagents. In variations of the disclosed methods, a robot (or manually with operator assistance) may engage each of the cartridges and reagent reservoirs. The reagent reservoir may interface with one or more sterile liquid transfer ports on the cartridge so that reagents or materials can be dispensed into the cartridge. Optionally, fluids can be added to or removed from the cartridge before, during, or after adding or removing reagents. In some variations, the system includes a sterile liquid transfer instrument similarly configured to transfer fluids into or out of the cartridge in an automated, manual, or semi-automated manner. An operator may manually stock the sterile liquid transfer instrument with reagents. The reagent reservoir may have automatic doors to allow access of sterile liquid transfer devices and / or other reagent containers, optionally each under independent closed-loop temperature control. Devices and containers may be configured for pick-and-place movement by a robot. In some variations, the reagent reservoir may include one or more sample pickup areas. For example, a robot may be configured to move one or more reagents to and from one or more of the sample pickup areas.

[0028] In some variations, the reagent reservoirs may be sized to accommodate multiple sterile fluid transfer devices needed to support a single patient process. Sterile fluid transfer devices may be manually provided to and / or replaced within the reagent reservoirs on a predetermined schedule (e.g., every three days or as required by the cell processing task).

[0029] Cell Selection System The CPMS described herein may include a cell selection system configured to separate cells based on predetermined criteria. For example, cells may be separated based on physical characteristics such as size and / or density, e.g., using counterflow centrifugal elutriation. Cells may also be separated based on the presence of predetermined antigens, e.g., using magnetically activated cell selection. In some variations, a CPMS with modules for these separation methods (e.g., cartridge module, docking station module) may facilitate one or more cell processing steps, including, but not limited to, cell concentration, cell dilution, cell washing, buffer exchange, and magnetic separation. The cell selection systems described herein may increase throughput and cell yield output in a compact and portable configuration. For example, before magnetically separating the cells, a suspension of cells may be mixed with an excess or predetermined concentration (e.g., cells / mL) of magnetic reagent. Similarly, after magnetically separating the cells, the cells may be washed in solution (e.g., a suitable buffer solution).

[0030] In some variations, a cell separation system (or cell separator) may include a rotor configured for countercurrent centrifugal elutriation of cells in a fluid, a first magnet configured to magnetically rotate the rotor and separate cells from the fluid in the rotor, a flow cell in fluid communication with the rotor and configured to receive cells from the rotor, and a second magnet configured to magnetically separate cells in the flow cell.

[0031] In some variations, the CCE module may be integrated into a cartridge to enable the cell processing and manufacturing system to separate cells based on cell size and / or density. In some variations, the cell separation system may include a housing comprising a rotor configured to separate cells from a fluid (e.g., separate cells of different size and / or density from cells remaining in the fluid) and a magnet configured to magnetically rotate the rotor. The housing may be configured to move relative to the magnet or vice versa (e.g., to move the magnet relative to the housing). The CCE modules described herein may provide cell separation in a compact and portable housing where the magnet may be located external to the housing (e.g., a magnet located within a corresponding CCE module of a docking station).

[0032] In some variations, a compact rotor, which may aid in cartridge integration, may include input and output fluid conduits extending from the rotor to opposite sides of the rotor housing. For example, the rotor may include a first side having a first fluid conduit and a second side having a second fluid conduit, the second side being opposite the first side. An elutriation chamber (e.g., a cone) may be coupled between the first and second fluid conduits.

[0033] In some variations, a method of separating cells from a fluid may include moving a rotor defining an axis of rotation toward a magnet, flowing a fluid through the rotor, rotating (e.g., magnetically) the rotor about the axis of rotation using a magnet while flowing the fluid through the rotor, and moving the rotor away from the magnet.

[0034] In some variations, a method of separating cells from a fluid can include flowing a fluid containing cells through a flow cell. The set of cells can be labeled with magnetic particles. The set of cells can be magnetically attracted toward a magnet array for a residence time, and the set of cells can flow out of the flow cell after the residence time.

[0035] In some variations, the flow cell may include an elongated cavity having a cavity height and a magnet array including a plurality of magnets, each of the magnets spaced apart by a spacing distance, A predetermined ratio between the cavity height and the spacing distance may optimize magnetic separation of cells within the flow cell.

[0036] Electroporation In some variations, the electroporation module (EP) or electroporator described herein can be configured to facilitate one or more of cell transduction and transfection. As described in more detail herein, the EP can be configured to physically separate a first volume of fluid containing cells (e.g., a first batch) from a second volume of fluid containing cells (e.g., a second batch, a third batch) using a gas (e.g., an air gap). The EP can also be configured to separately apply an electroporation signal (e.g., a voltage pulse, a waveform) to each discrete batch of fluid to improve electroporation efficiency and thus increase throughput. In some variations, active electric field compensation can similarly improve electroporation efficiency and throughput.

[0037] In some variations, the cell processor may include a fluid conduit configured to receive a first fluid containing cells and a second fluid (e.g., gas, oil), a set of electrodes coupled to the fluid conduit, a pump coupled to the fluid conduit, and a controller with a processor and memory. The controller may be configured to generate a first signal to introduce the first fluid into the fluid conduit using the pump, generate a second signal to introduce the second fluid into the fluid conduit such that the second fluid separates the first fluid from a third fluid, and generate an electroporation signal to electroporate cells in the fluid conduit using the set of electrodes.

[0038] In some variations, a method of electroporating cells may include receiving a first fluid containing cells in a fluid conduit, receiving a second fluid containing a gas in the fluid conduit to separate the first fluid from a third fluid, and applying an electroporation signal to the first fluid to electroporate the cells.

[0039] In some variations, a method of electroporating cells may include receiving a first fluid containing cells in a fluid conduit, applying a resistance measurement signal to the first fluid using a set of electrodes, measuring resistance between the first fluid and the set of electrodes, and applying an electroporation signal to the first fluid based on the measured resistance.

[0040] Bioreactor In some variations, the bioreactor may include an enclosure having a base and sidewalls, and a gas-permeable membrane coupled to one or more of the base and sidewalls of the enclosure. The gas-permeable membrane may support cell culture. In some variations, the cell processing and manufacturing system may include a bioreactor and an agitator coupled to the bioreactor. The agitator may be configured to agitate the bioreactor based on orbital motion.

[0041] Fluid Connector In particular, conventional cell processing systems lack automated, multi-use sterile fluid connector solutions for cell therapy production, with a set of sterile fluid connectors capable of multiple connection and disconnection cycles with a given system. For example, conventional sterile fluid connectors may typically be single-use devices and, therefore, may be expensive and labor-intensive in terms of manufacturing and performing cell processing procedures. Generally, the fluid connectors described herein may include multiple sealed enclosures between sterile portions (e.g., fluid connector lumens or cavities) and the external (e.g., non-sterile) ambient environment, thereby facilitating sterilization control of the fluid connector and devices coupled thereto. The fluid connectors described herein may be durable components that can be reused for multiple cycles while maintaining sterility and / or bioburden control. For example, the fluid connectors may be sterilized using sterilants without harming the cell product or other biological materials.

[0042] In some variations, the sterile manufacturing systems described herein utilize one or more sterile fluid connectors and may have a configuration suitable for being operated by a robot, such as a robotic arm. The sterile fluid connectors described herein may enable automated, sterile, and metered transfer of fluids to automate cell therapy manufacturing. Automating cell therapy manufacturing may thereby provide reduced manufacturing costs per patient, reduced risk of process failure, and the ability to meet commercial-scale patient demand for cell therapy. In some variations, the sterile fluid connectors may increase one or more of sterility, efficiency, and speed by removing human operators from the manufacturing process. The automated, integrated sterilization processes described herein may be applied to the fluid connectors to maintain system sterility. For example, the fluid connectors may maintain sterility through multiple connection / disconnection cycles between separate sterile, closed-volume fluidic devices (e.g., enclosures, containers, vessels, cartridges, docking station modules, cartridge modules, bioreactors, containment vessels, sealed chambers). Thus, the systems, devices, and methods described herein may reduce the complexity of the sterilization process, reduce energy usage, and increase sterilization efficiency.

[0043] In some variations, the fluid connector may include a first connector configured to mate with a second connector (e.g., a male connector and a female connector). The proximal end of each of the connectors may be configured to connect (e.g., be in fluid communication with, form a fluid pathway) with a respective fluidic device to transfer one or more of a fluid (e.g., a liquid and / or gas) and a biological material (e.g., a cellular product) between the fluidic devices. The distal ends of the connectors may include ports configured to mate with each other. The fluidic connectors may also include sterilant ports configured to facilitate sterilization of chambers within the distal ends of the first and second connectors. The fluidic connectors may be sterilized before or after connection, as desired, to ensure sterility. In this manner, the fluidic connectors may be reused for multiple connection and disconnection cycles.

[0044] In some variations, cell processing and manufacturing systems utilizing the fluidic connectors described herein may include a robot configured to operate the fluidic connector, and a controller configured to control the robot and manipulate (e.g., move, connect, open, close, disconnect) the first and second connectors together (without human interaction) while maintaining sterility of the fluidic connector and the plurality of fluidic devices, thereby further reducing the risk of contamination. The fluidic devices may be one or more of an instrument, a docking station module, a cartridge, a cartridge module, etc.

[0045] Cell Processing Control Described herein are systems and methods for manufacturing cellular products for biomedical applications using automated systems. Conventional semi-automated solutions for cell processing may not allow users to define the biological process. Instead, users may select from a limited set of predefined machine processes and process control parameters. Furthermore, conventional semi-automated solutions may not provide a scalable manufacturing solution for cell therapy production. For example, cell therapy manufacturing may traditionally be performed in a batch fashion (i.e., one product is manufactured in a single room / suite, with the necessary processing tools located internally). This may be guided by a technician following a standard operating procedure (SOP), or in some cases, processing tools (e.g., Miltenyi Prodigy, Lonza Cocoon) may perform a series of processing steps for a single patient product on a single multi-function processing tool. However, existing solutions (e.g., Miltenyi Prodigy) may not allow users to define the biological process. Furthermore, the manual labor required for conventional solutions may increase the risk of product contamination and human error.

[0046] In some variations of the present disclosure, a set of cell therapy biological manufacturing processes can be converted into a set of machine instructions suitable for automated execution using the systems described herein. For example, a method for converting user-defined cell processing operations into cell processing steps to be executed by a processor of an automated cell processing and manufacturing system can include receiving an ordered input list of cell processing operations and executing a transformation model on the ordered input list to create an ordered output list of cell processing steps that can be performed by the system. As used herein, a transformation model can refer to an algorithm, process, or transformation configured to convert a set of cell processing steps into a set of machine or hardware instructions for the system. Advantageously, in some variations, multiple cell processing steps can be performed on a cartridge positioned within a docking station of a cell processing and manufacturing system without the need to move or transport the cartridge from the docking station. In some variations, a docking station module can be controlled to perform a cell processing step for each cell product. In this manner, the systems and methods enable biologists to define a manufacturing process in biological terms and have the system convert this biological model (e.g., process definition) into a set of machine-executable instructions.

[0047] The end-to-end closed-system automation described herein may reduce process failure rates and costs. For example, end-to-end automation may shorten production time (e.g., residence time) and increase throughput compared to traditional manual methods. That is, multiple processes (e.g., 10 or more processes) may be performed simultaneously. The methods described herein may further reduce the opportunity for contamination and user error. Thus, the systems, devices, and methods described herein may increase one or more of cell processing automation, reproducibility, reliability, process flexibility, instrument throughput, and process scalability, and reduce one or both of labor costs and process duration.

[0048] I. System Described herein are systems and devices configured to perform cell processing steps for manufacturing a cellular product (e.g., a cell therapy product). In some variations, a CPS of a CPMS may include a docking station having multiple docking station modules, each independently configured to perform one or more cell processing operations on a cartridge (e.g., a fluidic device) of the CPMS. Use of a controller may facilitate one or more of the automation, efficiency, and sterility of the cell processing and manufacturing system.

[0049] In some variations, the cell processing and manufacturing system may include a cell processing station that includes an enclosure.

[0050] 1A is a block diagram of a cell processing and manufacturing system (CPMS) 100 including a cell processing station (CPS) 110 and a cartridge 114 (also referred to herein as a consumable). In some variations, the CPS 110 may include one or more of a controller 120, a dock 125, a pump 138, a reagent reservoir 118, a fluid connector 132, a sterilant source 134, a fluid source 136, at least one sensor 140, and a sterile liquid transfer device 142. In some variations, the controller 120 may include one or more of a processor 122, a memory 124, a communication device 126, an input device 128, and a display 130. In some variations, the dock 125 may include a plurality of docking station modules 112 and a docking station 115 configured to receive the cartridge 114, and each of the plurality of docking station modules 112 may correspond to a cartridge module of the cartridge 114.

[0051] In some variations, the CPS may include a fully or at least partially enclosed housing in which one or more cell processing steps are performed in a fully or at least partially automated process. In some variations, the CPS may be an open system lacking an enclosure, which may be configured for use in a clean room, biosafety cabinet, or other sterile location. In some variations, the CPS may be configured to perform sterile fluid transfer into and out of cartridges in a fully or partially automated process. For example, one or more fluids may be stored in the sterile fluid transfer device 142. In some variations, the sterile fluid transfer device 142 may be a portable consumable that can be moved within the CPMS 100. The sterile fluid transfer devices and fluid connectors described herein enable automated, sterile, and metered transfer of fluids for automating cell therapy manufacturing. In some variations, the enclosure of the CPS may be configured to meet International Organization for Standardization (ISO) standard ISO 7 or better (e.g., ISO 6 or ISO 5). An advantage of meeting ISO 7 or better standards is that the system can be used in facilities that do not meet ISO 7 standards (i.e., lack a clean room or other well-filtered air space). Optionally, the facility can be an ISO 8 or ISO 9 facility. In some variations, the CPS is designed to be approximately 800 m 3 Less than 700m 3 Less than 600m 3 Less than 500m 3 Less than 300m 3 Less than 250m 3 Less than 200m 3 Less than 150m 3 Less than 100m 3 Less than 50m 3 Less than 25m 3 Less than 10m 3 Less than and about 5m 3 The term "volume" may include volumes less than 10 ...

[0052] In some variations, cartridges 114 may receive cell products from different donors or may contain cell products intended for different recipients. Cell product from a single donor may be split among multiple cartridges 114 if needed to generate enough product for therapeutic use or if the donor provides cell product to several recipients (e.g., for allogeneic transplantation). In some variations, multiple cartridges 114 may be placed sequentially within a docking station 115 (e.g., a first cartridge may be processed within the docking station, then a second cartridge may be processed within the docking station). In yet other variations, a first cartridge may be placed within the docking station 115 of a first cell processing station 110, and a second cartridge may be placed within the docking station 115 of a second cell processing station 110. Cell product for a single recipient may advantageously be split among multiple cartridges 114 as needed and processed within separate CPSs to generate enough product for therapeutic use for the recipients. Similarly, a cell product for a single recipient can be split between multiple cartridges 114, processed in separate CPSs, and optionally recombined in a proportion for therapeutic use for the recipient. In other variations, a cell product from a single donor can be split between two separate bioreactors in the same cartridge, whereby they undergo separate processes. The cell products can then be combined and used as part of a therapeutic treatment.

[0053] As illustrated in FIG. 1B , cartridge 114 may optionally include multiple cartridge modules, including one or more of bioreactor 150, cell separator 152, electroporator 160, spinoculator 153, cell sorter 155, counterflow centrifugation elutriator 151, and other modules 168, as described in more detail herein. It should also be understood that one or more of the multiple cartridge modules may use shared equipment to accomplish the tasks of the modules. Cartridge 114 may also include a liquid transfer bus 162 (also referred to herein as a fluid bus), at least one sensor 164, and a fluid connector 166, as described in more detail herein. Cell separator 152 may include one or more of rotor 154, flow cell 156, and magnet 158. In some variations, magnet 158 ​​may include one or more magnets and / or magnet arrays. For example, the cell separation system 152 may include 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.

[0054] Cell Processing Station (CPS) In some variations, the CPS 110 may include an at least partially enclosed enclosure (e.g., housing) in which one or more automated cell processing steps are performed. For example, the CPS 110 may be configured to transfer sterile liquids into and out of the cartridge 114 in a fully or partially automated process. In some variations, the CPS 110 may not have an enclosure and may be configured for use in a clean room, biosafety cabinet, or other suitably clean or sterile location. In some variations, the CPS 110 may be part of a CPMS 100 that includes a feed-through access biosafety cabinet, quality control instrumentation, pumps, consumables (e.g., fluidic devices), fluid connectors, consumable feed-throughs, and a sterilization system (e.g., sterilant source and / or generator, fluid source, heater / dryer, aerator).

[0055] FIG. 2A is a block diagram of a cell processing and manufacturing system 200, including a cell processing station (CPS) 210 and cartridges 214. The CPS 210 may include an enclosure 211 with four walls, a base, and a top. The CPS 210 may be divided into an interior zone with feedthrough access 213 and, optionally, quality control (QC) instrumentation. An air filtration inlet (not shown) may provide high-efficiency particulate air (HEPA) filtration to provide ISO 7 or better air quality in the interior zone. This air filtration may maintain sterile cell processing in an ISO 8 or ISO 9 manufacturing environment. The CPS 210 may have an air filter at the air outlet to maintain the room's ISO rating. In some variations, the CPS 210 may further include a novel docking station 215 having multiple docking station modules, such as a bioreactor, a cell selector (e.g., MACS), an electroporator (EP), a counterflow centrifugal elutriator (CCE), a cell sorter (e.g., fluorescence activated cell sorting (FACS)), a spinoculator, etc. In variations, the CPS 210 may also include sterile fluid transfer equipment, reagent reservoirs, and a sterilization system. The reagent reservoirs may be accessible by the user through a sample pickup port 219.

[0056] In some variations of methods according to the present disclosure, a human operator may load a cartridge 214, which may be empty or may include any combination of modules described herein, into the feedthrough 213. The cartridge 214 may be pre-sterilized or may be sterilized using ultraviolet (UV) light or a chemical sterilant provided as a steam, spray, or cleaning solution after passing through the feedthrough 213 and entering the docking station 215. In some variations, the cartridge 214 may be pre-loaded with an input cell product by a user. After positioning the sterilized, loaded cartridge 214 into the docking station 215 via the feedthrough 213, the user may initiate automated cell processing using a computer processor (e.g., controller 220) in the computer server rack. Advantageously, in some variations, the cartridge may undergo an automated end-to-end process to produce a desired cell product while all remain in their docked positions within the docking station 215. At the end of cell processing, the cartridge 214 with the processed cell product can be retrieved by a user from the docking station 215 via the feedthrough 213. In some variations, the exterior surface of the enclosure 202 can include input / output devices 208 (e.g., display, touchscreen) communicatively coupled to the controller 220.

[0057] 2B is a perspective view of the CPMS 200 of FIG. 2B. In some variations, the CPS 210 can have a height of greater than about 1 meter, e.g., from about 1 m to about 1.1 m, from about 1 m to about 1.2 m, from about 1 m to about 1.3 m, from about 1 m to about 1.4 m, from about 1 m to about 1.5 m, from about 1 m to about 1.6 m, from about 1 m to about 1.7 m, from about 1 m to about 1.8 m, from about 1 m to about 1.9 m, from about 1 m to about 2 m, from about 1 m to about 2.5 m, from about 1 m to about 3 m, from about 1 m to about 5 m, from about 3 m to about 10 m, from about 5 m to about 20 m, from about 10 m to about 30 m, from about 20 m to 100 m, and greater than about 100 m (including all values ​​and ranges therebetween). For example, in some variations, the CPS can have a height of about 1.9 m. In some variations, the CPS 210 can have a length and / or width greater than about 1 meter, e.g., about 1 m to about 5 m, about 3 m to about 10 m, about 5 m to about 20 m, about 10 m to about 30 m, about 20 m to 100 m, and greater than about 100 m (including all values ​​and ranges therebetween). Conversely, in some variations, the CPS 210 can have a length and / or width less than about 1 meter, e.g., about 0.2 m to about 0.9 m, about 0.3 m to about 0.9 m, about 0.4 m to about 0.5 m, about 0.5 m to about 0.9 m, about 0.6 m to about 0.9 m, about 0.7 m to about 0.9 m, or about 0.8 m to about 0.9 m. For example, in some variations, the CPS can have a length and / or width of about 0.9 m.

[0058] 2B , the CPMS 200 may include a cartridge 214 and a CPS 210 having an enclosure 211. The cartridge 214 may be fed through a feedthrough 213 of the enclosure to be disposed within a docking station 215 of the CPS 210. The CPS 210 may also include a sample pickup port 219, which may also be a reagent reservoir 218 having one or more sterile liquid transfer devices 242. In variations, the CPS 210 may further include a rack module 221 for storing one or more of a controller 220, a power distribution unit (PDU), and a cooler, and the controller 220 may be controllable by at least a user interface of the input / output device 208. In some variations, the CPS 210 may be mobile. For example, the CPS 210 may be equipped with wheels or casters for repositioning and relocation. The CPS 210 may be easily transportable, allowing it to be transported to any room within a facility (e.g., a hospital), or it may be possible to transport two or more CPSs to a single room if desired.

[0059] FIG. 2C is a side view of the cell processing and manufacturing system 200 depicting a cartridge 214 (e.g., any of the cartridges described herein) being installed in a CPS 210 (e.g., any of the CPSs described herein). Each inserted cartridge 214 can undergo one or more cell processing operations. As shown in FIG. 2C, the cartridge 214 can be positioned on a docking station drawer 216 that is movable through a feedthrough 214 of the enclosure 211 of the CPS 210 to position the cartridge 214 within the docking station 215 of the CPS 210. The CPS 210 can further include an on-board reagent reservoir 218 configured to store one or more sterile liquid transfer devices 242 that can be inserted, removed, and / or replaced via a sample pickup port 219 (e.g., by an operator or by the CPS robotics). In some variations, the docking station 215 can be equipped with a sterile liquid transfer instrument 243 in addition to a docking station module configured to interface with a corresponding module of the cartridge 214. A sterile fluid transfer fixture 243, which may optionally be separate from but releasably coupleable to cartridge 214, may be configured to transfer fluids from and / or to sterile fluid transfer devices 242 of reagent reservoirs 218 (or other fluid containers configured similarly to fluid connectors, as described below, to be controllable by sterile fluid transfer fixture 243 to exchange fluids with cartridge 214). In some variations, sterile fluid transfer fixture 243 may include a controllable robot to interact with each of sterile fluid transfer devices 242 of reagent reservoirs 218 (which may be generally referred to herein as fluid transfer devices 242) and cartridge 214 to supply and remove fluids from modules of cartridge 214 as required by cell processing operations.

[0060] Similarly, FIG. 2D is a perspective view of the CPMS 200 depicting the cartridge 214 positioned on the docking station drawer 216 of the CPS 210. As shown, the drawer 216 may include a telescoping mechanism that can extend from the docking station 215 (e.g., external to the CPS to receive the cartridge 214) and retract into the docking station 215 (e.g., once the cartridge 214 has been received), thereby enclosing the entire drawer 216 within the docking station 215. Also depicted in FIG. 2D is a door 270 having a sample pickup port 219 and input / output devices 208. In some variations, the door 270 may be movable relative to the sample pickup port 219. In particular, the door 270 may be configured to be repositioned to block or allow access to the sample pickup port 219. The door 270 may be repositionable manually and / or automatically (e.g., by an operator and / or by the controller 220). For example, in some variations, door 270 may be slidable (e.g., translatable along its mounting) both toward (and in front of) and away from sample pickup port 219. Thus, door 270 may provide temporary access to sterile liquid transfer devices and / or other reagent containers at sample pickup port 219. Additionally, in some variations, door 270 may support or be an input / output device 208. For example, at least a portion of door 270 may include an input / output device 208 configured to receive user input directly (e.g., via a touchscreen and / or button control).

[0061] As described above, the CPS 210 may generally include at least one docking station module (e.g., docking station module 112 of FIG. 1A) configured to engage with the cartridge 214 and / or interface with a corresponding module of the cartridge 214. An internal view of the CPS 210 with some exemplary docking station modules is depicted in FIG. 2E. For example, in some variations, the CPS 210 may include a nest control module for ensuring that the cartridge is properly oriented within the docking station 215 and / or for connecting the cartridge 214 to the CPS 210. The nest control module may include one or both of an electrical interface 272 and a cartridge presence sensor 274. The electrical interface 272 may be, for example, a PCB having pogo pins configured to physically and electrically interface with corresponding electrical portions of the cartridge 214. Furthermore, the presence sensor 274 may include one or both of a cartridge seating sensor and a cartridge presence sensor. The cartridge seating sensor may be a diffuse reflectance sensor configured to direct a beam across the cartridge 214 and detect any light reflected off the sensor. If the beam is reflected, the cartridge may be mispositioned. Thus, the cartridge seating sensor may be electrically coupled to the controller 220 and / or the input / output device 208 to indicate to the operator (e.g., visually, audibly, tactilely, etc.) whether the cartridge 214 is properly oriented within the CPS 210. Thus, the operator may have an opportunity to reorient the cartridge 214 within the CPS 210 before clamping the cartridge 214 inside the CPS 210. Similarly, the cartridge presence sensor may be a diffuse reflectance sensor (as described above) that may be used to determine whether a cartridge is present within the docking station 215. In some variations, the nest control module may include one or more additional or alternative components to ensure that the cartridge 214 is properly oriented within and connected to the CPS 210.Non-limiting examples of one or more additional or alternative components may include a clamp actuator (e.g., for clamping the cartridge 214 within the docking station 215), a limit switch detector (e.g., for sensing physical contact between the cartridge and the docking station 215), and a barcode scanner.

[0062] Further, in some variations, the CPS may include a fluid control module having one or more of a valve actuator 276, a sensor 278, and a pump mechanism 280. The valve actuator 276 may be a pinch valve actuator that controls the flow of fluid (e.g., liquid, gas) through the fluidic bus (e.g., to and from the cartridge 214, as described throughout) by adjusting the amount of pressure applied to the valve via a pneumatic cylinder. The sensor 278 may detect air bubbles in one or more fluid paths of the fluidic bus. For example, the sensor 278 may be an air bubble sensor that uses optical fiber to visualize a change in the amplitude of the sensor output, which may indicate whether air, liquid, or air and liquid (i.e., air bubbles) are flowing through the fluidic bus. The pump mechanism 280 may be configured to move fluid through the cartridge 214. For example, the pump mechanism 280 may include one or more pump rollers coupled to one or more motors, allowing the one or more pump rollers to act as a peristaltic pump to move fluid through the cartridge 214. The pump mechanism 280 may engage with the cartridge 214 via tubing to form one or more fluid pathways. In some variations, the pump mechanism 280 may be considered a separate module (i.e., a pump module) configured to engage with multiple cartridge modules. That is, any module requiring gas or fluid flow (e.g., gas and / or liquid flow) may be engaged with the pump module.

[0063] Additionally or alternatively, the CPS may include a bioreactor module for culturing various cell types (described throughout this specification). Within docking station 215, the bioreactor module may include one or both of port 281 and viewing window 282 (also referred to herein as a fluid window). Port 281 may be a supply support (e.g., a gas, fluid, or solid supply port) configured to supply nutrients (e.g., CO, N, and / or air) into the bioreactor so that cells may be properly cultured. Viewing window 282 may, for example, allow a camera to detect the liquid level in one or more containers of the bioreactor.

[0064] Referring briefly to FIG. 2F, in some variations, the bioreactor module may include a manifold 283 external to the CPS 210. The manifold 283 may be configured to accommodate external sources, such as clean dry air (CDA), nitrogen (N), and carbon dioxide (CO), and to supply gases to the CPS 210 by providing the gases to a gas mixer 284 of the CPS 210. Thus, the bioreactor module may prepare gases for supply to the bioreactor within the cartridge 214. In some variations, the supplied gases may be filtered and then each provided to one or more mass flow meters 285 (depicted on the input side of the bioreactor in FIG. 2F). The one or more mass flow meters may be considered first flow meters configured to combine the gases and provide the resulting gas to a second mass flow meter, where it is evaluated by a carbon dioxide sensor, an oxygen sensor, and then provided to the bioreactor (e.g., via the base plate of the bioreactor). Mass flow meter 285 can be configured to provide information about the mass flow rate of gases (e.g., volume per second of each gas) to ensure that a suitable cell growth environment is provided. That is, cell growth requires a specific gas mixture (e.g., CO, O), and the required gas mixture can change depending on the growth state of the cells.

[0065] 2E, the CPS may include a magnetically activated cell selection module (MACS) for magnetically selecting cells having predetermined properties or components, such as antigens (described throughout this specification). Accordingly, the MACS may include a magnet 286 that may engage with the actuator and cartridge 214 to perform magnetic selection of cells. Additionally, in some variations, the MACS may include a viewing window 287 (also referred to herein as a fluid window) that may allow a camera (e.g., an external camera) to view the liquid volume and / or the extent of cell separation that has occurred during magnetic selection.

[0066] As another example, the CPS may include a countercurrent centrifugal elutriation (CEE) module configured to separate cells based on predetermined characteristics, such as size and / or density (as described throughout this specification). The CEE module may include one or more of a magnetic coupler 289, a camera 290, a tachometer 291, and a sensor 292. The magnetic coupler 289 may include a motor coupled to a magnet, which is configured to pair with a mating magnet in the cartridge 214. Thus, when the magnets are close enough to attract each other, the motor of the magnetic coupler 289 may rotate to operate the CEE module. The camera 290 may then be configured to view the elution chamber (e.g., a bicone or funnel where cell separation occurs) of the CEE in the cartridge 214. The tachometer 291 may be configured to calculate the RPM of the CCE as it rotates. In particular, tachometer 291 may include a sensor configured to direct a beam at the rotor of the CEE in cartridge 214, which may be reflective (e.g., may include reflective tape or coating) so that tachometer 291 can calculate the RPM of the CEE relative to the rotor. In some variations, tachometer 291 may be paired with camera 290 so that images may be taken to visualize cells within the CEE in cartridge 214. Additionally, sensor 292 may be a leak detection sensor configured to detect leaks in the CEE rotor using optical fiber. Sensor 292 may be configured to shut down the CEE process when a leak occurs. In some variations, sensor 292 may be electrically coupled to controller and / or input / output device 208 to indicate to an operator (e.g., visually, audibly, tactilely, etc.) whether a leak is occurring.

[0067] As yet another example, the CPS may include an electroporation (EP) module to facilitate intracellular delivery (i.e., transfection by electroporation) of macromolecules (as described throughout this specification). The EP module may include one or both of a sensor 294 and an actuator 296. The sensor 294 may be a liquid volume sensor configured to detect the volume of fluid in a flow cell of the EP module using optical fibers. Furthermore, the actuator 296 may be a high-voltage actuator configured to form a high-voltage coupling between the cartridge 214 and the CPS 210.

[0068] 2E may be shared between modules. For example, the electrical interface 272 described with respect to the nest control module may additionally or alternatively be a component of one or more of the docking station modules described above (e.g., the fluidic control module, the bioreactor module, the magnetic cell selection module, the CEE module, and the EP module) and / or one or more of the docking station modules described in more detail below (e.g., the electronics and control module).

[0069] FIG. 2G is a block diagram of the CPMS 200 described throughout this specification. In particular, the block diagram of FIG. 2G illustrates components of the CPS 210 of the CPMS 200, which can be grouped in various combinations to form a docking station module, as described above with respect to FIGS. 2E and 2F. For example, the CPS 210 can include one or more of an electronics and control module, a nest control module, a fluid control module, a gas control module, a bioreactor sensor, a bioreactor agitation module, a temperature control module, a pulser control module, a CCE control module, and a magnetic separation (MS) control module. In some variations, one or more of a pump mechanism, a valve, a viewing window, a magnetic coupling (e.g., for the CEE and / or bioreactor module), a gas gasket, a bioreactor sensor interface, an electroporator (EP) high voltage (HV) connection, and a CCE magnetic coupling can enable the docking station 215 of the CPS 210 to interface with the cartridge 214. In variations, CPS 210 may also include or be operably coupled to manifolds (e.g., gas manifolds), electrical supplies, network boxes, cooling supplies, gas supplies, and software (e.g., SW interfaces, orchestration) for automating cell processing operations within cartridge 24, which may be integrated with or separate from CPS 210. In some variations, the electronics and control module may include one or more of a programmable logic controller (PLC) and / or interprocess communication (IPC), as well as one or more of controller 220, input / output devices 208, and electrical interface 272. Thus, the electronics and control module may enable user interaction with CPS 210, thereby coordinating at least a subset of the operations and processes of CPMS 200.

[0070] 2E, the dotted lines in FIG. 2F illustrate how components of CPS 210 may be grouped to form docking station modules configured to interface with cartridge 214 and / or corresponding modules of cartridge 214. For example, a fluidic control module, which may include a valve actuator, a pump, and / or an air bubble sensor, may interface and / or engage with a fluidic bus, the exterior of cartridge 214, a fluidic connector of cartridge 214, and / or a fluidic window of cartridge 214. As another example, a nest control module may engage with an electrical portion of cartridge 214 and / or one or more exterior portions of cartridge 214 (e.g., to clamp cartridge 214 within docking station 215).

[0071] As yet another example, the bioreactor module of cartridge 214 may be controllable via magnetic coupling, gas gaskets, and / or a bioreactor sensor interface. In some variations, this control may be by or in coordination with one or more of a temperature control module, a bioreactor agitation module, a bioreactor sensor, and a gas control module. In variations, the temperature control module may include one or more of a heater, a cooler, a baseplate temperature sensor, etc. In variations, the bioreactor agitation module may include one or more of an agitation motor and encoder, a magnetic coupler, etc. In variations, the bioreactor sensors may include one or more of a thermistor reader, a dissolved oxygen (DO) reader, a pH reader, etc. In variations, the gas control module may include one or more of a gas mixer, a flow meter, a pressure sensor, a gas homogenizer, an oxygen sensor, a carbon dioxide sensor, etc. (as described with respect to FIG. 2G ). Similarly, the electroporator module of cartridge 214 may be controllable via an EP HV connection by a pulser control module, which in some variations may include one or more of a pulser, an HV connection actuator, a voltage and / or current reader, etc. Similarly, the CCE module and / or magnetic cell separation module, etc. of cartridge 214 may be controllable via a CCE magnetic coupling by a CCE control module and an MS control module (among other things), respectively. In some variations, the CCE control module may include one or more of a motor and encoder, a magnetic coupler, an optical strobe, a camera, an optical trigger, and a CCE leak sensor. In some variations, the MS control module may include one or more of a magnetic engagement actuator, a magnetic engagement sensor, etc.

[0072] FIG. 3 provides a two-dimensional (2D) schematic diagram of a docking station 315 of a CPS, taking into account the cartridge module, modules, instruments, and docking station modules described above. In particular, the 2D schematic diagram of FIG. 3 represents the docking station 315 of the CPS in a flattened version, so that the relative positions of the interfaces between corresponding modules are shown, and assuming that the front of the docking station 315 is open to receive a cartridge therein. In other words, assuming the docking station 315 is a rectangular prism, the panels of FIG. 3 are labeled "bottom," "rear," "top," "left," and "right," and modules (e.g., cartridge module and / or docking station module) and instruments are identified based on their positioning on the docking station 315. The 2D schematic diagram of FIG. 3 illustrates how a CPS can advantageously perform multiple functions on a cartridge docked within the docking station 315 by carefully utilizing panels to perform different cell processing steps within a partially or fully enclosed system that other systems cannot. FIG. 3 includes reference to a sterile liquid transfer instrument (SLTI), a bioreactor (BR), and a magnetic separator (MS) such as a MACS.

[0073] For example, a magnetic separator (MS) and barcode or barcode scanner (interfaced with the nest control module) are interfaced on the left wall of docking station 315. The top wall of docking station 315 includes an electroporator (EP) box and an air bubble sensor reader (of the fluidic control module). The right wall of docking station 315 includes an electroporator (EP) high voltage (HV) interface and fluidic bus, which interfaces with a pump module, valves, and fluidic windows to supply and remove fluid from each of the cartridge modules in the cartridge. The back wall of docking station 315 includes a strobe, CCE motor, and camera contained within the CCE control module to interface with the cartridge's CCE module. The bottom wall of docking station 315 includes a bioreactor bath, gas mixer, and gas sensor configured to interface with the cartridge's bioreactor module, and a clamp plate configured to secure the cartridge onto the docking station's 315 drawer.

[0074] The 2D schematic diagram of FIG. 3 is described in more detail below with reference to FIGS. 4A-5V, taking into account the previous figures.

[0075] FIG. 4A provides a perspective view of a rendering of a docking station 415, a sterile liquid transfer instrument 443 coupled thereto, and a cartridge 414 (e.g., consumables) positioned on a docking station drawer 416. As shown in FIG. 4A, the cartridge 414 has not yet been moved into a chamber of the docking station 415 via the feedthrough 413. In a variation, the sterile liquid transfer instrument 443 comprises a first robot 444 and a second robot 445 configured to provide a two-axis range of motion for the sterile liquid transfer instrument 443. In a variation, as shown in FIG. 4A, a sterile liquid transfer device 442 can be coupled to the sterile liquid transfer instrument 443 in a position such that it is adjacent to the cartridge 414 when the cartridge 414 is in the docking station 415. Thus, the sterile liquid transfer instrument 443 can be advantageously used to supply media (e.g., fluids and liquids) to or extract media from the cartridge while the cartridge is docked in the docking station 415. In a variation, the docking station drawer 416 is a telescoping drawer, as utilized in FIG. 4B.

[0076] As shown in FIG. 4B, cartridge 414 can be retracted into docking station 415 on docking station drawer 416 via feedthrough 413. Cartridge 414 can then be lifted from docking station drawer 416 by lift 417 (shown in FIG. 4C), and cartridge 414 can be positioned to contact, or alternatively, clamped to, the upper inner surface of docking station 415. Alternatively, contacting cartridge 414 with the upper inner surface of docking station 415 allows cartridge 414 to be engaged by a sterile liquid transfer instrument 443. For example, after engagement between cartridge 414 and sterile liquid transfer instrument 443, sterile liquid transfer device 442 can supply fluid to or extract fluid from cartridge 414 via sterile liquid transfer port 466 of sterile liquid transfer device 442.

[0077] Figures 5A-5V illustrate various aspects of the CPMS and the arrangement of components relative to the docking station of the CPS, including the docking station module and, in some variations, the cartridge module. The panels and related features shown in Figures 5A-5V may, according to some variations, be part of the same assembly shown in Figures 4A-4C.

[0078] Figures 5A and 5B illustrate an embodiment of an electroporation assembly of a CPMS of the present disclosure. Each of Figures 5A and 5B depicts a cartridge 514 within a docking station 515, which is coupled at its top surface to a sterile liquid transfer fixture 543. As shown in Figure 5A, the docking station 515 includes a docking station electroporation module, the electroporator actuator 561 of which is shown. A corresponding embodiment 571 of the cartridge electroporation module is shown in Figure 5B, and it can be seen that the electroporation actuator 561 of the docking station electroporation module interfaces with the embodiment 571 of the cartridge electroporation module.

[0079] 5C and 5D illustrate an embodiment of a magnetic cell sorting assembly of a CPMS of the present disclosure. Each of FIGS. 5C and 5D depicts a cartridge 514 within a docking station 515, with the docking station 515 coupled at its top surface to a sterile liquid transfer fixture 543. In an alternative embodiment, the docking station 515 includes a docking station magnetically activated cell selection (MACS) module, and the cartridge 514 includes a cartridge MACS module. FIG. 5C shows the docking station MACS module 559 with its actuator in a retracted position. FIG. 5D shows an embodiment 579 of the MACS assembly 559 with its actuator in an extended position, configured to provide a magnetic field to the corresponding cartridge MACS module of the cartridge 514. FIGS. 5E-5G provide further details regarding the docking station MACS module 559. In a variation, the docking station MACS module 559 includes at least one camera 573 for viewing the corresponding cartridge MACS module of the cartridge 514 through a viewing window 575. In a variation, the at least one camera 573 is positioned such that the mirror 574 is deployed to view the corresponding cartridge MACS module of the cartridge 514 through the viewing window 575.

[0080] 5H-5J illustrate a liquid transfer bus actuator 563 of a docking station 515 having a sterile liquid transfer fixture 543 thereon, the liquid transfer bus actuator 563 configured to interface with a liquid transfer bus of a cartridge disposed within the docking station 515 to direct fluid within the cartridge. FIG. 51 provides a more detailed examination of the liquid transfer bus actuator 563, and FIG. 5J provides a more detailed examination of its single actuator 562. In an alternative embodiment, the actuator of the liquid transfer bus actuator 563 may function to compress a corresponding pinch valve on the cartridge, allowing flow to be directed to different locations within the cartridge.

[0081] Operating in conjunction with the liquid transfer bus actuators of Figures 5H-5J, Figure 5K illustrates an air bubble sensor 533 disposed on an inner surface of the docking station 515 and configured to detect any air bubbles that may be present in the liquid transfer bus.

[0082] Fluid may be pumped throughout the cartridge by a motor 534 attached to a pump roller disposed on the rear wall of the docking station 515. This allows the pump roller to be used as a peristaltic pump to move fluid through the cartridge.

[0083] 5M and 5N illustrate engagement between a bioreactor base plate 549 of a docking station 515 and a cartridge 514 disposed therein. In an alternative embodiment, the bioreactor base plate 549 may be integrally formed with a lift (described above) that allows the cartridge 514 to be lifted out of the docking station drawer and into contact with the upper interior surface of the docking station 515. The bioreactor base plate 549 is, in an alternative embodiment, an embodiment of a docking station bioreactor module configured to interact with the cartridge bioreactor module of the cartridge 514. To this end, the docking station bioreactor module interacts with the bioreactor of the cartridge bioreactor module via a motor within the docking station bioreactor module, for example, driving an impeller within the bioreactor to agitate fluids and suspend liquids therein. Additionally, via the bioreactor base plate 549, premixed gas may be provided from the docking station bioreactor module to the bioreactor of the cartridge bioreactor module to support cell growth.

[0084] Figure 5O illustrates an embodiment of a docking station bioreactor module of the present disclosure. As shown in Figure 5O, the docking station bioreactor module may include a camera 548, a viewing window 546, and a mirror 547. Camera 548 is capable of monitoring the fluid level within the bioreactor of the cartridge bioreactor module through viewing window 546. In an alternative embodiment, camera 548 is positioned such that mirror 547 is deployed to allow viewing of the corresponding cartridge bioreactor module through viewing window 546.

[0085] Similarly, Figure 5P illustrates an embodiment of a docking station waste module configured to assess the waste volume level within the cartridge. To this end, the docking station waste module may include a camera 577, a viewing window 578, and optionally a mirror (not shown). Camera 577 can monitor the waste fluid level within the cartridge through viewing window 578. In a variation, camera 548 is positioned so that the mirror is deployed to allow viewing of the waste level within the cartridge through viewing window 546. In a variation, the cartridge may be plumbed directly to a waste tank, which may be emptied, cleaned, and / or replaced with each process.

[0086] 5Q-5S illustrate a sterile fluid transfer instrument 543 coupled to the upper surface of a docking station 515. The sterile fluid transfer instrument 543 may include a first robot 544 and a second robot 545 that, when actuated by the gantry 541, provide at least two axes of movement relative to the docking station 515. As previously described, a sterile fluid transfer device 542 may be coupled to the gantry 541 of the sterile fluid transfer instrument 543 so as to be positioned for engagement with the cartridge 514. To this end, as shown in FIG. 5S, engagement may include supplying or withdrawing fluid from the cartridge 514 through a sterile fluid transfer port 566 of the sterile fluid transfer device 542, such supply and withdrawal being effected by a motor 537 in combination with pump rollers 534, thereby providing peristaltic fluid flow into and out of the sterile fluid transfer device 542.

[0087] 5T-5V illustrate embodiments of a docking station CCE module. As shown in FIG. 5T, the docking station CCE module may be disposed on a rear (i.e., back) wall of the docking station 515 and may include at least a magnet 558 actuated by a motor 572 and configured to magnetically couple to the cartridge CCE module and rotate its CCE compartment. As the CCE compartment rotates, the CCE compartment may be imaged by imaging components of the docking station CCE module, including a camera light 576, a viewing window 575, and a camera 573.

[0088] FIG. 6 is a schematic, illustrative diagram of a cartridge 600, which may be a consumable item manufactured from materials at a cost that makes recycling or limited use practical. Cartridge 600 may include a small bioreactor module 614a, a large bioreactor module 614b, a cell selection module 616, a cell sorting module 618, an electroporation module 620, and a liquid transfer bus 624 fluidly coupled to a countercurrent centrifugal elutriation (CCE) module 622. In some variations, cell selection module 616 may be a magnetically activated cell sorting (MACS) module. Cell sorting module 618 may include a fluorescence-activated cell sorting (FACS) module. Cartridge 600 may include a housing 602 that makes the cartridge self-contained and optionally protects the contents from contamination. Sterile liquid transfer ports (SLTPs) 606a-606k may be fluidly coupled to reservoirs 607a-607k, each of which may independently be a flexible bag or a rigid container. In some variations, the flexible bag may be configured to hold a large volume and allow for the transfer of fluid without exchanging the transferred fluid with a liquid or gas to maintain pressure within the reservoir, as the bag may collapse when fluid is transferred out and expand when fluid is transferred in.

[0089] In some variations, the liquid transfer bus 624 may include valves V1-V28 and corresponding tubing fluidically connecting the valves to each other and to each of the modules. Valves shown coupled to four fluid lines are 4 / 2 (four-port, two-position) valves, and valves shown coupled to three fluid lines are 3 / 2 (three-port, two-position) valves. The internal flow paths of the valves are indicated in the legend. The cartridge may further include a first pump 632a and a second pump 632b, each of which exposes tubing on the exterior of the housing 602, allowing each pump to interface with pump actuators (e.g., rotors) in several docking station modules within the CPS. The liquid transfer bus 624 may be fluidly coupled to a reservoir 607d and a product bag fluidly coupled to the SLTP 606d and product input tubing lines 627a-627b. An operator may input a cell product into reservoir 607d by connecting product input tubing line 627a or 627b to an external source of cells (e.g., a bag of cells collected from a donor). SLTP 606d may be configured to allow a system (e.g., a CPS) according to the present disclosure to add fluid to reservoir 607d in an automated manner. For example, one or more fluid transfer containers, such as reservoirs 607a-607k, bags, etc., may receive fluid via the SLTP. Additionally or alternatively, the SLTP may be configured to allow periodic sampling of one or more of the fluid transfer containers. The cartridge may further include collection bags 626a-626c fluidly coupled to liquid transfer bus 624 via valves V17-V19. Cartridge 600 may be configured to allow an operator to remove collection bags 626a-626c after cell processing by the system is complete.

[0090] 7 is a schematic diagram of another variation of cartridge 700. For example, cartridge 700 may include a reduced feature set compared to cartridge 600. Cartridge 700 may include a bioreactor module 714, a countercurrent centrifugal elutriation (CCE) module 722, and a fluid transfer bus 724 fluidly coupled to at least one module 716 selected from a cell selection module, a cell sorting module, an electroporation module, a spinoculation module, or any other cell processing module. Cartridge 700 may include a housing 702 and sterile liquid transfer ports (SLTPs) 706a-706f (e.g., fluid connectors) fluidly coupled to reservoirs 707a-707f, each of which may independently be a flexible bag or a rigid container. SLTP 706g is fluidly coupled to bioreactor module 714, allowing direct access to the bioreactor by a system or operator. Reservoir 707c may be fluidly coupled to SLTP 706c and product input tubing line 727. In some variations, liquid transfer bus 724 may include 14 valves V1-V3, V9, V11-V12, V17-V23, and V28, and tubing fluidly coupling the valves to each other and / or to each of the modules. The cartridge may further include collection bags 726a-726c fluidly coupled to liquid transfer bus 724 via valves V17-V19. The cartridge may further include pumps 732 that expose tubing on the exterior of housing 702 to allow each pump to interface with a pump actuator within the system (e.g., a cell processing station).

[0091] Side and top views of another cartridge variation are shown in FIGS. 8A and 8B, respectively. In some variations, cartridge 800 may include bioreactor 814, pump 816, and counterflow centrifugal elutriation (CCE) module 822. Cartridge 800 may include blanks 818, 819, and 820 configured to house additional modules, such as a cell selection module, a cell sorting module, an electroporation module, a miniature bioreactor module, or a spinoculation module. In some variations, the blanks may define an empty volume of the cartridge reserved for housing modules at another time. In some variations, cartridge 800 may include two or more additional bioreactors and / or reservoirs within blanks 818, 819, and 820. Along the proximal surface of cartridge 800 may be sterile liquid transfer ports 806a-806j fluidly connected to reservoirs 807a-807f. Reservoirs 807b and 807e may contain fluid (e.g., buffer or media). Along the top surface are product input tubing lines 827a-d, which may be fluidly connected to reservoirs 807a, 807b, 807e, and 807f, respectively. Fluid transfer bus 824 may fluidly connect the SLTP, reservoirs, and product input tubing lines to the module via tubing.

[0092] In some variations, the housing 802 may have exterior dimensions of approximately 225 mm x approximately 280 mm x 385 mm, approximately 225 mm x approximately 295 mm x 385 mm, and approximately 450 mm x approximately 300 mm x approximately 250 mm, including all values ​​and subranges therebetween. In some variations, the cartridge 800 may be approximately 10%, approximately 20%, approximately 30%, or more smaller in volume, including all ranges and subranges therebetween. In some variations, the cartridge 800 may be approximately 10%, approximately 20%, approximately 30%, approximately 50%, approximately 100%, approximately 200%, or more smaller in volume, including all ranges and subranges therebetween. Of course, the external dimensions of cartridge 800 depend in part on the internal dimensions of the CPS's docking station, as cartridge 800 must be sized to be received within the docking station and engage with a corresponding docking station module interface disposed adjacent the interior wall of the docking station.

[0093] In some variations, cartridge 800, as shown in the side view of Figure 8C and the perspective view of Figure 8D, may include a MACS module 818. For example, bioreactor module 814 may include ports 815a-815f including pH and DO sensors (ports 815a and 815b), a gas input line 815c, an output line 815d each having a sterile filter after the connector, and a coolant input line 815e and an output line 815f from the docking station bioreactor module interface when interfacing with cartridge bioreactor module 814 (for heat exchange). For example, gas input line 815c may be configured for gas transfer to the fluid (e.g., through headspace gas control or a gas-permeable membrane).

[0094] 9 shows a cross-sectional side view of cartridge 900. In some variations, cartridge 900 may include an enclosure (e.g., housing), a bioreactor 914, one or more pumps 916, a valve 930, a cell selection module 917, and a countercurrent centrifugal elutriation (CCE) module 922. In some variations, cell selection module 616 may be a magnetically activated cell selection (MACS) module 917. The cartridge may further include a collection bag 926. Cartridge 900 may optionally include a blank configured to house additional modules, such as a cell selection module, a cell sorting module, or an electroporation module 918. In some variations, cartridge 900 may include one or more bioreactors and / or reservoirs within the blank.

[0095] In some variations, the cartridge may include one or more valves. In some variations, a valve 1000 on the cartridge may be configured to receive an actuator 1010 provided by a docking station module (as shown in FIG. 10A) of a docking station of a CPS. When the cartridge is inserted into the docking station, the valve 1000 may be configured to dock with the actuator 1010 of the docking station module (as shown in FIG. 10B), such that rotation of the actuator 1010 can switch the valve 1000 from one position to another. In some variations, the valve may be constructed to pinch a section of flexible tubing. A pinch valve may include a closing configuration, and an external actuator may be configured to interface with the pinch valve (e.g., utilizing a solenoid with linear motion) to open or close the valve. The valve itself may be configured to be disposable, while the actuator may be integrated into a docking station module configured to repeatedly process cartridges.

[0096] Countercurrent centrifugal elutriation Countercurrent centrifugal elutriation (CCE) is a technique used to separate cells based on characteristics such as size and / or density. Countercurrent centrifugal elutriation combines centrifugation with countercurrent elutriation, where centrifugation corresponds to the process of sedimentation under the influence of a centrifugal field and countercurrent elutriation corresponds to the process of separation by washing. Separation occurs in a conical (e.g., biconical, funnel) shaped elutriation chamber. Particles (e.g., cells) transported in a fluid within the elutriation chamber are acted upon by two opposing forces: centrifugal force, which drives the fluid away from the axis of rotation, and fluid velocity (e.g., countercurrent flow), which drives the fluid toward the axis of rotation. By varying the flow rate and centrifugal force, particle (e.g., cell) separation can be achieved. For example, as described in more detail herein, particles can be separated based on characteristics such as size and density.

[0097] Counterflow centrifugal elutriation can perform multiple operations useful in cell therapy manufacturing workflows, including, but not limited to, cell washing, cell concentration, media / buffer exchange, transduction, and separation of white blood cells from other blood components (e.g., platelets and red blood cells). In some variations, the fluid source for the cell separation process (e.g., an apheresis bag) can include a suspension of white blood cells, red blood cells, platelets, and plasma. To separate immune cells of interest, white blood cells can be isolated and then magnetically tagged for magnetic separation. The white blood cell separation step can be performed in a CCE module to separate cells based on size and density, while magnetic separation can be performed in a MACS module. In some variations, the CCE module can be integrated into a cartridge to enable the cell processing and manufacturing system to separate cells based on one or more of progression through the cell cycle (e.g., G1 / M phase cells are larger than G0, S, or G2 phase cells) and cell type (e.g., white blood cells from red blood cells and / or platelets).

[0098] Generally, a rotor configured to rotate may include an elutriation chamber (e.g., conical, biconical). A fluid containing a suspension of cells may be pumped into the rotor under continuous flow. When cells are introduced into the cone (e.g., biconical), they migrate according to their sedimentation velocity to a position in the gradient where the effects of two forces on them are equilibrated. Smaller cells (e.g., platelets) with low sedimentation velocities can be rapidly washed toward the axis of rotation by increasing the flow rate. Such smaller cells can be output (e.g., flushed) from the cone. Relatively larger (or denser) cells (e.g., red blood cells) flow through the cone relatively more slowly, reaching equilibrium at the elutriation boundary where centrifugal and drag forces are balanced, and the fluid velocity is relatively low because the cone is widening. The largest or densest cells (e.g., white blood cells) remain near the entrance to the chamber where centrifugal force and fluid velocity are high. By increasing the flow rate stepwise, successive fractions of cells of increasing size or density (e.g., platelets → red blood cells → white blood cells) can be output from the rotor. Successive incremental increases in fluid flow rate eventually flush all cells from the cone.

[0099] Returning now to the drawings, Figure 55 is a block diagram of a cell separation system 5600 including a cell processing station (CPS) 5610 and a cartridge 5620. In some variations, the CPS 5610 may comprise one or more of a docking station (DS) countercurrent centrifugal elutriation (CCE) module 5632 (e.g., first magnet), a DS magnetically activated cell selection (MACS) module 5642 (e.g., magnet array, second magnet), a fluid transfer connector 5652 which may be a sterile fluid transfer port or a connector to the fluid transfer bus of the cartridge, a pump 5654, an imaging system including an optical sensor 5660 and an illumination source 5662, a sensor 5664, and a processor 5670. In some variations, the cartridge 5620 may include one or more of a cartridge CCE module 5630 (e.g., a rotor), a cartridge MACS module 5640 (e.g., a flow cell), and a fluid transfer connector 5650, which may be a sterile liquid transfer port or a liquid transfer bus of the cartridge and may correspond to the fluid transfer connector 5652 of the CPS 5610. For example, a cartridge for cell processing may include a liquid transfer bus and multiple modules, each module fluidly coupled to the liquid transfer bus.

[0100] In some variations, the imaging system (e.g., optical sensor 5660, illumination source 5622) may be configured to generate image data corresponding to one or more of the cartridge CCE module 5630 and the cartridge MACS module 5640. For example, image data of fluid flow through the rotor of the cartridge CCE module 5630 may be analyzed and used to control the fluid flow rate and / or the rotational speed of the rotor, as described in more detail herein. In some variations, the optical sensor 5660 may be a CMOS / CCD sensor, for example, having a resolution of about 100 μm, a working distance of about 40 mm to about 100 mm, and a focal length of less than about 8 mm. The optical sensor 5660 may be configured to operate in synchronization with the illumination source 5662. In some variations, the optical sensor 5660 may comprise one or more of a colorimeter, a turbidity sensor, and an optical density sensor. In some variations, the illumination source 5662 may operate as a strobe light configured to output light pulses synchronized to the rotational speed of the rotor of the CCE module 5630.

[0101] In some variations, the sensor 5664 may comprise one or more of an optical density sensor configured to measure the strength of the fluid, a leak detector configured to detect moisture and / or leaks, an inertial sensor configured to measure vibrations, a pressure sensor (e.g., a photoelectric sensor) configured to measure pressure in the fluid line, an air bubble sensor configured to detect the presence of air bubbles in the fluid conduit, a colorimetric sensor, a vibration sensor, etc.

[0102] In some variations, the fluid transfer connector 5652 may include one or more valves configured to control fluid flow between the CPS 5610 and the cartridge 5620. The processor 5670 may correspond to a controller (e.g., a processor and memory) described in more detail herein. The processor 5670 may be configured to control one or more of the DS CCE module 5632, the DS MACS module 5642, the pump 5654, the fluid transfer connector 5652 (e.g., a valve), the optical sensor 5660, the illumination source 5662, and the sensor 5664.

[0103] In some variations, a CPMS 5600 for cell processing may include a cartridge 5600 including a rotor of a CCE module 5630 configured for counterflow centrifugal elutriation of cells in a fluid. A first magnet of a docking station CCE module 5632 may be configured to magnetically rotate the rotor and separate cells from the fluid in the rotor. The cartridge may further include a flow cell of a MACS module 5640 coupled to the rotor and configured to receive cells from the rotor. A second magnet of the docking station MACS module 5642 may be configured to magnetically separate cells in the flow cell.

[0104] In some variations, the illumination source 5662 may be configured to illuminate the cells. The optical sensor 5660 may be configured to generate image data corresponding to the cells. In some variations, the CPMS 5600 may include one or more of an oxygen depletion sensor, a leak sensor, an inertial sensor, a pressure sensor, and an air bubble sensor. In some variations, the CPMS 5600 may include one or more valves and pumps.

[0105] FIG. 56 is a cross-sectional side view of a countercurrent centrifugal elutriation (CCE) module 5700 comprising a housing 5710 (e.g., an enclosure), a rotor 5720 configured to rotate within and relative to the housing 5710, and one or more fluid ports 5730 (e.g., fluid inlet, fluid outlet).

[0106] FIG. 57 is a cross-sectional side view of a magnetically activated cell selection (MACS) module including a housing 5810 (e.g., enclosure), a first fluid port 5820 (e.g., fluid inlet), a second fluid port 5830 (e.g., fluid outlet), and a flow cell 5810 coupled between the first fluid port 5820 and the second fluid port 5830. As described in more detail herein, the flow cell 5810 may comprise a cavity (e.g., chamber) containing one or more channels (e.g., linear channels, laminar fluid flow channels). In some variations, the cavity of the flow cell 5810 may be substantially empty. For example, the flow cell 5810 may lack mesh, beads, serpentine channels, etc. In some variations, the flow cell 5810 may have a longitudinal axis aligned vertically with respect to the ground. That is, the flow cell 5810 may be oriented vertically, with the first fluid port 5820 positioned at a higher elevation than the second fluid port 5830 so that gravity may assist fluid flow through the flow cell 5810.

[0107] Figure 58 is a semi-transparent perspective view of a CPMS 5900 for cell processing including a CCE system. Figure 59A provides a relatively opaque perspective view of a CPMS 5900 including a CCE system. As shown in Figures 58 and 59A, the CCE system includes a cartridge CCE module 5930 including a housing 5931 and a rotor 5910, and a docking station CCE module 5932 including an optical sensor 5960 and an illumination source 5962. In some variations, the docking station CCE module 5932 may include a magnet configured to magnetically rotate the rotor 5910 within the cartridge CCE module 5930. One or more portions of the housing 5931 and the rotor 5910 may be optically transparent to facilitate illumination by the illumination source 5962 and image data generation by the optical sensor 5960.

[0108] In some variations, a CPMS 5900 for cell processing may include a cartridge 5930 including a housing 5931 and a rotor 5910 configured to separate cells from a fluid therein. A corresponding docking station module 5932 including a magnet may be configured to interface with the cartridge 5930 to magnetically rotate the rotor 5910. The cartridge 5930 may be configured to transfer cellular products between multiple cartridge modules, docking station modules, and / or other instruments. In some variations, the housing 5931 may enclose the rotor 5910. In some variations, the housing 5931 may include one or more openings 5937 configured to facilitate visualization (e.g., imaging) of the rotor 5910. FIGS. 58 and 59A depict the magnet 5932 proximate to, but not attached to, the housing 5931. FIG. 59B is a perspective view of rotor 5910 and housing 5931 without magnet 5932, optical sensor 5960, and illumination source 5962.

[0109] In some variations, the cartridge 5930 (e.g., housing 5931, 5910) may comprise a consumable component, such as a disposable component, a limited use component, or a single use component. In some variations, the magnet 5932 may comprise a durable component that may be reused multiple times. In some variations, the magnet 5932 may be releasably coupled to the housing 5931. For example, the housing 5931 may be moved relative to the magnet 5932 to facilitate magnetic coupling between the magnet 5932 and multiple cartridges 5930. Additionally or alternatively, the magnet 5932 may be configured to move relative to the housing 5931.

[0110] 59C is a side cross-sectional view of a cartridge CCE module 5930. In some variations, the housing 5931 of the rotor 5910 may have a first side 5933 including a first fluid port 5912 (e.g., a first fluid conduit) and a second side 5935 including a second fluid port 5914, the second side 5935 being opposite the first side 5933. The rotor 5910 (including a cone or a bi-cone, as described in more detail herein) may be coupled between the first fluid port 5912 and the second fluid port 5914. In some variations, the cartridge CCE module 5930 may include an air gap 5902 between the housing 5931 and the magnet 5932. That is, the cartridge 5930 and the magnet 5932 may be coupled in a contactless manner. Thus, the cartridge does not need to be mechanically coupled to the magnet 5932 to perform countercurrent centrifugal elutriation. Thus, rotor 5910 can have low alignment sensitivity with magnet 5932, as well as low vibration between rotor 5910 and magnet 5932. Furthermore, the space between rotor 5910 and magnet 5932 allows second fluid port 5914 to extend toward second side 5935 of housing 5931, thus allowing fluid to flow on each side of rotor 5910.

[0111] In some variations, countercurrent centrifugal elutriation may be performed by the CPMS 5900 by moving the magnet 5932 toward (or away from) the rotor 5910. The rotor may define an axis of rotation (e.g., coaxial with the first fluid port 5912 and the second fluid port 5914). Fluid may flow through the rotor via the first fluid port 5912 and the second fluid port 5914. The magnet 5932 may magnetically rotate the rotor about the axis of rotation while flowing fluid through the rotor 5910. The rotor may move away from the magnet. For example, moving the rotor 5910 may include advancing and retracting the rotor 5910 relative to the magnet 5932.

[0112] In some variations, fluid may flow into rotor 5910 through first fluid port 5912 along a first side 5933 of rotor 5910. After countercurrent centrifugal elutriation through rotor 5910, fluid may exit rotor 5910 through second fluid port 5914 along a second side 5935 of rotor 5910.

[0113] In some variations, the countercurrent centrifugation elutriation may be visualized by an optical sensor 5960 and an illumination source 5962 to monitor and correct cell separation in real time based on predetermined criteria in a closed-loop manner to maximize elutriation efficiency. In some variations, the optical sensor 5960 may be configured to image any portion of the rotor through which fluid flows (e.g., the first fluid conduit, the second fluid conduit, the third fluid conduit, the first bi-cone, the second bi-cone). For example, image data of one or more of the fluid and cells in the rotor 5910 may be generated using the optical sensor 5960. In some variations, one or more of the fluid and cells may be illuminated using the illumination source 5962. For example, the output of the cone may be imaged by the optical sensor to identify non-target cells being elutriated.

[0114] In some variations, one or more of the rotor rotational speed and the fluid flow rate may be selected based at least in part on the image data. For example, the rotor may include a rotational speed of up to 6,000 RPM. For example, the fluid may include a flow rate of up to about 150 ml / min while rotating the rotor. In some variations, the rotor may be moved toward the illumination source 5962 and the optical sensor 5960. Additionally or alternatively, the rotor 5910 may be moved away from the illumination source 5962 and the optical sensor 5960.

[0115] FIG. 59D is a cross-sectional side view of a rotor 5910 including a first fluid port 5912 (e.g., a fluid conduit, inlet) and a second fluid port 5914 (e.g., a fluid conduit, outlet). In some variations, the first fluid port 5912 and the second fluid port 5914 may extend parallel to each other and / or the axis of rotation of the rotor 5910. In some variations, the first fluid port 5912 and the second fluid port 5914 may be located on opposite sides of the rotor 5910, which may simplify fluid routing, cartridge design, and reduce manufacturing costs. For example, fluid seals may be simplified because they each include only a single lumen. Conventionally, complex fluid flow paths (including inlets and outlets) are formed on the first side of the rotor due to the fixed mechanical coupling of the drive motor to the second side of the rotor. FIGS. 59E and 59F are cross-sectional side views of the rotor 5910 disposed within a housing 5931.

[0116] FIG. 60A is a plan view of a rotor 6000 that may be used with any of the CCE systems, CCE modules, cartridges, housings, combinations thereof, etc. described herein. The rotor 6000 may comprise a first fluid conduit 6010, a cone 6020 (e.g., a bicone), a second fluid conduit 6030, a magnetic portion 6040 (e.g., a magnet), and a housing 6050. Fluid may flow sequentially through the first fluid conduit 6010, the cone 6020, and the second fluid conduit 6030. In some variations, the magnetic portion 6040 may comprise one or more magnets. In some variations, the rotor 6000 may define an axis of rotation 6060. In some variations, at least a portion of the first fluid conduit 6010 and at least a portion of the second fluid conduit 6030 may extend parallel to the axis of rotation (e.g., into or out of the page with respect to FIG. 60A ). In some variations, at least a portion of the first fluid conduit 6010 and at least a portion of the second fluid conduit 6030 may be coaxial.

[0117] In some variations, the cone 6020 may comprise a bi-cone having a first cone including a first base and a second cone including a second base, the first base facing the second base. In some variations, the bi-cone may comprise a cylinder (or some other shape) between and / or in fluid communication with the first and second cones. For example, one or more cones of the rotor may comprise a generally stepped shape. For example, one or more cones may comprise stacked circular steps. In some variations, the cones of the rotor may comprise a single cone.

[0118] In some variations, at least a portion of the rotor may be optically transparent to facilitate visualization and / or imaging of rotor 6000 and / or fluid (e.g., cells) within rotor 6000. For example, cone 6020 may be transparent, as may portions of first fluid conduit 6010 and second fluid conduit 6030.

[0119] In some variations, the cone may have a volume of about 10 ml to about 40 ml. In some variations, the cone may have a cone angle of about 40 degrees to about 60 degrees.

[0120] In some variations, the cone may comprise a first cone (e.g., a distal cone) and a second cone (e.g., a proximal cone), where the first cone is larger than the second cone. In some variations, the first cone length may be between about 60 mm and about 90 mm. In some variations, the proximal cone length may be between about 15 mm and about 40 mm. In some variations, the cone diameter (e.g., the maximum diameter of the cone) may be between about 15 mm and about 40 mm.

[0121] In some variations, rotor 6000 may comprise an asymmetric shape, in which a first portion (e.g., a first end) of rotor 6000 may comprise a cone 6020 and a second portion (e.g., a second end) may comprise a paddle shape.

[0122] In some variations, the cone may have a length of at least about 4 cm (e.g., about 9 cm to about 12 cm), a cone diameter of about 5 cm or less (e.g., about 3 cm to about 5 cm), a fluid flow rate of up to about 100 ml / min (e.g., about 60 ml / min to about 100 ml / min), and a rotational speed of less than about 3000 RPM. The shape of the first and second cones may be generally linear (as opposed to convex or concave).

[0123] Figures 60B and 60C are perspective views, and Figure 60D is a side view of rotor 6002, including first fluid conduit 6012, cone 6022, second fluid conduit 6032, and housing 6052. Figure 60E is a perspective view of rotor 6002 disposed within housing 6090.

[0124] 60F is a plan view of a rotor 6004 having two cones (e.g., two bi-cones) configured to elutriate cells (e.g., red blood cells, leukoreduction product) in the second cone to recirculate the buffer for reuse. The rotor 6004 may comprise a housing 6052, a first fluid conduit 6012, a first cone 6022 coupled to the first fluid conduit 6012, a second fluid conduit 6023 coupled to the first cone 6022, a second cone 6024 coupled to the second cone 6023, and a third fluid conduit 6032 coupled to the second cone 6024. The first cone 6022 may comprise a first volume, and the second cone 6024 may comprise a second volume that is larger than the first volume. In some variations, the ratio of the second volume to the first volume can be from about 2:1 to about 5:1. Fluid can flow sequentially through first fluid conduit 6012, first cone 6022, second fluid conduit 6023, second cone 6024, and third fluid conduit 6032. In some variations, rotor 6004 can include a magnetic portion 6042.

[0125] In some variations, the first cone 6022 may comprise a first bicone and the second cone 6024 may comprise a second bicone. In some variations, the first bicone may comprise a third cone including a first base and a fourth cone including a second base, where the first base faces the second base. In some variations, the second bicone may comprise a fifth cone including a third base and a sixth cone including a fourth base, where the third base faces the fourth base.

[0126] In some variations, a portion of the rotor 6004 may be optically transparent, such as the first cone 6022, the second cone 6024, and at least a portion of the first fluid conduit 6012, the second fluid conduit 6023, and the third fluid conduit 6032. In some variations, the first fluid conduit 6012 may include an inlet and the third fluid conduit 6032 may include an outlet.

[0127] In some variations, cells may enter the first cone 6022, and red blood cells (RBCs) 6030 may be elutriated into the second cone 6024. Because the second cone 6024 is further out from the axis of rotation (the center of the housing 6052), the RBCs 6030 may be concentrated at the inlet 6025 of the second cone 6024 due to centrifugation. The larger volume of the second cone 6024 may further reduce the velocity of the fluid (e.g., buffer solution), thereby reducing the force on the RBCs 6030 within the second cone 6024. By recirculating the fluid (e.g., buffer solution), a higher concentration of RBCs may be elutriated with less fluid (e.g., buffer solution). In some variations, white blood cells 6040 may be collected from the first cone 6022. The optical sensor may be configured to image the first cone 6022 to generate imaging data used to identify the boundary between the WBCs 6040 and the RBCs 6030. In some variations, the recirculating fluid may be passed through a filter to remove small particles (e.g., platelets) using less fluid (e.g., buffer).

[0128] FIG. 60G is a plan view and FIG. 60H is a side view of a rotor 6005 having two cones (e.g., two bi-cones) configured to elutriate cells (e.g., red blood cells) in the second cone. A rotor with two cones may facilitate recycling of the buffer solution for reuse. The rotor 6006 may include a housing 6052, a first fluid conduit 6012, a first cone 6022 coupled to the first fluid conduit 6012, a second cone 6024 coupled to the first cone 6022, and a fluid conduit 6032 (e.g., an outlet) coupled to the second cone 6024.

[0129] Figure 601 is a perspective view of rotor 6006 with cone 6024 and housing 6054. Figure 60J is a perspective view of rotor 6007 with cone 6026 and housing 6056. Figure 60K is a schematic plan view of rotor 6008 and corresponding dimensions. Figure 60L is an image of a set of rotors with various dimensions.

[0130] 11A-11C depict another variation of a countercurrent centrifugal elutriation (CCE) module 1100. FIG. 11A is a perspective view of a cartridge 1110 including the CCE module 1100 in an extended configuration configured to receive a docking station (DS) CCE module. FIGS. 11B and 11C are cross-sectional side views of the cartridge CCE module 1100 in retracted and extended configurations, respectively. In some variations, the cartridge CCE module may include a conical element having an inner and outer surface fixedly attached to the distal end of a linear member having an inner and outer surface. The proximal end of the linear member may be rotatably attached to a fulcrum to enable extension, retraction, and / or rotation of the linear member. For example, FIG. 11C depicts a linear member extending outside the cartridge housing and then rotated to generate centrifugal force. The cell product can be conveyed to the conical element (optionally within a tube) between the inner and outer surfaces of the linear member and fed to an opening at the distal end of the inner surface of the conical element, resulting in a flow of cell product that can flow against the centrifugal force generated by the rotation of the linear member. Cells in the cell product can be separated based on the ratio of their hydrodynamic cross-sectional area to their mass due to the counterflow of the solution and the sedimentation of the cells subjected to the centrifugal force. The flow rate can then be increased and / or the rotation of the linear member can be decreased to selectively allow the cells to return to the proximal end of the linear member through the gaps in the inner surface of the linear member. The selected cells can be directed to a tube that returns the selected cells to the cartridge. After the concentration / washing step is performed, the linear member can be retracted into the housing to the retracted configuration, as shown in FIG. 11B.

[0131] Magnetic cell selection Generally, the systems and methods described herein can select cells based on magnetically labeled cells corresponding to cells bearing a predetermined antigen. For example, a cell suspension of interest can be immunologically labeled with magnetic particles (e.g., magnetic beads) configured to selectively bind to the surface of the cells of interest. The labeled cells can generate a large magnetic moment as the cell suspension flows through a flow cell. The flow cell can be placed in proximity to a magnet array (e.g., permanent magnets, electromagnets) that generates a magnetic field with a gradient across the flow cell to attract the labeled cells for separation, capture, recovery, and / or purification. The magnet array can be configured to generate a non-uniform magnetic field at the edges and interfaces of the individual magnets to cover the entire volume of the flow cell so that the magnetophoretic force is equal to the resistive force exerted by the fluid flowing through the flow cell.

[0132] 61A-61C are schematic diagrams of a magnetic cell separation (e.g., magnetically activated cell selection) system and process. The magnetic cell separation system may include a flow cell 6110 including an inlet 6130 and an outlet 6132, a magnet array 6120, a first fluid source 6140 (e.g., an input sample source), a second fluid source 6142 (e.g., a buffer source), a third fluid source 6150 (e.g., a target cell reservoir), a fourth fluid source 6152 (e.g., a waste reservoir), and a set of valves 6134. As shown in step 6100, a set of cells 6160, 6170 may include labeled cells 6160 (e.g., magnetically labeled cells), and unlabeled cells 6170 may flow into the flow cell 6110. For example, the set of cells 6160 may be labeled with a magnetically activated cell selection (MACS) reagent. The MACS reagent may be incubated with the set of cells to label (e.g., attach, couple) the cells to the MACS reagent. As described in more detail herein, the magnet array 6120 may be disposed external to the flow cell 6110 such that the magnet array 6120 may be movable relative to the flow cell 6110. For example, the magnet array 6120 may be moved away from the flow cell 6110 to facilitate the flow of the set of cells 6160 out of the flow cell 6110. Conventional flow cells include a tortuous path that includes a mesh and / or beads to capture cells. However, recovery of labeled cells from conventional flow cell configurations is difficult. In contrast, the flow cell 6110 described herein may lack a tortuous path of beads, mesh, etc., thus allowing for efficient performance of sequential separations using either positive or negative selection. In some variations, the flow cell may generally comprise layered channels, as described in more detail herein.

[0133] In step 6102, the magnet array 6120 may magnetically attract the set of cells 6160 toward the magnet array 6120 for a predetermined dwell time and / or based on the measured amount of magnetically separated cells. In some variations, the dwell time may be at least 1 minute (e.g., at least 2 minutes, at least 3 minutes, at least 5 minutes). Unlabeled cells 6170 are not magnetically attracted to the magnet array 6120 and may flow out of the outlet 6132 of the flow cell 6110 and into the fourth fluid source 6152. In some variations, the fluid within the flow cell (e.g., cells 6160, 6170) may be held statically within the flow cell 6110 for the dwell time before the fluid (e.g., cells 6170) flows out of the outlet 6132. In some variations, the longitudinal axis of the flow cell 6110 may be oriented substantially perpendicular to the ground for gravity-assisted fluid flow through the flow cell 6110. In step 6104, the magnetic coupling between the magnet array 6120 and the cells 6160 may be released after a residence time, and the cells 6160 may flow into the third reservoir 6150.

[0134] In some variations, stiction can cause cells to remain attached to the surface of the flow cell even after removal of the magnet array 6120. Thus, gas can flow through the flow cell 6110 and assist in cell collection into the third reservoir 6150. Gas flow through the flow cell can provide improved cell recovery over liquid flushing through the flow cell. The interface created by the gas (e.g., bubbles, voids) can be maintained by gravity, thereby allowing for the implementation of relatively wide flow cells, which further improves cell recovery compared to horizontally oriented flow cells. The MACS modules described herein can be configured for positive and / or negative selection by altering the order of steps.

[0135] Additionally or alternatively, an optical sensor may be configured to image the flow cell to generate imaging data used to identify the amount of cells magnetically attracted to the magnet array, and the fluid containing the labeled cells may be flowed out of the flow cell when a predetermined amount of cells is measured by the optical sensor.

[0136] FIG. 62A is a perspective view of a cartridge MACS module 6200 in a first configuration. The cartridge MACS module 6200 may be a component of any of the cartridges described herein. For example, a cartridge for cell processing may include a fluid transfer bus and multiple modules, each fluidly coupled to the fluid transfer bus. The cartridge MACS module 6200 may include a flow cell 6210 including an elongated cavity having a cavity height, an inlet 6230, and an outlet 6232. The MACS module 6200 may further include a magnet array 6220 including multiple magnets. Each of the magnets may be spaced apart by a spacing distance, as illustrated in FIGS. 62G, 63D, and 63E, although FIGS. 62A-62E illustrate a magnet array 6220 having magnets that contact adjacent magnets.

[0137] FIG. 62G is a schematic diagram of a flow cell 6210 and a magnet array 6220. In some variations, the flow cell 6210 may have a cavity height 6202 and a cavity width 6204. Fluid may be configured to flow through the flow cell 6210 in a first direction 6206. The magnet array 6220 may include a plurality of magnets, each having a respective width 6222. In some variations, adjacent magnets may be separated by a predetermined spacing distance 6224. Each magnet pair may have the same or a different spacing distance 6224. As shown in FIG. 62G, the orientation (e.g., poles) of the magnets in the magnet array 6220 may include a predetermined pattern. In some variations, the ratio of the cavity height 6202 to the spacing distance 6224 is between about 20:1 and about 1:20, between about 10:1 and about 1:10, between about 5:1 and about 1:5, and between about 3:1 and about 1:3, including all values ​​and subranges therebetween. In some variations, the actuator 6240 (e.g., linear, rotational) may be configured to move the magnet array 6220 relative to the flow cell 6210. In some variations, the orientation (e.g., poles) of the magnets in the magnet array 6220 may include a predetermined pattern (e.g., a Halbach array).

[0138] In some variations, the magnet array 6220 may move relative to the flow cell 6210, or vice versa. Figure 62A illustrates the cartridge MACS module 6200 in an open configuration, and Figure 62B illustrates the cartridge MACS module 6200 in a closed configuration. Figure 62B is a perspective view of the cartridge MACS module 6200 in a second configuration in which labeled cells may be magnetically attracted toward the magnet array 6220. In the second configuration, magnetic field lines generated by the magnet array traverse the flow channel and exert a magnetophoretic force on magnetically tagged cells injected into the channel. Figure 62C is a cross-sectional side view of the cartridge MACS module 6200 including the magnet array 6220. Figure 62D is a perspective view of the cartridge MACS module 6200 in the second configuration. Figure 62E is a plan view of the flow cell 6210 and magnet array 6220 of the MACS system. Figure 62F is a plan view of the flow cell 6210 of the cartridge MACS module.

[0139] 63A-63E are perspective views of a set of magnet arrays 6300, 6310, 6320, 6330, and 6340. One or more of the size, strength, shape, spacing, and orientation of the magnets in the magnet array can be configured to generate a magnetic field to attract magnetically labeled cells. Additionally or alternatively, the magnet array can include a high-permeability material configured to enhance or reduce the magnetic field strength and magnetic field gradient within the flow cell. The material can be disposed between the magnet and the flow cell. Additionally or alternatively, the material can be disposed within the flow cell and / or on one or more sides of the flow cell.

[0140] 64A and 64B are perspective and cross-sectional side views, respectively, of a cartridge MACS module 6400 comprising a flow cell 6410 and a magnet array 6420. The flow cell 6410 may comprise a set of linear channels 6412, 6414, 6416 comprising a first channel 6412 parallel to a second channel 6414 and a third channel 6416 in fluid communication with each of the first channel 6412 and the second channel 6416. As shown in FIG. 64B, the third channel 6416 may be disposed between the first channel 6412 and the second channel 6416 and define a volume in which fluids from the first channel 6412 and the second channel 6416 interact (e.g., mix). In some variations, flow cell 6410 may include a first inlet 6430 coupled to first channel 6412 and configured to receive a first fluid 6460 (e.g., cells). A second inlet 6431 may be coupled to second channel 6414 and configured to receive a second fluid 6470 (e.g., a buffer solution). Flow cell 6410 may include a first outlet 6432 coupled to first channel 6412 and a second outlet 6433 coupled to second channel 6414.

[0141] The magnet array 6420 may be located external to the flow cell 6400 and may be moved relative to the flow cell 6400 as described herein. In some variations, the longitudinal axis of the flow cell 6410 may be perpendicular to the ground so that the fluid flows in a generally vertical direction.

[0142] In some variations, the first channel 6412 can have different dimensions than the second channel 6414. For example, the first cavity height of the first channel 6412 can be greater than the second cavity height of the second channel 6414. For example, the ratio of the first cavity height to the second cavity height can be about 1:1 to about 3:7, about 1:1 to about 2:3, and about 2:3 to about 3:7, including all values ​​and subranges therebetween. Fluid flowing through the first channel 6412 can have a slower flow rate compared to the second channel 6414 due to the greater cavity height of the first channel 6412 compared to the second channel 6414. In some variations, the third channel 6416 can have a ratio of the length of the third channel 6416 to the diameter of the third channel 6416 of about 2:1 to about 6:1, about 2:1 to about 3:1, about 3:1 to about 4:1, about 4:1 to about 5:1, about 5:1 to about 6:1, and about 3:1 to about 5:1, including all values ​​and subranges therebetween.

[0143] As shown in FIG. 64B , a first fluid 6462 may flow through the flow cell 6410 generally according to a first direction. Magnetically labeled cells 6416 within the first fluid 6462 may separate from the remainder of the first fluid 6462 in the third channel 6416 as the magnetic attractive force generated by the magnet array 6420 pulls the cells 6416 away from the first channel 6412 toward the second channel 6414 (e.g., toward the magnet array 6420). Similarly, a second fluid 6470 (e.g., a buffer solution) may flow through the second channel 6414. As the cells 6416 flow toward the magnet array 6420, they may displace the second fluid 6470 flowing through the third channel 6416, causing a portion of the second fluid 6470 to flow into the first channel 6412. In this way, magnetically labeled cells 6416 may be magnetically separated from first fluid 6462 and second fluid 6470 may assist in the removal of first fluid 642 that does not contain cells 6416.

[0144] In some variations, a set of fluid loops can be coupled to a flow cell to enable multiple cell separation cycles. Figure 64C is a schematic diagram of a cartridge MACS module including a flow cell 6410 and a first fluid conduit 6480 coupled to an inlet 6430 of the flow cell 6410 and an outlet 6432 of the flow cell 6410. The first fluid conduit 6480 can be configured to receive a set of cells from the outlet 6432 of the flow cell 6410 for collection and / or recirculation through the inlet 6430 of the flow cell 6410. A second fluid conduit 6490 can be coupled to the inlet 6431 of the flow cell 6410 and the outlet 6433 of the flow cell 6410 to recirculate fluids such as buffer and uncollected magnetically labeled cells. The second fluid conduit 6490 can be configured to receive fluid from the flow cell 6410 that does not contain the set of cells. Higher purity labeled cells can be recovered based on the number of cycles performed. For example, one cell separation cycle may result in a cell purity of about 80%, a second cell separation cycle may result in a cell purity of about 96%, a third cell separation cycle may result in a cell purity of about 99.2%, and a fourth cell separation cycle may result in a cell purity of about 99.84%.

[0145] In some variations, applying centrifugal force to the magnetic cell separation process may further attract labeled cells toward the magnetic array, independent of fluid flow rate, to maintain throughput. Figures 65A-65C are schematic diagrams of a cartridge MACS module 6500 that utilizes centrifugal force to assist the cell separation process. Figure 65A depicts a flattened flow cell 6510 configured to be wrapped to form a generally cylindrical shape 6512. The flow cell 6510 may include a curved flow channel 6520.

[0146] Figure 65B illustrates a cylindrical flow cell 6510 concentrically surrounded by (e.g., nested within) a cylindrical magnet array 6530. In Figure 65B, for clarity, only a cross section of the magnet array 6530 is shown. The flow cell 6510 can be spaced a predetermined spacing distance from the magnet array 6530. Thus, the flow cell 6510 can be configured to rotate (6550) about its longitudinal axis to generate a centrifugal force on the fluid 6540 in the flow path 6520 in an outward direction, toward the magnet array 6530. During the cell separation process, the fluid may be subjected to a set of forces depicted in FIG. 65C , including a bulk fluid force 6560 in the axial direction (e.g., bulk flow), a centrifugal force 6570 radially outward from the center of rotation (e.g., proportional to the net particle-system buoyant force), and a magnetic force 6580 extending radially outward from the center of rotation (e.g., proportional to the net particle-system magnetic attractive force). In some variations, labeled cells may comprise a higher density than unlabeled cells. Thus, the centrifugal force may preferentially push labeled cells toward the magnet 6530, further increasing the specificity and efficiency of cell separation.

[0147] 66A-66C are schematic diagrams of a cell separation system and process. The magnetic cell separation system may include a flow cell 6610 with a flow path 6620 (shown schematically and flat for clarity) and a magnet array 6630. As shown in step 6600, a set of cells 6640, 6642 may include labeled cells 6640 (e.g., magnetically labeled cells), and unlabeled cells 6642 may flow into the flow path 6620 of the flow cell 6610. For example, the set of cells 6640 may be labeled with a magnetically activated cell selection (MACS) reagent. The magnet array 6630 may be positioned outside the flow cell 6610 such that the magnet array 6630 may be movable relative to the flow cell 6610. For example, the magnet array 6630 may be moved away from the flow cell 6610 to facilitate the flow of the set of cells 6640 out of the flow cell 6610.

[0148] In step 6602, the flow cell 6650 may be rotated to generate centrifugal force, pushing the cells 6640, 6642 toward the magnet array 6630. In some variations, the longitudinal axis of the flow cell 6610 may be oriented substantially perpendicular to the ground for gravity-assisted fluid flow through the flow cell 6610. In step 6604, the magnet array 6630 may magnetically attract the set of cells 6640 toward the magnet array 6630 for a predetermined dwell time, as described herein. Unlabeled cells 6642 are not magnetically attracted to the magnet array 6630 and may flow out of the flow cell 6610, for example, into a waste container. In some variations, the fluid within the flow cell (e.g., cells 6160, 6170) may be held statically within the flow cell 6110 for a dwell time before the fluid (e.g., cells 6170) flows out of the outlet 6132. In some variations, the magnetic coupling between the magnet array 6630 and the cells 6640 may be released after a dwell time, and the cells 6640 may be retrieved.

[0149] Figures 12A and 12B illustrate the magnet of a docking station MACS module 1200 that includes a magnet and cartridge MACS module 1210. The magnet is shown in an on configuration in Figure 12A and in an off configuration in Figure 12B.

[0150] Bioreactor The bioreactors described herein may include a vessel configured to culture mammalian cells. Generally, cells and gene therapy products may be grown in the bioreactor to generate a clinical dose, which may subsequently be administered to a patient. Many biological and environmental factors may be controlled to optimize the growth rate and success of cell growth. The bioreactor modules described herein allow for one or more of monitoring, adjusting, and / or controlling cell growth (e.g., to facilitate consistent and efficient cell growth).

[0151] 67A is a schematic diagram of a bioreactor system 6700 including one or more cartridge bioreactor modules 6750 comprising a bioreactor 6710 and a docking station bioreactor module 6712 including one or more sensors 6720, an agitator 6730, a temperature regulator 6740, and a gas regulator 6750. In some variations, the sensor 6720 may be configured to monitor (e.g., measure, sense, determine) one or more characteristics of the cartridge bioreactor module, including the cells within the bioreactor 6710. For example, the sensor 6720 may include one or more of a pH sensor, a DO sensor, a temperature sensor, a glucose sensor, a lactose sensor, a cell density sensor, a humidity sensor, combinations thereof, etc., which may be used to probe the environment within the bioreactor 6710. One or more of the sensors may be a non-invasive optical sensor.

[0152] 67B is a schematic diagram of a cell processing and manufacturing system including a CPS 6760 and a bioreactor system 6700 therein, the bioreactor system 6700 including a docking station bioreactor module 6712, a cartridge 6714, and a cartridge bioreactor module 6750. The docking station bioreactor module 6712 may include an agitator 6730, as well as a temperature regulator, sensors, and gas regulators (not shown). The CPS 6760 may further include a fluid transfer connector 6780. In some variations, the cartridge 6714 for cell processing may include a liquid transfer bus and multiple modules (e.g., a CCE module, a MACS module, an EP module) in addition to the cartridge bioreactor module 6750. Each module may be fluidically coupled to the liquid transfer bus. In variations, the cartridge bioreactor module may include at least one bioreactor.

[0153] 67B, the docking station bioreactor module 6712 can be configured to interface with the cartridge bioreactor module 6750 of the cartridge 6770, which includes a bioreactor therein. In some variations, the docking station bioreactor module 612 can include an agitator 6730 configured to couple to the bioreactor of the cartridge bioreactor module 6750. The agitator 6730 can be configured to agitate cell culture medium containing cells within the bioreactor. In some variations, a fluid transfer connector 6780 can be configured to couple the docking station bioreactor module 6700 with the cartridge bioreactor module 6750.

[0154] 67B, the cartridge 6714 is disposed within the CPS 6760. When the fluid transfer connector 6780 couples the docking station bioreactor module 6712 of the CPS 6760 to the cartridge bioreactor module 6750 of the cartridge 6714 (e.g., to create a sterile flow path), the cell culture medium in the bioreactor of the cartridge bioreactor module 6750 can be agitated by the agitator 6730 of the docking station bioreactor module 6712. In some variations, the fluid transfer connector 6780 can comprise a set of foldable sidewalls (e.g., accordion-like) configured to receive and dissipate the agitation of the agitator 6730 without transmitting such motion to the CPS 6760. That is, the fluid transfer connector 6780 can function as a bellows to maintain the connection between the docking station bioreactor module 6712 of the CPS 6760 and the cartridge bioreactor module 6780 without agitating the CPS 6760.

[0155] In some variations, the agitator may be configured to generate motion (e.g., orbital, rotational, linear) in the bioreactor of the cartridge bioreactor module 6750 to mix the culture as required to facilitate interaction with the reagents and cells. For example, orbital motion may be used to create a homogenous culture volume so that a small sample taken from the culture may represent the entire culture. In some variations, the agitator 6730 may comprise one or more impellers. The agitator 6730 may be configured to provide variable intensity mixing for defined periods of time during the culture.

[0156] In some variations, the orbital motion comprises a geometry that can encourage a continuous and gentle flow of fluid around the bioreactor, thereby encouraging increased interactions within the cell culture, such as in the toroidal bioreactor described herein, which can aid in homogenous mixing with minimal shear stress transmitted to the cells.

[0157] In some variations, a temperature regulator may be configured to control the temperature of the bioreactor and corresponding process. The temperature regulator may be coupled to the bioreactor. For example, the temperature regulator may control the temperature of the cell culture from about 2°C to about 40°C, thereby ensuring that the culture is heated to physiological conditions and, if desired, cooled to slow metabolic processes (e.g., to keep the cells quiescent). For example, the thermal regulator may comprise a circulating coolant coupled to a heat exchanger that is coupled to a thermal interface (e.g., the heating / cooling plate of the docking station bioreactor module 6712).

[0158] In some variations, a gas regulator may be coupled to the bioreactor and configured to control the gas composition of the bioreactor and corresponding process using one or more of clean dry air (CDA), carbon dioxide, and nitrogen, as shown in FIG. 2E. The gas regulator may be coupled to the bioreactor within the cartridge bioreactor module 6750. For example, the sensor and gas regulator may provide closed-loop gas control of the cartridge bioreactor module 6750. In some variations, the CDA may include oxygen, such as pure oxygen. In some variations, the gas regulator may comprise a manifold coupled to one or more gas sources. The manifold may include a solenoid coupled to a valve (e.g., a restrictive orifice) configured to control gas flow through the bioreactor. The solenoid may be configured to emit pulses to control the amount and composition of gas received through the manifold. Additionally or alternatively, one or more of a proportional valve and a mass flow controller (MFC) may be configured to meter and control the flow of gas to the manifold. In some variations, the gas regulator may include one or more sensors for measuring the gas mixture and / or flow rate. Additionally or alternatively, the sensors may be configured for closed-loop control of gas flow through the gas regulator.

[0159] In some variations, the measured pH from the pH sensor can be used to control the pH of the bioreactor using a gas regulator. For example, depending on the measured pH, the gas regulator can control the CO2 concentration of the gas contacting the cell culture to control the free hydrogen ions and pH of the culture. In some variations, the pH of the bioreactor can be about 5.5 to about 8.5. To adjust the pH, one or more of the CO2 composition of the gas in the bioreactor, a buffer, and a reagent (e.g., acid, base) can be used. In some variations, the dissolved oxygen concentration of the bioreactor can be about 0% to about 21%. The nitrogen composition of the gas in the bioreactor can be used to adjust the dissolved oxygen concentration. For example, the dissolved carbon dioxide concentration can be adjusted by controlling both the agitator in the bioreactor and the flow rate and composition of the gas contacting the cell culture.

[0160] In some variations, the measured dissolved oxygen from the dissolved oxygen sensor can be used to control the oxygen concentration in the bioreactor (e.g., below atmospheric levels) using a gas regulator. For example, the gas regulator can control the nitrogen concentration of the gas contacting the cell culture to create hypoxic conditions.

[0161] 68A and 68B are cross-sectional perspective views of a bioreactor 6800 including an enclosure 6810 including a base 6812, a sidewall 6814, and a top 6816. A gas permeable membrane 6820 may be coupled to one or more of the base 6812 and the sidewall 6814 of the enclosure 6810. In some variations, the enclosure 6810 may comprise a first chamber 6830 having a first volume and a second chamber 6832 having a second volume, wherein the first chamber 6830 is separated from the second chamber 6832 and the first volume is smaller than the second volume. In some variations, the first chamber 6830 may be concentrically nested within the second chamber 6832. For example, nesting the chambers may allow for a larger overall working volume range (e.g., 100:1). The first chamber 6830 may comprise a well shape with an angled base surface to encourage fluid pooling in the center of the first chamber 6830 during aspiration. In some variations, the base 6812 may be disposed on a thermal regulator (not shown), such as a thermoelectric device. In some variations, the enclosure 6810 may be constructed of a thermally conductive material, such as a metal (e.g., aluminum).

[0162] In some variations, the bioreactor 6800 may be coupled to a gas regulator (not shown) to facilitate gas transfer (e.g., into and out of the culture) through a gas permeable membrane 6820. The gas permeable membrane 6820 may be configured to retain the cell culture. Gas may diffuse through the surface of the culture in contact with the gas permeable membrane, allowing for increased oxygenation of the cell culture and removal of gaseous metabolic by-products of the cell culture, thus increasing the potential for metabolic activity. For example, the gas permeable membrane 6820 allows dissolved oxygen to diffuse into the culture in close proximity to the cell bed where oxygen can be consumed. In some variations, the bioreactor may be coupled to both a first gas regulator to facilitate gas transfer through the gas permeable membrane and a second gas regulator to facilitate control of the headspace gas composition.

[0163] In addition to gas transfer, the bioreactors described herein can be configured to efficiently control the temperature of the cell culture using conductive thermal interfaces (e.g., gas permeable membrane 6820, enclosure 6810) along both the base and sidewalls of the bioreactor.

[0164] In some variations, the first chamber 6830 can have a working volume of about 10 ml to about 100 ml. In some variations, the first chamber 6830 can have a total volume of about 10 ml to about 130 ml. In some variations, the second chamber 6832 can have a working volume of about 100 ml to about 1000 ml. In some variations, the second chamber 6832 can have a total volume of about 100 ml to about 1400 ml. In some variations, the first chamber 6830 can have a diameter of about 10 mm to about 100 mm and a height of about 10 mm to about 100 mm. In some variations, the second chamber 6832 can have a diameter of about 100 mm to about 250 mm and a height of about 10 mm to about 100 mm.

[0165] As shown in FIG. 68B, the base 6822 of the gas permeable membrane 6820 can be angled at about 3 degrees to about 10 degrees relative to the base 6812 of the enclosure 6810. Similarly, FIGS. 69A and 69B depict a sloped base. For example, due to the slope of the base 6822, the chambers 6830, 6832 are deeper toward the center of the bioreactor 6800. This can promote cell growth toward the center of the bioreactor 6800, which can assist with one or more of cell sampling, cell transfer, cell recovery, etc. In some variations, orbital motion of the bioreactor 6800 can promote cell aggregation toward the center of the bioreactor 6800, thereby increasing cell-to-cell interactions.

[0166] In some variations, the gas-permeable membrane 680 can include curved surfaces. In some variations, the gas-permeable membrane can include a set of patterned curved surfaces. For example, the set of patterned curved surfaces can include a radius of curvature of about 50 mm to about 500 mm.

[0167] In some variations, the bioreactor can be configured to facilitate monitoring (e.g., temperature, pH, dissolved oxygen) and fluid flow (e.g., gas composition, fluid transfer) between chambers. As shown in FIG. 68C, the enclosure 6810 can comprise one or more nested surfaces curved around a longitudinal axis (e.g., center) of the enclosure 6810. For example, the nested surfaces can comprise a set of concentric toroids. The enclosure 6810 can comprise a toroidal shape. FIG. 68C is a perspective view of the enclosure 6810 including a set of openings 6818 (e.g., holes, openings, slits, slots), and FIG. 68D is a bottom view thereof. In some variations, the openings 6818 can allow gas and / or heat transfer between components of the bioreactor 6800 and the chambers. Additionally or alternatively, one or more sensors can be coupled to the openings 6818. For example, opening 6818 can be coupled to a non-contact sensor (e.g., pH, DO), such as an optical sensor (not shown) configured to determine a fluorescent spot disposed on the surface of the bioreactor. In some variations, one or more of the sensor and fluid connector can be introduced through opening 6818.

[0168] In some variations, the gas permeable membrane extends along the base 6812 and sidewall 6814 of the enclosure 6810, as shown in FIG. 68B. In some variations, the gas permeable membrane extends only along the base 6812 of the enclosure 6810. FIG. 68E is a perspective view of the gas permeable membrane 6820, and FIG. 68F is a side view of the gas permeable membrane 6820, the outer surface of which comprises one or more protrusions 6824 (e.g., protrusions, spacers, ribs). The protrusions 6824 are also depicted in the perspective view of FIG. 68G and the bottom view of FIG. 68H. The protrusions 6824 contact the enclosure 6810 and define a cavity between the enclosure 6810 and the gas permeable membrane 6820. That is, the protrusions 6824 can be configured to mechanically space the enclosure 6810 from a portion of the gas permeable membrane 6820 to facilitate heat transfer from the enclosure 6810 to the cell culture. In some variations, the gas permeable membrane can include polydimethylsiloxane (PDMS) (e.g., silicone), fluorinated ethylene propylene (FEP), polyolefin (PO), polystyrene (PS), ethyl vinyl acetate (EVA), and can have a thickness of about 0.1 mm to about 0.4 mm, about 0.2 mm to about 0.3 mm, and about 0.25 mm, including all ranges and subvalues ​​therebetween.

[0169] Figure 69A is a cross-sectional side view of a bioreactor enclosure 6910 comprising a first chamber 6912, a second chamber 6914, and a column 6916 extending along the longitudinal axis of the enclosure 6910. Figure 69B is a cross-sectional perspective view of the enclosure 6910 showing the nested curves of the enclosure 6910. The column 6916 can be configured to facilitate cell culture in combination with agitation, such as orbital motion.

[0170] Figure 70 is an exploded perspective view of a bioreactor 7000 including an enclosure 7010, a gas permeable membrane 7020, and an upper portion 7030. The upper portion 7030 may be constructed from a material such as polyethylene.

[0171] Figure 71A is a plan view of a bioreactor 7100 comprising a first chamber 7110 and a second chamber 7120. Figure 71B is a cross-sectional side view of the bioreactor 7100.

[0172] 13 and 14 are perspective views of a cartridge 1300 and a docking station bioreactor module interface 1310. The docking station bioreactor module interface 1310 is coupled to the cartridge 1300 of FIG.

[0173] Electroporation Module In some variations, the electroporation module can be configured to facilitate intracellular delivery of macromolecules (i.e., transfection by electroporation). The electroporation module can contain a continuous flow or batch mode chamber and one or more electrode sets for applying direct or alternating current to the chamber. Discharge from one or more capacitors or current sources can generate sufficient current within the chamber to facilitate the transfer of polynucleotides, proteins, nucleoprotein complexes, or other macromolecular cellular products into cells. As with other modules described herein, one or more components used in a process step (herein, electroporation) can be provided on the cartridge or in a docking station to which the cartridge interfaces. For example, a capacitor and / or battery can be provided in a docking station module on the cartridge module. In some variations, the electroporation module can be configured to apply an electric field to a cell suspension undergoing continuous flow within a microfluidic device, as described, for example, in Garcia et al. Sci. Rep. 6:21238 (2016).

[0174] Additionally or alternatively, intracellular delivery of macromolecules can also be achieved by other methods (e.g., mechanoporation). It should be understood that throughout this disclosure, variations that include an electroporation module can instead or additionally include a mechanoporation module, or another module configured to implement any suitable method of delivering macromolecules into cells. Mechanoporation can be achieved, for example, by applying transient fluid pressure to a solution containing cells or by applying physical pressure to the cells (e.g., with a microneedle). Illustrative methods of mechanoporation by passing a cell suspension through a constriction are provided, for example, in International Patent Publications 2017 / 041051 and 2017 / 123663, which are incorporated herein by reference. Mechanoporation can also be achieved by applying a vortex to a cell suspension in a microfluidic device.

[0175] Figure 72 is a schematic diagram of an electroporation module 7200 (e.g., an electroporation system) comprising an electroporation chamber 7210 (which may comprise a fluid conduit), a pump 7220, an inlet 7230, an outlet 7232, a set of pinch valves 7234, a first fluid source 7240 (e.g., a fluid reservoir, a cell reservoir), a second fluid source 7242 (e.g., a vent, a gas source), a set of sensors 7250 (e.g., a bubble sensor), and a controller (e.g., a processor and memory) configured to control the module 7200, and a signal generator 7270 configured to deliver an electroporation signal (e.g., a voltage pulse) to the electroporation chamber 7210.

[0176] In some variations, the fluid conduit 7210 may be configured to receive a first fluid containing cells and a second fluid. A set of electrodes may be coupled to the fluid conduit 7210. A pump may be coupled to the fluid conduit 7210. The controller 7260 may be configured to generate a first signal to introduce the first fluid into the fluid conduit 7210 using the pump 7220, generate a second signal to introduce the second fluid into the fluid conduit 7210 such that the second fluid separates the first fluid from the third fluid, and generate an electroporation signal to electroporate the cells in the fluid conduit 7210 using the set of electrodes.

[0177] In some variations, the second fluid may include a gas or oil. In some variations, the controller may be configured to generate a third signal to introduce a third fluid into the fluid conduit 7210. The third fluid may be separated from the first fluid by the second fluid. In some variations, a cartridge for cell processing may include a liquid transfer bus and multiple modules, such as electroporation module 7200. Each module may be fluidically coupled to the liquid transfer bus.

[0178] The set of sensors 7250 may be configured to measure a fluid change in the fluid conduit, such as a change from a first fluid to a second fluid (e.g., from liquid to air) in the fluid conduit. The module 7200 may further comprise a set of valves configured to ensure that fluid does not flow back into the electroporation chamber 7210 and / or fluid source 7240. The electroporation chamber 7210 may comprise a cavity configured to hold the fluid to be electroporated and a set of electrodes for applying an electroporation signal to the fluid. For example, the signal generator 7270 may generate square valve pulses, as described in more detail herein.

[0179] In some variations, the electroporation module 7200 (e.g., valve 7234, pump 7220, sensor 7250, and controller 7260) can be configured to control fluid flow through the electroporation chamber 7210 in a discontinuous (e.g., batch process) manner. For example, a first batch of cells can be electroporated and physically separated from a second batch of cells by an intermediate fluid such as air or a fluid such as oil. Separating the cell batches can reduce mixing of transfected and non-transfected cells and also ensure a fixed batch volume. That is, a fluid gap can form a visually verifiable boundary between cell batches to reduce diffusion and mixing between electroporated and non-electroporated cells. Separating the cell batches can reduce the time cells are exposed to certain cytotoxic reagents (e.g., electroporation buffer), thereby improving performance.

[0180] In some variations, a batch of cells can be electroporated in a substantially static state (e.g., with substantially no fluid flow). In contrast, conventional continuous-flow electroporation has an upper fluid flow rate limit that correlates with transfection efficiency. In the batch processing described herein, a batch of cells can be transported into and out of the electroporation chamber 7210 at a predetermined rate to increase the overall throughput of the system 7200 without reducing electroporation efficiency. Furthermore, the electroporation system 7200 does not utilize precisely controlled flow rates / pulse rates as required for continuous-flow electroporation systems.

[0181] FIG. 73 is an exploded perspective view of a cartridge electroporation module 7300, which may include an electrode 7310, a fluid conduit 7320 (e.g., an electroporation chamber), a substrate 7330 (e.g., an alloy bus bar), a housing 7340, and a fastener 7350. In some variations, the fluid conduit 7320 may be configured to hold a fluid volume of about 0.4 ml to about 3.5 ml. The electroporation module 7300 is a parallel plate design. In some variations, the electrodes may comprise stainless steel and may be separated by an insulating gasket. In some variations, the electrodes may be polished and / or coated with a non-reactive material (e.g., gold, platinum) to reduce the gradual accumulation of biological material (e.g., charged molecules, DNA, proteins) on the electrode surface.

[0182] Generally, a method of electroporating cells may include receiving a first fluid containing cells in a fluid conduit, receiving a second fluid in the fluid conduit to separate the first fluid from a third fluid, and applying an electroporation signal to the first fluid to electroporate the cells. In some variations, the third fluid may be received in the fluid conduit separated from the first fluid by the second fluid. In some variations, the first fluid may be substantially static when applying the electroporation signal.

[0183] 74A-74B are schematic diagrams of variations of electroporation processes 7400, 7402. Method 7400 may include loading 7410 cells into an electroporation chamber 7450. For example, in step 7412, a first fluid may be pumped into the electroporation chamber by opening valve v1, and the pump creates negative pressure (valves v2 and v3 are closed). In step 7414, a second fluid (e.g., gas, oil) may separate the first fluid from the third fluid and create a first batch of cells for electroporation. For example, valves v1 and v3 may be closed with valve v2 open, and the pump creates negative pressure. In some variations, the loading volume may be about 1 ml to about 3 ml, with a pumping time of about 8 seconds to about 15 seconds (at a rate of about 20 ml / min). In step 7420, cells of the first fluid may be electroporated with each of the valves closed and the pump off. In step 7430, cells of the first fluid may be forced out of electroporation chamber 7450 and into an output where valves v1 and v2 are closed, valve v3 is open, and the pump generates positive pressure.

[0184] 74B depicts another configuration in which a pump is disposed between the input and the electroporation chamber such that the pump can be configured to pump unidirectionally. Method 7402 can include loading 7411 cells into electroporation chamber 7450. For example, in step 7416, a first fluid can be pumped into the electroporation chamber by opening valves v1 and v4, and the pump generates positive pressure (valves v2 and v3 are closed). In step 7418, a second fluid (e.g., gas, oil) can separate the first fluid from a third fluid and create a first batch of cells for electroporation. For example, valves v1 and v3 can be closed with valves v2 and v4 open, and the pump generates positive pressure. In step 7422, cells in the first fluid can be electroporated with each of the valves closed and the pump off. In step 7432, cells in the first fluid can be forced out of the electroporation chamber 7450 and flow to the output where valves v1 and v4 are closed, valves v2 and v3 are opened, and the pump creates positive pressure.

[0185] In some variations, the impedance / resistance across the electrodes of an electroporation system may increase over time due to electrode passivation / degradation caused by charged biological material (e.g., charged molecules, DNA, proteins) adhering to the electrode surface. Active field compensation can be applied to ensure consistent electric field strength applied to cells across multiple batches of cells. This may reduce the need for electrode surface modification to reduce passivation.

[0186] Figure 75 is a circuit diagram of a resistor divider network for an electroporation process 7500. For example, a set of cells may be connected to a voltage V チップ The fluid resistance R can be applied to the electroporation chamber 7510. b corresponds to the fluid (e.g., cell mixture) resistance. Assuming a uniform cell distribution, the fluid resistance R b should be consistent, and assume that the volume of each fluid batch electroporated is the same. R i corresponds to the resistance between the fluid and the electrode, which increases over time throughout the electroporation process. In a conventional electroporation process, the voltage V ps However, the R i Due to this, the voltage applied to the fluid decreases over time, leading to a lower field strength.

[0187] Fluid resistance R b Due to variations in and the small number of pulses that can be applied, interpolation to compensate for the reduced field strength may not accurately compensate for electrode passivation.

[0188] In some variations, a method of electroporating cells may include receiving a first fluid containing cells in a fluid conduit, applying a resistance measurement signal to the first fluid using a set of electrodes, measuring the resistance between the first fluid and the set of electrodes, and applying an electroporation signal to the first fluid based on the measured resistance. In some variations, a second fluid containing a gas may be received in the fluid conduit before applying the electroporation signal to the fluid. The first fluid may be separated from a third fluid by the second fluid.

[0189] Figures 76A-76D are plots 7600, 7602, 7604, and 7606 of measurement and electroporation waveforms. Figure 76A illustrates a first resistance measurement pulse 7620 having a low voltage and a wide pulse width. Figure 76B illustrates a second resistance measurement pulse 7622 having a high voltage and a short pulse width. Figure 76C illustrates a third resistance measurement pulse 7624 having a continuous low voltage waveform for continuously monitoring impedance changes over time. Figure 76D illustrates a fourth resistance measurement pulse 7626 having a low AC voltage waveform for continuously monitoring impedance changes over time. Each resistance measurement pulse reduces the voltage and / or pulse width to avoid inducing electroporation in the cells. By monitoring the voltage and current of the applied resistance measurement pulses, changes in resistance can be measured and the electroporation pulses applied to the cell batch can be compensated accordingly.

[0190] In some variations, the electroporation signal may include about 1 pulse to about 50 pulses, a voltage of about 100 V to about 700 V, a pulse width of about 100 μs to about 1 ms, a pulse interval of about 5 seconds to about 30 seconds, a resistive pulse voltage of about 10 V to about 40 V, and a resistive pulse width of about 10 μs to about 50 μs.

[0191] For example, an eight-batch electroporation run may receive one electroporation pulse per batch. Each electroporation pulse may have a field strength of about 0.5 kV / cm to about 2.0 kV / cm. The resistance measurement pulse applied before each batch may have a field strength of less than about 0.2 kV / cm so that electroporation is not induced by the resistance measurement pulse.

[0192] Sterile Liquid Transfer Devices Generally, the sterile fluid transfer devices described herein can be configured to store fluids for transfer to another component of a cell processing and manufacturing system, such as a cartridge, a bioreactor, etc. In some variations, the sterile fluid transfer device can comprise a portable consumable that is configured to be moved using a robot. For example, a robot can be configured to move the sterile fluid transfer device from a reagent reservoir to an ISO7 space to a sterile fluid transfer fixture within the cell processing and manufacturing system. The sterile fluid transfer device enables the transfer of fluids in an automated, sterile, metered manner to automate cell therapy manufacturing.

[0193] 103A and 103B are perspective views of a sterile liquid transfer device 10300 including a fluid cavity 10310 (e.g., container, vessel), a fluid connector 10320, and a pump 10330. Fluid stored in the fluid cavity 10310 can be transferred into or out of the sterile liquid transfer device 10300 through the fluid connector 10320 using the pump 10330. In some variations, the sterile liquid transfer device 10300 can include engagement features 10340 (e.g., a robotic mount) to facilitate robotic arm control.

[0194] Fluid Connector Generally, the sterile fluid connectors described herein can form a sterile fluid pathway between at least two fluidic devices to enable fluid transfer that can be one or more of: sterile, fully automated, and precisely metered (e.g., precise control of the fluid volume transferred). In some variations, a robot can be configured to couple the fluid connectors between at least two sterile liquid transfer devices and a plurality of cartridge modules. In some variations, the robot can be configured to operate a fluid controller to open and close a set of ports and valves in the fluidic connectors. The use of a robot and controller to operate the fluidic connectors can facilitate automation and sterilization of cell processing and manufacturing systems.

[0195] In some variations, a system may include a robot configured to operate a fluid connector as described herein and a controller including a memory and a processor. The controller may be coupled to the robot. The controller may be configured to generate a port signal to couple a first port to a second port using the robot arm, generate a first valve signal to translate a first valve relative to a second valve using the robot arm, and generate a second valve signal to transition the first valve and the second valve to an open configuration.

[0196] In some variations, the fluid pump may be coupled to a sterilant source, and the controller may be configured to generate a first fluid signal to circulate fluid into the chamber through the sterilant port, the controller may be configured to generate a second fluid signal to circulate sterilant into the chamber through the sterilant port to at least sterilize the chamber, and the controller may be configured to generate a third fluid signal to remove sterilant from the chamber.

[0197] In some variations, the controller may be configured to generate a port signal to couple the first port to the second port using the robot, generate a first valve signal to translate the first valve relative to the second valve using the robot, and generate a second valve signal to transition the first valve and the second valve to an open configuration.

[0198] The fluid connectors may also allow for multiple connection cycles within a sterilization system and may be controlled without human intervention. For example, the fluid connectors may include one or more engagement features to facilitate robotic control and alignment features to ensure proper connection between the connector components. Figure 15 is a block diagram of an illustrative variation of a fluid connector system 1500 including a fluid connector 1510, a first fluidic device 1520 (e.g., a cartridge, cartridge module, sterile liquid transfer device (SLTD)), a second fluidic device 1522 (e.g., a cartridge, cartridge module, SLTD), a sterilant source 1530, a fluid source 1532, a robot 1540, and a controller 1550.

[0199] The fluid connector 1510 may be removably coupled (e.g., connected / disconnected, attached / detached) to each of the first fluidic device 1520, the second fluidic device 1522, the sterilant source 1532, the fluid source 1532, and the robot 1540. In some variations, the fluidic device may comprise one or more of a cartridge and a sterile liquid transfer device. For example, the sterile liquid transfer device may be in fluid communication with the cartridge via the fluidic connector.

[0200] As described in more detail herein, the separate portions (e.g., male connector, female connector) of the fluid connector 1510 may be removably coupled to one another. The robot 1540 may be configured to physically manipulate (e.g., removably couple) one or more of the fluid connector 1510, the first fluidic device 1520, the second fluidic device 1522, the sterilant source 1530, and the fluid source 1532 in a predetermined manner. For example, the robot 1540 may connect the fluid connector 1510 between the first fluidic device 1520 and the second fluidic device 1522. The robot 1540 may also connect the sterilant source 1530 and / or the fluid source 1532 to a sterilant port of the fluid connector 1510. In some variations, the robot 1540 may control one or more valves and / or ports of the fluid connector 1510, thereby initiating a sterilization process of one or more portions of the fluid connector 1510, for example, using sterilant from the sterilant source 1530. The controller 1550 may be coupled to one or more of the robot 1540, the sterilant source 1530, and the fluid source 1532 to control one or more of the fluid transfer and sterilization.

[0201] 16A is a schematic diagram of an illustrative variation of a fluid connector 1600. The fluid connector 1600 may include a lumen extending along its length and may be disposed between a first fluid device 1630 and a second fluid device 1640 to allow fluid flow therethrough. In some variations, the first fluid device 1630 and the second fluid device 1640 may be aseptically connected and disconnected using the fluid connector 1600. The fluid devices 1630, 1640 may comprise closed sterile devices and may be the same or different types of fluid devices. For example, the fluid devices 1630, 1640 may comprise one or more of a sterile liquid transfer device and a consumable (e.g., cartridge). In some variations, the fluid connector 1600 may include a first connector 1610 including a first proximal end 1612 and a first distal end 1614. The first proximal end 1612 may be configured to couple to a first fluid device 1630. The first distal end 1614 may include a first port 1616, a first housing 1617, and a first valve 1618. The first housing 1617 may be configured to receive the first port 1616 in a closed configuration, as described in more detail herein.

[0202] The fluid connector 1600 may further include a second connector 1620 including a second proximal end 1622 and a second distal end 1624. The second proximal end 1622 may be configured to couple to a second fluid device 1640. The second distal end 1624 may include a second port 1626, a second housing 1627, and a second valve 1628. The second housing 1627 may be configured to receive the second port 1626 in a closed configuration. In FIG. 16A , the first connector 1610 includes a sterilant port 1650 configured to couple to a sterilant source (not shown). Additionally or alternatively, the second connector 1620 may include the sterilant port 1650. The sterilant port 1650 may be configured to be in fluid communication with the first distal end 1614 and the second distal end 1624 when the second port 1626 is coupled to the first port 1616, as described in more detail herein.

[0203] In some variations, the fluidic devices 1630, 1640 may include a sterilant chamber and a sterilant port configured to receive the sterilant. The sterilant chamber may enclose a fluidic device connector (not shown) configured to mate with the proximal end of the first connector 1610 or the second connector 1620. The fluidic devices 1630, 1640 may receive the sterilant in a manner similar to the fluidic connector 1600.

[0204] 16B is a detailed schematic diagram of a first connector 1610 including a first port housing 1617 and a chamber 1615. The chamber 1615 may be defined by a cavity surrounded by one or more of the distal ends 1614, 1624. For example, the chamber 1615 in FIG. 16B may comprise a portion of the first connector 1610 (e.g., the first distal end 1614) between the first valve 1618 and the first port 1616 in a closed configuration. In some variations, the first chamber 1615 is approximately 1 cm 3 ~about 5cm 3 When the first connector 1610 is coupled to the second connector 1620 and the ports 1616, 1626 are in an open configuration (as shown in FIG. 16D ), the chamber 1616 may comprise a portion of the fluid connector 1600 between the first valve 1618 and the second valve 1628 (e.g., the first distal end 1614 and the second distal end 1624). The chamber 1615 may comprise an enclosed volume configured to receive a fluid, such as a sterilant, from a sterilant port 1650. In some variations, the sterilant port 1650 may comprise an inlet 1652 and an outlet 1654. Methods of using the fluid connector are described in more detail with respect to FIGS. 16C-16L and 27 .

[0205] In some variations, the fluid connector 1600 may include one or more alignment and robotic engagement features configured to facilitate robotic manipulation, as described in more detail herein. In some variations, the fluid connector 1600 may be coupled to one or more sensors, pumps, and valves to facilitate fluid transfer and monitoring.

[0206] In some variations, the components of the fluidic connector that come into contact with fluids may be USP Class VI compatible for cell processing and / or GMP applications. In some variations, the components of the fluidic connector may be constructed from materials including, but not limited to, one or more of cyclic olefin copolymer (COC), polychlorotrifluoroethylene, polyetherimide, polysulfone, polystyrene, polycarbonate, polypropylene, silicone, polyetheretherketone, polymethylmethacrylate, nylon, acrylic, polyvinyl chloride, vinyl, phenolic resins, petroleum-derived polymers, glass, polyethylene terephthalate, metals, stainless steel, titanium, aluminum, cobalt chromium, chromium, silicates, glasses, alloys, ceramics, carbohydrate polymers, minerals, and combinations or composites thereof.

[0207] 17A-18D depict exterior and interior views of variations of a fluid connector. FIG. 17A is a front perspective view of fluid connector 1700 in a closed port configuration. FIG. 17B is a rear perspective view of fluid connector 1700, and FIG. 17C is a rear view of fluid connector 1700. Generally, the fluid connector may include multiple internal seals to reduce contamination and aid in sterilization, as well as alignment features to aid in proper alignment of the fluid connector components.

[0208] The fluid connector 1700 may comprise a lumen extending along its length. In some variations, the fluid connector 1700 may comprise a first connector 1710 including a first proximal end 1712 and a first distal end 1714. The first proximal end 1712 may be configured to couple to a first fluid device (not shown for clarity). The first proximal end 1712 may comprise a Luer connector or any other suitable connector. The first distal end 1714 may include a first port 1716 and a first housing 1717. The first housing 1717 is shown in FIG. 17A and holds the first port 1716 in a closed configuration. The first connector 1710 further comprises sterilant ports 1750, 1752 configured to couple to a sterilant source (not shown for clarity). In some variations, the sterilant ports may comprise an inlet and an outlet. In some variations, the sterilant port may optionally include one or more of a check valve and a particle filter configured to reduce contamination to the sterilant port when not connected to a robot or actuator. The first connector 1710 may include a first alignment feature 1760, such as a set of protrusions on the first distal end 1714 of the first connector 1710. The alignment feature may ensure that small positioning errors due to robotic manipulation do not affect the operation of the fluid connector.

[0209] The fluid connector 1700 may further comprise a second connector 1720 including a second proximal end 1722 and a second distal end 1724. The second proximal end 1722 may be configured to couple to a second fluidic device (not shown for clarity). The second proximal end 1722 may comprise a Luer connector or any other suitable connector. The second distal end 1724 may include a second port 1726 and a second housing 1727. The second housing 1727 is shown in FIG. 17A and holds the second port 1726 in a closed configuration. The second connector 1720 may comprise a second alignment feature 1762, such as a set of holes, on the second distal end 1724 of the second connector 1720. The second alignment feature 1762 can be configured to mate with the first alignment feature 1760 in a predetermined axial and rotational configuration to assist in mating of the first connector 1710 and the second connector 1720 .

[0210] The first port 1716 and the second port 1726 held within the first housing 1717 at the first distal end 1714 and the second housing 1727 at the second distal end 1724, respectively, facilitate robotic control as the ports 1716, 1726 are inseparable from the fluid connector 1700, thus reducing the risk of failure of automated handling by the robot.

[0211] In some variations, the first connector 1710 may include a first robotic engagement feature 1770, and the second connector 1720 may include a second robotic engagement feature 1772. The robotic engagement features 1770, 1772 may be configured to be manipulated by a robot (e.g., robot 1540). In some variations, the robotic engagement features 1770, 1772 may be operably coupled to the respective first and second ports 1716, 1726 and configured to actuate the ports 1716, 1726 between a closed port configuration and an open port configuration, as shown in FIGS. 17A-17F. Additionally or alternatively, a user may manually actuate the robotic engagement features 1770, 1772 to actuate the respective ports 1716, 1726.

[0212] Figure 17D is a front perspective view of the fluid connector 1700 in the open port configuration. Figure 17E is a rear perspective view of the fluid connector 1700 in the open port configuration, and Figure 17F is a rear view of the fluid connector 1700 in the open port configuration. In the open port configuration, the first valve 1718 of the first connector 1710 and the second valve 1728 of the second connector 1720 are shown in Figure 17D.

[0213] 18A is a side view of the fluid connector 1800 in an unmated configuration, and FIG. 18B is a cross-sectional side view thereof. In some variations, the fluid connector 1800 may include a first connector 1810 including a first housing 1817 with a first port 1816, a sterilant port 1850 configured to couple to a sterilant source (not shown), and a first alignment feature 1860 configured to mate with a corresponding alignment feature (not shown) of the second connector 1820. The fluid connector 1800 may include a second connector 1820 including a second housing 1827 with a second port 1826. The first connector 1810 and the second connector 1820 may be axially aligned, and the alignment feature may assist in rotational alignment of the first connector 1810 to the second connector 1820. The first valve 1818 may include a first valve stem 1819 and the second valve 1828 may include a second valve stem 1829 .

[0214] 18C is a side view, and FIG. 18D is a cross-sectional side view, of the fluid connector 1800 in a coupled configuration in which the first housing 1817 and the second housing 527 are brought together but the first connector 1810 and the second connector 1820 are not in fluid communication because the first port 1816 and the second port 1826 are both in a closed configuration. First alignment features on each connector 1810, 1820 can be configured to ensure axial and / or rotational alignment between the first connector 1810 and the second connector 1820.

[0215] Figure 18E is a side view of the fluid connector 1800 in an open port configuration, and Figure 18F is a cross-sectional side view thereof. Each of the first port 1817 and the second port 1827 is transitioned from a closed configuration to an open configuration. This creates a closed internal volume within the distal end of each connector 1810, 1820. Each of the first valve 1818 and the second valve 1828 is in a closed configuration such that fluid flow is prevented between the first connector 1810 and the second connector 1820, restricted in each half due to the automatic shut-off valves on both sides.

[0216] 18G is a side view, and FIG. 18H is a cross-sectional side view, of the fluid connector 1800 in an open valve configuration in which the first valve 1818 is coupled to the second valve 1828. For example, the second valve 1828 can be translated toward the first valve 1818 along the longitudinal axis of the second connector 1820. As shown in FIGS. 18G and 18H, the second connector 1820 can be axially compressed to translate the second valve 1828 toward the first valve 1818. The first valve 1818 coupled to the second valve 1828 can form a radial seal, and the first valve stem 1819 and the second valve stem 1829 can contact to allow fluid communication between the first connector 1810 and the second connector 1820.

[0217] 19-26B are schematic diagrams of variations of fluid connector systems for connecting fluidic devices. In some variations, the fluidic connector may include a first connector configured to couple to any one of a plurality of second connectors. FIG. 19 is a schematic diagram of an illustrative variation of a fluidic connector system 1900 including a first connector 1910, a plurality of second connectors 1920, 1921, 1922, a first fluidic device 1930 (e.g., a sterile liquid transfer device), a second fluidic device 1940 (e.g., a consumable), and a robot 1960 (e.g., a robotic arm, a 3DOF robot). The first connector 1910 may be fluidly coupled to the first fluidic device 1930, and the second connectors 1920, 1921, 1922 may be fluidly coupled to the second fluidic device 1940. The first connector 1910 and the second connectors 1920, 1921, 1922 may each include a port 1916 configured to couple to a corresponding port, as described in more detail herein. The robot 1960 may include one or more end effectors 1962, 1964 configured to manipulate and / or couple to one or more of the first fluidic device 1930 and the first connector 1910. For example, the first connector 1910 may include one or more sterilization ports 1950 configured to couple to the end effector 1962 (e.g., a gripper). Similarly, the first fluidic device 1930 may include one or more fluid ports 1952 configured to couple to the end effector 1964.

[0218] In some variations, the robot 1960 may be configured to couple to one or more of a sterilant source, a fluid source, and a pump to facilitate efficient and shared fluid connections between the fluidic device, the fluid connector, and the sterilization system. For example, FIG. 96A is a plan view of a fluidic device 9600 (e.g., a sterile liquid transfer device) including a fluid port 9610 configured to couple to a fluid source (not shown) and a sterilization port 9620 configured to couple to a sterilant source (not shown). FIGS. 96B and 96C are side and perspective views, respectively, of the fluidic device 9600 coupled to the robot 9650. In some variations, the robot 9650 may include one or more fluid conduits 9660 configured to couple to one or more of the fluid port 9610 and the sterilization port 9620 of the fluidic device 9600.

[0219] In some variations, the fluid connector may include a third connector disposed between the first and second connectors. Figure 20A is a schematic diagram of an illustrative variation of a fluid connector system 2000 including a first connector 2010, a plurality of second connectors 2020, 2021, 2022, a third connector 2070 (e.g., an instrument, a sterile enclosure), a first fluidic device 2030 (e.g., a sterile liquid transfer device), a second fluidic device 2040 (e.g., a cartridge), and a robot 2060 (e.g., a 3DOF robot, a 1DOF robot). The first connector 2010 may be fluidly coupled to the first fluidic device 2030, and the second connectors 2020, 2021, 2022 may be fluidly coupled to the second fluidic device 2040. The third connector 2070 may be coupled between the first connector 2010 and one of the second connectors 2020, 2021, 2022. The third connector 2070 may include a lumen configured to receive and circulate a sterilant through one or more portions of the first connector 2010, the second connectors 2020, 2021, 2022, and the third connector 2070. In some variations, the sterilization port 2052 may be non-removably coupled to a sterilant source and / or a fluid source, thereby simplifying one or more of the first fluidic device 2030 and the first connector 2010.

[0220] The robot 2060 may include one or more end effectors 2062, 2064, 2066 configured to manipulate and / or couple to one or more of the first fluidic device 2030, the first connector 2010, and the third connector 2070. For example, the first fluidic device 2030 may include one or more fluid ports 2050 configured to couple to the end effector 2062. Similarly, the third connector 2070 may include one or more sterilization ports 2052 configured to couple to the robot 2060 (e.g., the end effector 2064). In some variations, the robot 2060 may be configured to couple to one or more of a sterilant source, a fluid source, and a pump to facilitate efficient and shared fluid connections between the fluidic devices, the fluid connectors, and the sterilization system.

[0221] 20B and 20C are schematic illustrations of a fluid connector connection process. In FIG. 20B, a third connector 2070 may be coupled to a distal end of a first connector 2010 at 2002. A distal end of a second connector 2020 may be coupled to the third connector 2070 at 2004. At 2006, the second connector 2020 may be translated through the third connector 2070 to directly couple the second connector 2020 to the first connector 2010.

[0222] 20C , at 2002, a third connector 2070 may be coupled to a distal end of the first connector 2010 and a distal end of the second connector 2020. Each of the first connector 2010 and the second connector 2020 may be translated toward one another through the third connector 2070 at 2005. At 2007, the second connector 2020 may be further translated toward the first connector 2010 to directly couple the first connector 2010 to the second connector 2010. FIG. 20C further illustrates the first port 2090 and the second port 2092 that may transition between a closed port configuration and an open port configuration.

[0223] In some variations, the fluid connector may include a third connector disposed between the first connector and the second connector. The third connector may be coupled to a second robot different from the first robot coupled to the first connector. FIG. 21 is a block diagram of an illustrative variation of a fluid connector system 2100 including a first connector 2110, a plurality of second connectors 2120, 2121, 2122, a third connector 2170 (e.g., an instrument, a sterile enclosure), a first fluidic device 2130 (e.g., a sterile liquid transfer device), a second fluidic device 2140 (e.g., a consumable), a first robot 2160, and a second robot 2166. The first connector 2110 may be coupled in fluid communication with the first fluidic device 2130, and the second connectors 2120, 2121, 2122 may be coupled in fluid communication with the second fluidic device 2140. The third connector 2170 may be coupled between the first connector 2110 and one of the second connectors 2120, 2121, 2122. The third connector 2170 may comprise a lumen configured to receive and circulate a sterilant through one or more portions of the first connector 2110, the second connectors 2120, 2121, 2122, and the third connector 2170. In some variations, the third connector 2170 may be non-removably coupled to a sterilant source and / or a fluid source, thereby simplifying one or more of the first fluidic device 2130 and the first connector 2110.

[0224] The first robot 2160 may include one or more end effectors 2162, 2164 configured to manipulate and / or couple to one or more of the first fluidic device 2130 and the first connector 2110. For example, the first fluidic device 2130 may include one or more fluid ports 2150 configured to couple to the end effector 2162. The third connector 2170 may be coupled to the second robot 2166 (e.g., a 3DOF robot). In some variations, the robots 2160, 2166 may be configured to couple to one or more of a sterilant source, a fluid source, and a pump to facilitate efficient and shared fluid connections between the fluidic devices, the fluid connectors, and the sterilization system.

[0225] In some variations, the fluid connector may include a sterilant source coupled to a plurality of second connectors. Figure 22 is a block diagram of an illustrative variation of a fluid connector system 2200 including a first connector 2210, a plurality of second connectors 2220, 2221, 2222, a first fluidic device 2230 (e.g., a sterile liquid transfer device), a second fluidic device 2240 (e.g., a consumable), a robot 2260, a sterilant source 2290 including one or more valves, and a sterilant switch 2292. The first connector 2210 may be coupled in fluid communication with the first fluidic device 2230, and the second connectors 2220, 2221, 2222 may be coupled in fluid communication with the second fluidic device 2240. The robot 2260 may include one or more end effectors 2262, 2264 configured to manipulate and / or couple to one or more of the first fluid device 2230 and the first connector 2210. For example, the first fluid device 2230 may include one or more fluid ports 2250 configured to couple to the end effector 2262. In some embodiments, the sterilant source 2290 may be coupled to a switch 2292. The switch 2292 may be coupled to each of the second connectors 2220, 2221, 2222 to facilitate an efficient and shared fluid connection between the fluidic device, the fluid connector, and the sterilization system. In some variations, a sterilant conduit may be routed from the switch 2292 through the second fluidic device 2240 to the respective second connectors 2220, 2221, 2222.

[0226] In some variations, the fluidic device may include one or more sterilant valves coupled to a plurality of second connectors. Figure 23 is a block diagram of an illustrative variation of a fluidic connector system. Figure 23 is a block diagram of an illustrative variation of a fluidic connector system 2300 including a first connector 2310, a plurality of second connectors 2320, 2321, 2322, a first fluidic device 2330 (e.g., a sterile liquid transfer device), a second fluidic device 2340 (e.g., a consumable), a robot 2360, a set of sterilant valves 2390 disposed within the housing of the second fluidic device 2340, and a sterilant switch 2392. The first connector 2310 may be coupled in fluid communication with the first fluidic device 2330, and the second connectors 2320, 2321, 2322 may be coupled in fluid communication with the second fluidic device 2340. The robot 2360 may include one or more end effectors 2362, 2364 configured to manipulate and / or couple to one or more of the first fluidic device 2330 and the first connector 2310. For example, the first fluidic device 2330 may include one or more fluid ports 2350 configured to couple to the end effector 2362. In some embodiments, the sterilant valve 2390 may be coupled to a switch 2392. The switch 2392 may be coupled to each of the second connectors 2320, 2321, 2322 via the sterilant valve 2390 to facilitate an efficient and shared fluid connection between the fluidic device, the fluid connector, and the sterilization system. In some variations, a sterilant conduit may be routed from the switch 2392 through the second fluidic device 2340 to the respective second connectors 2320, 2321, 2322.

[0227] In some variations, the fluid connector may include a sterilant source coupled to multiple second connectors, each having a sterilant port (e.g., a sterilant valve) and a sterilant conduit through the fluidic device. Figure 24A is a block diagram of an illustrative variation of a fluid connector system 2400 including a first connector 2410, multiple second connectors 2420, 2421, 2422, a first fluidic device 2430 (e.g., a sterile liquid transfer device), a second fluidic device 2440 (e.g., a cartridge), a robot 2460, and a sterilant switch 2492 coupled to a sterilant source (not shown). The first connector 2410 may be coupled in fluid communication with the first fluidic device 2430, and the second connectors 2420, 2421, 2422 may be coupled in fluid communication with the second fluidic device 2440. The robot 2460 may include one or more end effectors 2462, 2464 configured to manipulate and / or couple to one or more of the first fluid device 2430 and the first connector 2410. For example, the first fluid device 2430 may include one or more fluid ports 2450 configured to couple to the end effector 2462.

[0228] In some variations, each of the second connectors 2420, 2421, 2422 may include a respective sterilant port 2494, 2496, 2498 that includes a valve coupled to the distal end of the second connector 2420, 2421, 2422. In some variations, a sterilant conduit may be routed from the switch 2492 through the second fluidic device 2440 to the respective sterilant port 2494, 2496, 2498. In some variations, a sterilant source (not shown) may be coupled to the switch 2492. The switch 2492 may be coupled to each of the second connectors 2420, 2421, 2422 via the sterilant ports 2494, 2496, 2498 to facilitate an efficient and shared fluid connection between the fluidic device, the fluid connector, and the sterilization system.

[0229] Figure 24B is a schematic diagram of a fluid connector connection process 2402, 2404, 2406 in which a first connector 2410 is coupled to a second connector 2420. For example, the sterilant port 2494 is in a closed valve configuration when the first connector 2410 and the second connector 2420 are separated and uncoupled 2402. Figure 24C is a detailed schematic diagram of the sterilant valve 2494. In some variations, the valve 2494 can transition to an open valve configuration when the first connector 2410 is coupled to the second connector 2420 at 2404 and 2406.

[0230] In some variations, the plurality of second connectors may include one or more pneumatic sterilant valves and a sterilant pathway through the fluidic device. Figure 25A is a block diagram of an illustrative variation of a fluid connector system 2500 including a first connector 2510, a plurality of second connectors 2520, 2521, 2522, a first fluidic device 2530 (e.g., a sterile liquid transfer device), a second fluidic device 2540 (e.g., a cartridge), a robot 2560, and a sterilant switch 2592 coupled to a sterilant source (not shown). The first connector 2510 may be coupled in fluid communication with the first fluidic device 2530, and the second connectors 2520, 2521, 2522 may be coupled in fluid communication with the second fluidic device 2540.

[0231] In some variations, each of the second connectors 2520, 2521, 2522 may include a respective pneumatic sterilant port 2594, 2596, 2598 that includes a valve coupled to the distal end of the second connector 2520, 2521, 2522. In some variations, a sterilant conduit may be routed from the switch 2592 through the second fluidic device 2540 to the respective sterilant port 2594, 2596, 2598. In some variations, a sterilant source (not shown) may be coupled to the switch 2592. The switch 2592 may be coupled to each of the second connectors 2520, 2521, 2522 via the sterilant ports 2594, 2596, 2598 to facilitate an efficient and shared fluid connection between the fluidic device, the fluid connector, and the sterilization system.

[0232] The robot 2560 may include one or more end effectors 2562, 2564 configured to manipulate and / or couple to one or more of the first fluid device 2530, the first connector 2510, and the sterilant ports 2594, 2596, 2598. For example, the first fluid device 2530 may include one or more fluid ports 2550 configured to couple to the end effector 2562. Similarly, the sterilant ports 2594, 2596, 2598 may be coupled to the end effector 2562 and configured to pneumatically actuate the sterilant ports 2594, 2596, 2598. Pneumatically actuated sterilant ports may allow for the formation of sterilant conduits with a fewer number of check valves between the sterilant ports 2594, 2596, 2598 and the switch 2592.

[0233] Figure 25B is a schematic diagram of a fluid connector connection process 2502 and 2504 in which a first connector 2510 is coupled to a second connector 2520. For example, the sterilant port 2594 is in a closed valve configuration when the first connector 2510 and the second connector 2520 are separated and uncoupled 2502. Figure 25C is a detailed schematic diagram of the sterilant valve 2594. In some variations, when the first connector 2510 is coupled to the second connector 2520 and the valve 2594 is pneumatically actuated in 2504, the valve 2594 can transition to an open valve configuration.

[0234] Liquid Transfer Bath Generally, the modules of the cartridge may be fluidly coupled to one another directly or via one or more fluid transfer buses to enable transfer of one or more of cellular products (i.e., solutions containing cellular products), fluids, and reagents between the modules. In some variations, the fluid transfer buses may comprise portions of the cartridge configured to control the flow and distribution of cellular products between the modules and reservoirs. The fluid transfer buses may comprise one or more of a fluid manifold, fluid conduits (e.g., tubing), and one or more valves (including, but not limited to, 2 / 2 valves, 3 / 2 valves, 3 / 3 valves, 4 / 2 valves, and rotary selector valves).

[0235] Transfer of cell products, reagents, or fluids within the cartridge can be achieved by any pump or other structure that generates a pressure differential between fluids in one portion of the cartridge and fluids in another portion of the cartridge. For example, the cartridge can include one or more pumps, can be pre-filled with pressurized fluid contained behind a valve, and can be connected to a fluid source or fluid sink. The cartridge can include one or more mechanical pumps (e.g., linear pumps, peristaltic pumps, gear pumps, screw pumps, plunger pumps) or portions of pumps (i.e., the pumps can interface with pump actuators). External pressure can be applied to the cartridge, tubing within the cartridge, or a bag within the cartridge (i.e., applying pressure to either the liquid within the bag or the headspace gas of the bag). In some variations, the arrangement of cartridge components can facilitate gravity-based fluid transfer within the cartridge (e.g., gravity-fed pumping). While one advantage of the disclosed variations may be reduced operator intervention, the disclosed systems and methods can use manual operation within a designed workflow or as a supplement to automated operation in the case of incomplete automated system operation. For example, a process step may include manual intervention, such as input or output of a fluid. An operator may intervene in an automated process to modify device operation (e.g., manually compress a bag to force remaining fluid into the system). Fluids may include liquids and / or gases, as compressed gas, supplied externally or provided within a pressurized chamber, may be used to generate a liquid flow, e.g., the transfer of a solution containing a cell product from one module to another.

[0236] In some variations, the fluid transfer bus may be configured to deliver the cell product to each of a series of modules in an order set by the cartridge design or in an order determined by operation of the system by a processor or processors. Similarly, some variations of the cartridge may have the advantage that the order of the cell processing steps, as well as the process parameters for any of the cell therapy processing steps, may not be set by the cartridge, but rather controlled by a controller. In some variations, the fluid transfer bus may be controlled (e.g., by configuring the state of valves attached to the fluid bus) to deliver the cell product to the modules in any of a variety of sequences or to bypass one or more modules. In some variations, a module may be used more than once in the cell processing method. Optionally, the method may include performing one or more wash steps. For example, a countercurrent centrifugal elutriation (CCE) module may be used more than once. In an illustrative method, the method includes culturing a cellular product in a first bioreactor module, transferring the cellular product to a CCE module for enrichment for a desired cell type, transferring the cellular product to a second bioreactor module for a second culturing step, washing the CCE module using a washing solution, and transferring the cellular product to the CCE module for a second enrichment step.

[0237] In some variations, the liquid transfer bus or buses may be fluidly coupled to multiple bags or reservoirs used to provide solutions or reagents, store cellular products, or collect waste or reagents.

[0238] In some variations, the cartridge may include one or more pumps that may be fluidly coupled to the liquid transfer bus and / or one or more modules. The pump may include a motor operably coupled to control circuitry and a power source (e.g., a battery or an electrical connector for an off-cartridge power source). In some variations, the pump may be split into a pump on the cartridge and a pump actuator on one or more docking station modules of the system. The pump may be an opening in the cartridge, with tubing disposed around the circumference of the opening and configured to receive a pump actuator (e.g., a peristaltic rotor). By separating the pump components that contact the cellular product (i.e., tubing) from the pump components that perform the action of the cellular product (i.e., pump actuator, e.g., peristaltic rotor), the cartridge may be compact and simplified. For example, Figures 26A and 26B illustrate a pump head 2610 and pump 2610 of a cartridge in an uncoupled configuration (Figure 13A) and a coupled configuration.

[0239] In some variations, one or more pumps 146 (e.g., fluid pumps) may generate a predetermined fluid flow rate to circulate the sterilant and / or fluid. In some variations, the pumps may comprise one or more positive displacement pumps (e.g., peristaltic pumps, diaphragm pumps, syringe pumps), centrifugal pumps, combinations thereof, etc. One or more fluid sources may be coupled to the pumps.

[0240] In some variations, the pump may be configured to receive a pump signal (generated by the controller) configured to circulate the sterilant for a dwell time sufficient to sterilize at least a portion of the fluid connector. For example, the pump may be configured to circulate the sterilant for at least 10 seconds. In some variations, the pump may be configured to receive a pump signal configured to circulate a non-sterilant gas (e.g., inert gas, air) to remove the sterilant.

[0241] In some variations, a discontinuous flow pump (e.g., a peristaltic pump) may generate pulsatile flow, for example, as the tubing contracts and relaxes between rollers. In some variations, closed-loop feedback from a flow sensor may be used to compensate for the pulsatile flow and generate a substantially continuous flow rate. For example, the flow sensor may be coupled to the fluid conduit to measure the flow rate. A controller may receive the measured flow rate and generate a pump signal to the pump based on a proportional correction function configured to reduce the "ripple" measured by the flow sensor. Additionally or alternatively, the controller may apply a cyclic error correction to the pump signal to reduce cyclic errors that may be unique to each pump. For example, the flow sensor may measure and determine the cyclic error of the pump. A pump signal including the cyclic error correction may correspond to a waveform including the inverse shape of the error. The resulting pump flow may compensate for fluctuations in the flow rate.

[0242] controller In some variations, the system 100 may include a controller 120 (e.g., a computing device) that includes one or more of a processor 122, a 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 CPS 110. The controller 120 may include multiple devices. For example, the CPS 110 may encapsulate one or more components of the controller 120 (e.g., the processor 122, the memory 124, the communication device 126), while one or more components of the controller 120 may be provided remotely to the CPS 110 (e.g., the input device 128, the display 130).

[0243] Processor A processor described herein (e.g., processor 122) may process data and / or other signals to control one or more components of a system (e.g., CPS 110, controller 120). The processor may be configured to receive, process, compile, calculate, store, access, read, write, and / or transmit data and / or other signals. Additionally or alternatively, the processor may be configured to control one or more components of a device and / or one or more components of a controller (e.g., a console, touchscreen, personal computer, laptop, tablet, server).

[0244] In some variations, the processor may be configured to access or receive data and / or other signals from one or more of the CPS 110, the server, the controller 120, and a storage medium (e.g., memory, flash drive, memory card, database). In some variations, 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 rates and / or memory requirements), encryption 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, etc. The processor may be configured to run and / or execute application processes and / or other modules, processes and / or functions associated with the system.The underlying device technology can be provided in a variety of component types (e.g., metal-oxide semiconductor field-effect transistor (MOSFET) technologies such as complementary metal-oxide semiconductor (CMOS), bipolar technologies such as emitter-coupled logic (ECL), polymer technologies (e.g., silicone-conjugated polymers and metal-conjugated polymer-metal structures), mixed analog and digital technologies, etc.

[0245] The systems, devices, and / or methods described herein may be implemented by software (executed 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 (executed 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 produced by a compiler, code used to create 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.

[0246] memory The cell processing and manufacturing systems and devices described herein may include memory (e.g., memory 124) configured to store data and / or information. In some variations, the memory may include one or more of 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, combinations thereof, etc. In some variations, the memory may store instructions that cause a processor to perform modules, processes, and / or functions associated with the device, such as image processing, image display, sensor data, data and / or signal transmission, data and / or signal reception, and / or communications. Some variations described herein relate to computer storage products with non-transitory computer-readable media (which may also be referred to as non-transitory processor-readable media) having instructions or computer code for performing various computer-implemented operations. The computer-readable media (or processor-readable media) is non-transitory in the sense that it does not include transient propagating signals (e.g., propagating electromagnetic waves that carry information over a transmission medium such as space or a cable) themselves. The computer code (which may also be referred to as code or algorithms) may be designed and constructed for one or more specific purposes. In some variations, the memory may be configured to store any received data and / or data generated by the controller and / or cell processing station. In some variations, the memory may be configured to store data temporarily or permanently.

[0247] Input devices In some variations, the display may include and / or be operably coupled to an input device 128 (e.g., a touchscreen) configured to receive input data from a user. For example, user input to the input device 128 (e.g., a keyboard, buttons, touchscreen) may be received and processed by a processor (e.g., processor 122) and memory (e.g., memory 124) of the CPMS 100. The input device may include at least one switch configured to generate a user input. For example, the input device may include a touch surface for a user to provide an input corresponding to the user input (e.g., a finger touch on 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 number of touch sensitivity technologies, including capacitive, resistive, infrared, optical imaging, dispersive signal, acoustic pulse recognition, and surface acoustic wave technologies. In variations of input devices that include at least one switch, the switch may comprise, for example, at least one of a button (e.g., hard key, 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 may receive acoustic data and recognize the user's voice as user input.

[0248] In some variations, the cell processing and manufacturing system may optionally include one or more output devices, such as, for example, an audio device and a tactile device, in addition to a display. The acoustic device may audibly output any system data, alarms, and / or notifications. For example, the acoustic device may output an audible alarm when a malfunction is detected. In some variations, the acoustic device may include at least one of a speaker, a piezoelectric acoustic device, a magnetostrictive speaker, and / or a digital speaker. In some variations, a user may communicate with other users using the acoustic device and a communication channel. For example, a user may form a voice communication channel (e.g., a VoIP call).

[0249] Additionally or alternatively, the system may include a haptic device configured to provide an additional sensory output (e.g., force feedback) to the user. For example, the haptic device may generate a haptic response (e.g., vibration) to cause the input device (e.g., touch surface) to confirm the user input. As another example, the haptic feedback may indicate that the user input is overridden by the processor.

[0250] communication devices In some variations, 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 cell processing station) via a wired or wireless connection. In some variations, the system may communicate with other devices over one or more wired and / or wireless networks. In some variations, 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 wired and / or wirelessly.

[0251] A communication device may include RF circuitry configured to receive and transmit RF signals. This RF circuitry may convert electrical signals to and from electromagnetic signals and communicate with communication networks and other communication devices via electromagnetic signals. The RF circuitry may include known circuits for performing these functions, including, but not limited to, an antenna system, an RF transceiver, one or more amplifiers, a tuner, one or more oscillators, a digital signal processor, a CODEC chipset, a subscriber identity module (SIM) card, memory, etc.

[0252] Wireless communication through any of the devices may use any of a number of communication standards, protocols, and technologies, including Global System for Mobile Communications (GSM), Enhanced Data GSM Environment (EDGE), high-speed downlink packet access (HSDPA), high-speed uplink packet access (HSUPA), Evolution, Data-Only (EV-DO), HSPA, HSPA+, Dual-Cell HSPA (DC-HSPA), long term evolution (LTE), near field communication (NFC), wideband code division multiple access (W-CDMA), code division multiple access (CDMA), time division multiple access (TDMA), Bluetooth, Wireless Fidelity (WFI), and the like. Fidelity, WiFi) (e.g., IEEE802.11a, IEEE802.11b, IEEE802.11g, IEEE802.11n, etc.), voice over Internet Protocol (VoIP), Wi-MAX, protocols for email (e.g., Internet message access protocol (IMAP) and / or post office protocol (POP)), instant messaging (e.g., extensible messaging and presence protocol (XMPP), Session Initiation Protocol for Instant Messaging and Presence Leveraging Extension (SIMPLE), Instant Messaging and Presence Service (IMPS)), and / or Short Message Service (SMS), EtherCAT, OPC Unified Architecture, or any other suitable communication protocol. In some variations, devices herein may communicate directly with each other without transmitting data over a network (e.g., via NFC, Bluetooth, WiFi, RFID, etc.).

[0253] In some variations, the systems, devices, and methods described herein may communicate with other wireless devices, for example, via one or more networks, each of which may be any type of network (e.g., wired network, wireless network). Communications may be encrypted or unencrypted. A wireless network may refer to any digital network that is not connected by any type of cable. Examples of wireless communications for a wireless network include, but are not limited to, cellular, radio, satellite, and microwave communications. However, a wireless network may connect to a wired network to interface with the Internet, other carriers' voice and data networks, business networks, and personal networks. Wired networks are typically carried over copper twisted pair, coaxial cable, and / or fiber optic cable. Many different types of wired networks exist, including wide area networks (WANs), metropolitan area networks (MANs), local area networks (LANs), Internet area networks (IANs), campus area networks (MAYs), Internet-like global area networks (GANs), and virtual private networks (VPNs). Hereinafter, a network refers to any combination of wireless, wired, public, and private data networks that are typically interconnected via the Internet to provide an integrated networking and information access system.

[0254] Mobile communications may encompass technologies such as GSM, PCS, CDMA or GPRS, W-CDMA, EDGE or CDMA2000, LTE, WiMAX, and 5G networking standards. Some wireless network deployments combine networks from multiple mobile radio networks or use a mix of mobile radio, Wi-Fi, and satellite communications.

[0255] display The image data may be output on a display of the cell processing and manufacturing system, such as display 130. In some variations, the display may include at least one of a light emitting diode (LED), a liquid crystal display (LCD), an electroluminescent display (ELD), a plasma display panel (PDP), a thin film transistor (TFT), an organic light emitting diode (OLED), an e-paper / e-ink display, a laser display, and / or a holographic display.

[0256] II. Method Generally, the systems and devices described herein can perform one or more cell processing steps to produce a cell product. FIG. 28 is a flowchart of a method of cell processing 2800. Method 2800 can include concentrating a selected cell population in a solution (e.g., fluid) 2802. For example, the solution can be transferred to a cartridge CCE module via a fluid transfer bus. The cartridge can be moved by a user to a docking station of a cell processing station (CPS). The docking station can include multiple docking station modules corresponding to one or more modules of the cartridge. For example, docking The station module can include a docking station CCE module configured to interface with the cartridge CCE module to perform a cellular process on the cells in the cartridge, where the cellular process includes enriching a selected population of cells via counterflow centrifugal elutriation.

[0257] Additionally or alternatively, the cell product may be introduced into and out of the cartridge (manually or automatically) via a sterile liquid transfer port for any of the steps described herein. In some variations, the cartridge may be sterilized (either manually or automatically) within the feed-through port.

[0258] In some variations, the selected cell population in the solution can be washed 2804. For example, the solution can be transported via a fluid transfer bus to a CCE module of a cartridge in a docking station. The docking station CCE module can be operated to cause the cartridge CCE module to remove media from the solution, introduce media into the solution, and / or exchange media in the solution.

[0259] In some variations, a population of cells in a solution may be selected 2806. For example, the solution may be transported to a cartridge selection module via a fluid transfer bus. The docking station selection module may interface with the cartridge selection module, and the docking station selection module may be operated to cause the cartridge selection module to select the selected cell population.

[0260] In some variations, a population of cells in a solution can be sorted 2808. For example, the solution can be transported to a cartridge sorting module via a fluid transfer bus. The docking station sorting module can interface with the cartridge sorting module, and the docking station sorting module can be operated to cause the cartridge sorting module to sort the cell population.

[0261] In some variations, the solution may be delivered to and quiesced in the cartridge bioreactor module via a fluid transfer bus 2810. A docking station bioreactor module may interface with the cartridge bioreactor module, and the docking station bioreactor module may be operated to cause the cartridge bioreactor module to maintain the cells at a predetermined set of conditions.

[0262] In some variations, cells may be grown in solution 2812. For example, the solution may be transported to the cartridge bioreactor module via a fluid transfer bus. A docking station bioreactor module may interface with the cartridge bioreactor module and be operated to grow cells in the cartridge bioreactor module by cell replication.

[0263] In some variations, the tissue may be digested 2814 by delivering an enzymatic reagent via a fluid transfer bus to a cartridge module containing a solution containing the tissue, such that the tissue releases selected cell populations into the solution.

[0264] In some variations, the selected cell population in solution may be activated 2816 by delivering an activation reagent via a fluid transfer bus to a cartridge module containing a solution containing the cell product.

[0265] In some variations, the solution may be delivered to the cartridge's electroporation module via a fluid transfer bus and receive an electroporation signal to electroporate cells in the solution 2818. For example, a docking station electroporation module may interface with the cartridge electroporation module and be operated to cause the cartridge electroporation module to electroporate a selected cell population in the presence of the genetic material.

[0266] In some variations, an effective amount of vector may be delivered via a fluid transfer bus to a cartridge module containing a solution containing the cell product, thereby transducing a selected cell population in the solution 2820.

[0267] In some variations, the formulation solution may be delivered via a fluid transfer bus to a cartridge module containing the cell product to produce a finished cell product 2822. For example, the finished cell product may be delivered to one or more product collection bags. In some variations, finishing the cell product may include one or more of washing the cells, concentrating the cells, exchanging the cell buffer with a formulation buffer, and dispensing the cells in the formulation buffer in a predetermined amount into one or more product collection bags and / or containers.

[0268] In some variations, the cell product may be removed 2824 from the cartridge, either manually or automatically, to harvest the cells.

[0269] In some variations, the cellular product may include one or more of the following immune cells: engineered chimeric antigen receptor T cells, engineered T cell receptor (TCR) cells, hematopoietic stem cells (HSCs), and tumor infiltrating lymphocytes (TILs). In some variations, the immune cells may include natural killer (NK) cells.

[0270] A method of cell processing may include a subset of cell processing steps in any suitable order. For example, a method of cell processing may include, in order, enrichment step 2802, selection step 2806, activation step 2816, transduction step 2820, expansion step 2812, and harvesting step 2824. In some variations, a method of cell processing may include, in order, enrichment step 2802, selection step 2806, quiescence step 2810, transduction step 2820, and harvesting step 2824. In some variations, a method of cell processing may include, in order, tissue digestion step 2820, washing step 2804, activation step 2816, expansion step 2812, and harvesting step 2824. Additionally, one or more of enrichment step 2802, wash step 2804, selection step 2806, sorting step 2808, resting step 2810, growth step 2812, digestion step 2814, activation step 2816, electroporation step 2818, transduction step 2820, polishing step 2822, and removal step 2824 may be performed while the cartridge remains stationary within the docking station.

[0271] Generally, the methods described herein offload complex steps performed in a cell processing operation to a set of docking station modules, thereby reducing the cost of cartridges (which may be consumables). In some variations, the cartridge may contain a cell product (e.g., a cell-containing solution) throughout the manufacturing process, and different docking station modules interfaced with the cartridge are engaged at appropriate times to perform one or more cell processing steps. For example, a cell processing step may include transferring cells and reagents to each of the modules within the cartridge. The set of docking station modules that interface with the cartridge facilitates process flexibility, and the CPS and docking stations therein may be customized with a given set of docking station modules for a given cell therapy product. For example, the order of cell processing steps may be customized for each cell product, as described in more detail herein with respect to Figures 35-55.

[0272] In some variations, the cell product may be retained within the cartridge throughout the manufacturing process (e.g., workflow). Additionally or alternatively, the cell product may be removed from the cartridge for one or more cell processing steps, either manually by an operator or automatically via a fluidic connector or other access port system on the cartridge. The cell product may then be returned to the same cartridge, transferred to another cartridge, or split among several cartridges. In some variations, one or more cell processing steps may be performed outside of the cartridge. In some variations, processing within the CPS may facilitate sterile cell processing within the cartridge.

[0273] 29 is a flowchart of a method of cell processing illustrating cell processing steps performed on a cartridge (e.g., consumable) within a CPS, including a docking station (DS) CCE module, a sterile liquid transfer (SLT) instrument, and a DS bioreactor module. The cartridge can be configured to interface with any of the DS CCE module, the SLT instrument, and the DS bioreactor module to perform one or more cell processing steps.

[0274] In some variations, the DS CCE module may include a pump and a centrifuge configured to interface with the cartridge (e.g., consumables). The SLT instrument may include one or more fluid connectors configured to interface with one or more of the bags and bioreactor of the cartridge. The DS bioreactor module may include one or more sensors, temperature regulators, pumps, agitators, etc., and may be configured to interface with the cartridge. In some variations, the cell product may be contained within the cartridge throughout cell processing.

[0275] The method of cell processing depicted in FIG. 29 may include using a pump to move fluid in a product bag (e.g., cells in solution) to a CCE module (e.g., rotor) of a cartridge (e.g., consumable) 2910. In some variations, the fluid may be concentrated using the cartridge CCE module 2912. For example, blood components may be collected in a waste bag 2913. In some variations, the fluid may be washed using the cartridge CCE module 2914. For example, buffer may be collected in a waste bag 2915. In some variations, media may be exchanged using the cartridge CCE module 2916. For example, one or more of a buffer (e.g., formulation buffer) and media may be collected in a waste bag 2917. In some variations, the fluid may be moved to a bioreactor of the cartridge 2918.

[0276] In some variations, the fluidic connector may fill the bag with a reagent 2920. In some variations, a reagent (e.g., beads, vector) may be added to the bioreactor of the cartridge 2922. In some variations, the fluidic connector removes waste from the bag 2924. In some variations, the fluidic connector may optionally remove sample from the bioreactor.

[0277] In some variations, the cells may be moved to a bioreactor 2930. In some variations, the cells may undergo activation or genetic modification 2932. In some variations, the cells may undergo incubation 2934. In some variations, the cells may undergo perfusion using a pump 2936. For example, spent media may be collected in a waste bag 2937. In some variations, the cells may undergo proliferation 2938. In some variations, the cells may be harvested after a media change 2940.

[0278] Figure 30A is a flowchart of a method of cell processing for autologous CAR T cells or engineered TCR cells. Method 3000 may include steps of enrichment, selection, activation, genetic modification, expansion, harvesting / formulation, and cryopreservation. Figure 30B is a flowchart of a method of cell processing for allogeneic CAR T cells or engineered TCR cells. Method 3010 may include steps of enrichment, activation, genetic modification (e.g., transduction, transfection), alpha / beta T cell depletion, expansion, harvesting / pooling / formulation, and cryopreservation.

[0279] 31 is a flowchart of a method of cell processing for hematopoietic stem cell (HSC) cells. Method 3100 can include the steps of enrichment, selection, quiescence, genetic modification, harvesting / formulation, and cryopreservation.

[0280] 32 is a flowchart of a method of cell processing for tumor infiltrating lymphocyte (TIL) cells. Method 3200 may include steps of tissue digestion, washing, selection, activation, expansion, harvesting / formulation, and cryopreservation.

[0281] Figure 33 is a flowchart of a method of cell processing for natural killer (NK) CAR cells. Method 3300 may include the steps of enrichment, selection, activation, genetic modification, expansion, harvesting / formulation, and cryopreservation.

[0282] 34A to 34C show the controllability T (T reg 34 is a flowchart of a method of cell processing of (a) cells. Method 3400 may include the steps of enrichment, selection, harvesting / formulation, and cryopreservation. Method 3402 may include the steps of enrichment, selection, activation, genetic modification, expansion, selection (optional), harvesting / formulation, and cryopreservation. Method 3404 may include the steps of introducing a feeder cell culture for enrichment, selection, activation / expansion, and harvesting / irradiation. Another set of cells may be subjected to enrichment, selection, co-culture with the treated feeder cells, harvesting, and cryopreservation.

[0283] 98-101 are flowcharts of methods of cell processing for cell therapy workflows including split (e.g., parallel) processing. Method 9800 may include steps of enrichment, selection, activation, genetic modification, expansion, formulation, and cryopreservation. For example, cell processing method 9800 (e.g., workflow) may include splitting the cell product into two or more portions after an enrichment step. The portions may be processed in parallel within a single cartridge. In some variations, one or more portions may be transferred to two or more cartridges and processed in parallel. One or more cell processing parameters (e.g., timing of process steps, types of reagents added, transfection constructs, etc.) may be configured independently for each portion of the cell product. In some variations, the portions may be pooled after an expansion step.

[0284] The method 9900 may include steps of enrichment, selection, activation, genetic modification, expansion, formulation, and cryopreservation. For example, the cell processing method 9900 (e.g., workflow) may include dividing the cell product into two or more portions after the activation step. The portions may be processed in parallel within a single cartridge. One or more cell processing parameters (e.g., timing of process steps, types of reagents added, transfection constructs, etc.) may be configured independently for each portion of the cell product. In some variations, the portions may be pooled after the expansion and / or genetic modification steps.

[0285] Method 10000 may include steps of enrichment, selection, activation, genetic modification, expansion, formulation, and cryopreservation. For example, cell processing method 10000 (e.g., workflow) may include splitting the cell product into two or more portions after a selection step. The portions may be processed in parallel within a single cartridge. One or more cell processing parameters (e.g., timing of process steps, type of reagent added, transfection construct, etc.) may be configured independently for each portion of the cell product. In some variations, the portions may not be pooled.

[0286] Method 10100 may include steps of enrichment, selection, activation, genetic modification, expansion, formulation, and cryopreservation. For example, cell processing method 10100 (e.g., workflow) may include splitting a cell product into two or more portions as a starting material. The separate products may be processed in parallel, remaining separated as aliquots within a single cartridge. One or more cell processing parameters (e.g., timing of processing steps, types of reagents added, transfection constructs, etc.) may be configured independently for each aliquot. In some variations, the aliquots may be pooled after the expansion step.

[0287] Figure 102 is a schematic diagram of a cell processing and manufacturing system 10200 configured for split processing within a single cartridge. For example, methods 9800-10100 described with respect to Figures 98-101 may be performed within a cartridge 10210. In some variations, the system 10200 may include a sterile liquid transfer device 10220 containing a reagent 10222, and the cartridge 10210 including multiple bioreactor modules 10230, a pump module 10240, a thermal module 10245, a pressure-driven flow module 10250, a MACS module 10255, an electroporation module 10260, a FACS module 10265, a CCE module 10270, and a blank module 10280. The cartridge 10210 may further include a reagent reservoir 10285, multiple product bags 10290, and a liquid transfer bus 10295. The liquid transfer bus 10295 can be configured to couple the components of the cartridge 10210 for fluid communication.

[0288] In some variations, loading and removal of the cell product into and from the cartridge can be performed on-system or off-system. In some variations, the cartridge is loaded at the patient's or donor's bedside and then delivered to a cell processing and manufacturing system in or near a hospital or shipped to a facility where the cell processing and manufacturing system is located. Similarly, the cell product can be removed from the cartridge after processing at a facility or near the intended recipient (patient) of the cell product. Optionally, the cell product is frozen before, during, or after the disclosed methods, optionally after adding one or more cryoprotectants to the cell product. In some variations, the system includes a freezer and / or a liquid nitrogen source. In some variations, the system includes a water bath or warming chamber containing gas at a controlled temperature (e.g., a water bath set between about 20°C and about 40°C) to enable controlled thawing of the cell product. In some variations, the cartridge is made of a material that resists mechanical damage when frozen.

[0289] Automated Cell Processing Described herein are methods for translating user-defined cell processing operations into cell processing steps using the automated cell processing and manufacturing systems and devices described herein. In some variations, cell processing operations are received and translated into cell processing steps to be performed by the system given a set of predetermined constraints. For example, a user may input a set of biological process steps and corresponding biological process parameters to be performed by the cell processing and manufacturing system. Optionally, the process parameters may be customized for each cartridge or set of cartridges.

[0290] FIG. 35 is a flowchart generally describing a variation of a method for automated cell processing. Method 3500 may include receiving an ordered input list of cell processing operations 3502. For example, more than one ordered input list set of cell processing operations may be received to be performed on more than one cartridge on an automated cell processing and manufacturing system. For example, as shown in GUI 4900 of FIG. 49 and described in more detail herein, one or more biological process inputs (e.g., available operations), such as enrichment, MACS selection, activation, transduction, transfection, proliferation, and in-line analysis, may be selected as an ordered input list of cell processing operations. Additionally, GUI 5200 of FIG. 52 illustrates a complete ordered input list of cell processing operations (e.g., a set of selected operations) 5220 selected by a user.

[0291] In some variations, one or more sets of cell processing parameters may be received 3504. Each set of cell processing parameters may be associated with one of the cell processing operations. Each set of cell processing parameters may specify characteristics of the cell processing step to be performed by the docking station module in that cell processing step. For example, GUI 4000 of FIG. 40 illustrates reagent and container parameters, GUI 4200 of FIG. 42 illustrates example process parameters, GUI 4400 of FIG. 44 illustrates example pre-processing analysis, and GUI 4800 of FIG. 48 illustrates an example set of activation settings.

[0292] In some variations, the transformation model may be executed on an ordered input list 3506. In some variations, the transformation model may include constraints on the ordered output list determined by a given configuration of the automated cell processing and manufacturing system. For example, the constraints may include information about the configuration of the automated cell processing and manufacturing system.

[0293] In some variations, the constraints may include one or more of the type and / or number and / or status of the docking station modules, the type and / or number and / or status of the modules on the cartridge, the type and / or number of reservoirs on the cartridge, the type and / or number of sterile liquid transfer ports on the cartridge, and the number and location of fluid paths between the modules, reservoirs, and sterile liquid transfer ports on the cartridge.

[0294] In some variations, a set of predetermined constraints may be placed on the set of process control parameters. For example, the amount and / or type of reagent used may be constrained based on the size of the system and / or the product being manufactured. Other process parameter constraints may include, but are not limited to, one or more of temperature, volume, time, pH, cell size, cell count, cell density, cell viability, dissolved oxygen, glucose level, on-board reagent storage and waste volume, combinations thereof, etc. For example, GUI 4000 in FIG. 40 illustrates that the reagents have a per-unit volume of 30 ml and a required volume of 54 ml, and the consumable container has a per-unit volume of 75 ml. GUI 4800 in FIG. 48 illustrates that the activation concentration is 12 mg / L, the activation incubation time is 1600 seconds, the activation temperature is 18°C, and the gas mixture includes 21% oxygen, 78.06% nitrogen, and 0.04% carbon dioxide. These constraints can be applied by the transformation model to generate an ordered output list of cell processing steps that affect how one or more of the robot, docking station module, cartridge, and cartridge module are operated and how the cell product is produced.

[0295] In some variations, the order of operations may be constrained based on hardware constraints.

[0296] In some variations, the product loading operation must be the first operation performed and may be performed once for each process, as illustrated in GUI 4900 of Figure 49. The fill and finish operation may always be the last operation performed before product completion and may be performed once for each process.

[0297] In some variations, the system may prevent a user from performing a set of actions in an order that may not be enforced by the system.

[0298] In some variations, a notification (e.g., a warning, an alert) may be output when a user commands a set of actions in a "non-standard" manner. For example, a notification may be output when the same type of action is repeated sequentially (e.g., enrichment immediately followed by enrichment). Similarly, a notification may be output when an action (e.g., selection, activation) is used more than once within a given process, even though such an action is typically used only once within a given process.

[0299] In some variations, the output of the transformation model may correspond to an ordered output list of cell processing steps that can be performed by the system 3508. For example, the transformation model may be run on an ordered set of input lists to produce an ordered output list of cell processing steps. The output list of cell processing steps may control a robot, a cartridge, a cartridge module, and one or more docking station modules.

[0300] In some variations, the ordered output list is further implemented by the system to control 3512 one or more of the docking station modules to perform one or more cell processing steps on one or more cellular products in the respective cartridges. For example, the compute server rack 210 (e.g., controller 120) may be configured to control a cartridge electroporation module 220 configured to apply a pulsed electric field to a cell suspension in the cartridge 250. In some variations, the ordered output list may include instructions for a docking station module (e.g., a bioreactor) to process a product (e.g., transfer a cellular product from a small cartridge bioreactor module to a large cartridge bioreactor module). Furthermore, the docking station module may be further configured to operate under a set of process parameters (e.g., 9-hour duration, pH 6.7, temperature 37.3°C to 37.8°C, mix mode 3). As another example, the ordered output list may include instructions for operating a sterile liquid transfer module to perform one or more of removing waste from the cartridge, adding media to the cartridge, and adding MACS reagents to the cartridge.

[0301] In some variations, one or more electronic batch records may be generated based on the process parameters and data collected from sensors during process execution 3514. The batch records generated by the system may include process parameters, time logs, sensor measurements from the docking station module, QC parameters determined by QC instrumentation, and other records.

[0302] FIG. 36 is a flowchart generally illustrating variations of a method 3600 for executing a transformation model. In some variations, one or more biological functions may be generated and output to a user. For example, a set of configurable biological function blocks may be displayed on a graphical user interface for user selection. The GUI may allow a user to select and order the biological function blocks and define biological control parameters. One or more control parameters of the biological function blocks may be modified by the user, if desired. In some variations, one or more biological function templates may be generated that include a predetermined sequence of biological function blocks. One or more biological control parameters of the biological function templates may be modified by the user, if desired.

[0303] In some variations, the cell processing and manufacturing system may be configured to receive and / or store one or more biological function (e.g., process) inputs from a user 3604. For example, a user may select one or more pre-defined biological function templates.

[0304] In some variations, a biological process model (e.g., process definition) may be generated based on the biological process inputs 3606. In some variations, the biological process model may include one or more of enrichment, isolation, MACS selection, FACS selection, activation, genetic modification, gene transfer, transduction, transfection, expansion, formulation (e.g., harvesting, pooling), cryopreservation, T cell depletion, resting, tissue digestion, washing, irradiation, co-culture, combinations thereof, and the like.

[0305] In some variations, the biological process model may be transformed into a docking station (DS) module execution process model 3608. For example, each biological function block in the biological process model may correspond to an ordered list of cell processing and manufacturing system operations with corresponding hardware control parameters. The DS module execution process model may include a sequence of hardware operations corresponding to the biological process model. As described herein, the transformation model may include one or more constraints.

[0306] Optionally, in some variations, the cell processing and manufacturing system may be configured to receive and / or store one or more DS module execution process inputs from a user 3610. For example, a user may modify the converted DS module execution process model if desired. The user may select particular hardware components to perform particular steps, change timing parameters, etc.

[0307] In some variations, the DS module execution process may be executed to produce a cellular product 3612. For example, a cellular processing and manufacturing system at runtime may process a cellular product through the system as defined by a DS module execution process model.

[0308] In some variations, a DS module execution process may be executed 3612. In some variations, the DS module execution process model may be converted back to a biological process model 3614. This progress of the biological process model may be output (e.g., displayed) to a user for monitoring. For example, the DS module execution process model may include one or more references (e.g., pointers) back to the biological process model, so that runtime execution progress can be reported to the biological process model.

[0309] In some variations, cellular products may be monitored 3616. For example, GUIs 5300 and 5400 in Figures 53 and 54, respectively, illustrate sensor data monitored by the system for multiple products. For example, the number of viable cells and the status of the process (e.g., as a function of percentage complete) may be illustrated graphically for the user.

[0310] In some variations, electronic records may be generated based on the monitored data 3618. For example, one or more electronic batch records may be generated in accordance with, for example, 21 CFR regulations.

[0311] Graphical User Interface In some variations, a graphical user interface (GUI) may be configured for designing a process and monitoring products. FIG. 37 is a variation of a GUI 3700 that includes an initial process design interface. For example, GUI 3700 may be a process design home page. GUI 3700 may indicate that no process has been selected or loaded. A create icon 3710 (e.g., "Create a Process") may be selectable by a user to begin the process design process. In some variations, one or more of the GUIs described herein may include a search bar.

[0312] Figure 38 is a variation of a GUI 3800 for creating a process. The GUI 3800 may be displayed following selection of the create icon 3710 of Figure 37. For example, the GUI 3800 may include a process creation window 3810 that allows a user to enter and / or select one or more of a process name, a process description, and a template. In some variations, the user may select from a list of pre-defined templates. For example, the user may create a process and save it as a template for later selection.

[0313] 39 is a variation of GUI 3900, including that associated with an empty process. GUI 3900 may be displayed following confirmation in GUI 3800 that a process will be created. GUI 3900 may show the process name (e.g., Car T Therapy) and highlight a process setup icon 3910, allowing process-specific parameters such as process reagents and containers, process parameters, and pre-processing analyses to be added. GUI 3900 may further include an add process reagents and containers icon 3920, an add process parameter icon 3930, and an add pre-processing analysis icon 3940. Once the process setup is complete, one or more process elements may be specified.

[0314] In some variations, the GUI 3900 may include one or more pre-defined templates for a set of biological processes (e.g., CAR-T, NK cells, HSC, TIL, etc.). For example, the templates may aid in process development and validate starting points for process development. The templates may be further modified (e.g., customized) based on the user's requirements.

[0315] FIG. 40 illustrates a variation of a GUI 4000, including those relating to adding reagents and consumable containers. The GUI 4000 may be displayed following selection of the add process reagents and containers icon 3920 of FIG. 39. For example, the GUI 4000 may include an add reagents and containers window 4010 that allows a user to input and / or select one or more reagents, including reagent type, manufacturer, part number, volume per unit, required volume, and required reagent inputs (e.g., lot number, expiration date, required container transfer). The add reagents and containers window 4010 may include one or more input fields, selection boxes, drop-down selectors, etc. Furthermore, the add reagents and containers window 3810 may allow a user to input and / or select one or more consumable containers, including manufacturer, part number, volume per unit, and required container inputs (e.g., lot number, expiration date). In some variations, a user may select from a list of pre-defined templates. For example, a user may create a process and save it as a template.

[0316] FIG. 41 is a variation of a GUI 4100 that includes association with a process parameter. The GUI 4100 may be displayed following selection of the add process reagents and containers icon 3930 of FIG. 39. For example, the GUI 4100 may include an add process parameter window 4110 that allows a user to enter and / or select one or more parameters, including a name, parameter identification, description, data type, units, and parameter type. The add process parameter window 4110 may include one or more of an input field, a selection box, a drop-down selector, or the like. In some variations, the user may select from a list of pre-defined templates. For example, the user may create a parameter and save it as a template. FIG. 42 is a variation of a GUI 4200 that includes association with a patient weight process parameter. For example, the GUI 4200 may include an add process parameter window 4110 filled in with parameter information, including patient weight, data type (e.g., integer), units (e.g., kg), and parameter type (e.g., input).

[0317] FIG. 43 is a variation of a GUI 4300 for a preprocessing analysis. The GUI 4300 may be displayed following selection of the add preprocessing analysis icon 3940 of FIG. 39. For example, the GUI 4300 may include an add preprocessing analysis window 4310 that allows a user to enter and / or select one or more parameters, including a name, identifier, description, data type, and display group. The add preprocessing analysis window 4310 may include one or more of an input field, a selection box, a drop-down selector, or the like. In some variations, the user may select from a list of predefined templates. For example, the user may create parameters and save them as a template.

[0318] 44 is a variation of a GUI 4400 for a white blood cell count preprocessing analysis. For example, the GUI 4400 may include an add preprocessing analysis window 4410 that is populated with preprocessing analysis information, including a name (e.g., CBC white blood cell count), an identifier (e.g., CBC white blood cell count), a description (e.g., the number of white blood cells in the sample), a data type (e.g., float), and a display group (e.g., WBC).

[0319] FIG. 45 is a variation of a GUI 4500 for process parameter calculations. The GUI 4500 may be displayed following selection of the add preprocessing analysis icon 3940 of FIG. 39 and selection of the “calculation” parameter type. For example, the GUI 4500 may include an add preprocessing analysis window 4510 that allows a user to enter and / or select one or more parameters, including a name, identifier, description, data type, display group, units, and parameter type. Additionally, a calculation builder may allow a user to define a formula (e.g., algorithm, equation) for performing a given calculation. For example, the calculation builder may include one or more of a set of available parameters (e.g., patient weight), constant values, expressions, and operands.

[0320] 46 is a variation of a GUI 4600 for a completed process setup. For example, the GUI 4600 may include a process setup window 4610 that lists process reagents, containers, process parameters, and pre-processing analyses. Once the process setup is complete, one or more process elements may be specified.

[0321] Figure 47 is a variation of a GUI 4700 for process operation activation settings. GUI 4700 may be displayed following selection of process element icon 4620 of Figure 46. For example, GUI 4700 may include an activation settings window 4710 that allows a user to enter and / or select one or more of an activation concentration (e.g., mg / L), an activation incubation time (e.g., seconds), an activation temperature (e.g., °C), and a gas mixing mode. In some variations, a user may select from a list of pre-defined templates. For example, a user may create a set of activation settings and save it as a template for later selection.

[0322] 48 is a variation of a GUI 4800 associated with completing process operation activation settings. For example, GUI 4800 may include an activation settings window 4810 in which activation setting information is entered. In some variations, a set of gases (e.g., O, N, CO) and corresponding concentrations may be defined.

[0323] 49 is a variation of a GUI 4900 associated with a process operations interface. GUI 4900 may include an available operations window 4910 and a selected operations window 4920. The available options for selection may include one or more biological process inputs as described herein, including, but not limited to, enrichment, MACS selection, activation, transduction, transfection, proliferation, and in-line analysis. One or more of the operations may be selected and dragged into the selected operations window 4920. The selected operations may be reordered within the selected operations window 4920.

[0324] 50 is a variation of a GUI 5000 for dragging process actions. GUI 5000 may include an available actions window 5010, a selected actions window 5020, and a selected (e.g., dragged) action 5030 that can be dragged and dropped between available actions window 5010 and selected actions window 5020. Selected actions window 5020 may include multiple selected actions.

[0325] 51 is a variation of a GUI 5100 for dragging process actions. GUI 5100 may include an available actions window 5110, a selected actions window 5120, and a selected (e.g., dragged) action 5130 that can be dragged and dropped between available actions window 5110 and selected actions window 5120. Selected actions window 5120 may include multiple selected actions.

[0326] 52 is a variation of a GUI 5200 associated with completed process actions. For example, the GUI 5200 may include an available actions window 5210 and a selected actions window 5220 containing the completed set of selected actions. In some variations, the settings (e.g., parameters) of each action may be selectively modified by the user by selecting a corresponding icon (e.g., a gear icon).

[0327] 53 and 54 illustrate variations of GUIs 5300 and 5400 related to product monitoring. GUIs 5300 and 5400 may include respective monitoring windows 5310 and 5410. For example, GUI 5310 may monitor multiple products 5320 and output one or more product characteristics 5330, including, but not limited to, a summary, process data, online analysis, imaging, a process audit log, process parameters, and a process schedule. Monitoring window 5410 may monitor one or more product characteristics of one or more products. For example, the product characteristics may include, but are not limited to, one or more of the process name, identification, process identification, progress, estimated completion, current step, and message.

[0328] Figure 77A is a flowchart of a method 7700 of separating cells using a cartridge CCE module. Figure 77B is a flowchart of a method 7710 of concentrating cells using a cartridge CCE module. Figure 77C is a flowchart of a method 7720 of buffer exchange using a cartridge CCE module.

[0329] FIG. 78 is a flowchart of a method 7800 of separating cells. The method 7800 of counterflow centrifugal elutriation (CCE) may include moving a rotor toward a magnet 7802. The rotor may define an axis of rotation. In some variations, moving the rotor includes advancing and retracting a magnet relative to the rotor using a robot. The rotor may optionally be moved toward an illumination source and an optical sensor 7804. A fluid may be flowed through the rotor 7806. In some variations, flowing the fluid includes a flow rate of up to about 150 ml / min while rotating the rotor. The rotor may be magnetically rotated about the axis of rotation using a magnet 7808 while flowing the fluid through the rotor. In some variations, rotating the rotor includes a rotational speed of up to 6,000 RPM. One or more of the fluid and the cells may optionally be illuminated using an illumination source 7810. Image data of one or more of the fluid and biological material (e.g., particles, cellular material) within the rotor may optionally be generated using an optical sensor 7812. One or more of a rotational speed of the rotor and a flow rate of the fluid may optionally be selected based at least in part on the image data 7814. The fluid may be drained from the rotor 7816. The rotor may be moved away from the magnet 7818. The rotor may optionally be moved away from the illumination source and the optical sensor 7820.

[0330] Figure 79A is a flow chart of a closed-loop method 7900 for separating cells. Figure 79B is a flow chart of a closed-loop method 7910 for elutriating cells. Figure 79C is a flow chart of a closed-loop method 7920 for harvesting cells.

[0331] Figure 80A is a flowchart of a method 8000 for isolating cells. Figure 80B is a flowchart of a method 8010 for selecting cells.

[0332] FIG. 81 is a flowchart of a method 8100 for separating cells. The method of magnetically activated cell selection (MACS) may include labeling cells with a reagent 8102. In some variations, the magnetically activated cell selection (MACS) reagent may be incubated with the input cells to label the set of cells with the MACS reagent. In some variations, the step of incubating the MACS reagent includes a temperature of about 1° C. to about 10° C. A fluid containing the input cells may be flowed into a flow cell 8104. The set of cells is labeled with the MACS reagent. In some variations, a magnet array may optionally be moved relative to the flow cell 8106. In some variations, the set of cells may be magnetically attracted toward the magnet array for a dwell time 8108. In some variations, the dwell time may be at least about 1 minute. In some variations, the magnet array may be disposed outside the flow cell. In some variations, the longitudinal axis of the flow cell is perpendicular to the ground. In some variations, the flow cell may be devoid of beads. In some variations, the magnet array may optionally be moved away from the flow cell to facilitate flowing the set of cells from the flow cell 8110. The set of cells may be flowed out of the flow cell after a residence time 8112. For example, flowing the set of cells from the flow cell may include flowing a gas through the flow cell. The fluid without the set of cells may optionally be flowed out of the flow cell after a residence time 8114.

[0333] Figure 82A is a flowchart of a method 8200 of preparing a bioreactor. Figure 82B is a flowchart of a method 8210 of loading a bioreactor. Figure 82C is a flowchart of a method 8220 of preparing a bioreactor. Figure 82D is a flowchart of a method 8230 of calibrating a bioreactor. Figure 82E is a flowchart of a method 8240 of mixing reagents. Figure 82F is a flowchart of a method 8250 of mixing reagents. Figure 82G is a flowchart of a method 8260 of culturing cells. Figure 82H is a flowchart of a method 8270 of freezing cells. Figure 821 is a flowchart of a method 8270 of taking samples. Figure 82J is a flowchart of a method 8280 of culturing cells. Figure 82K is a flowchart of a method 8290 of medium exchange. Figure 82L is a flowchart of a method 8292 of controlling gas. Figure 82M is a flowchart of a method 8294 of controlling pH.

[0334] 83 is a flowchart of a method 8300 of electroporating cells using a cartridge electroporation module. In some variations, the cartridge electroporation module may include fluid conduits configured to receive a first fluid and a second fluid containing cells, a set of electrodes coupled to the fluid conduits, a pump coupled to the fluid conduits, and a controller comprising a processor and a memory.

[0335] The method of electroporating cells may optionally include generating a first signal to introduce a first fluid into a fluid conduit using a pump 8302. The first fluid containing cells in the fluid conduit may be received 8304. In some variations, a second signal may optionally be generated to introduce a second fluid into the fluid conduit such that the second fluid separates the first fluid from a third fluid 8306. In some variations, the second fluid may include a gas or oil. The second fluid in the fluid conduit may be received 8308 to separate the first fluid from the third fluid. An electroporation signal may optionally be generated to electroporate the cells in the fluid conduit using a set of electrodes 8310. The electroporation signal may be applied to the first fluid to electroporate the cells 8312. In some variations, the first fluid may be substantially static when applying the electroporation signal. In some variations, a third signal may optionally be generated to introduce a third fluid into the fluid conduit 8314. The third fluid may be separated from the first fluid by a second fluid. The third fluid may optionally be received in the fluid conduit separated from the first fluid by the second fluid 8316.

[0336] Figure 84 is a flowchart of a method 8400 of electroporating cells. The method of electroporating cells may include receiving a first fluid containing cells in a fluid conduit 8402. A resistance measurement signal may be applied to the first fluid using a set of electrodes 8404. Resistance between the first fluid and the set of electrodes may be measured 8406. An electroporation signal may be applied to the first fluid based on the measured resistance 8408. In some variations, a second fluid containing a gas may optionally be received in the fluid conduit before applying the electroporation signal to the fluid. The first fluid may be separated from a third fluid by the second fluid.

[0337] Fluid Connector A method 2700 of transferring fluids using a fluid connector is described in the flowchart of FIG. 27 and illustrated generally in the corresponding steps depicted in FIGS. 16B-16L. The method 2700 may include a step 2702 of coupling a sterilant source to the fluid connector. For example, as shown in FIG. 16B, an inlet 1652 and an outlet 1654 are coupled to the sterilant source to form a fluid pathway or connection. In some variations, the robot may be configured to couple and uncouple the sterilant source to the sterilant port 1650 using a fluid conduit, such as a tube. In some variations, the fluid connector 1600 may include multiple sterilant ports 1650. As described herein, in some variations, the sterilant port may optionally include one or more of a check valve and a particle filter configured to reduce the ingress of debris (e.g., after disconnecting the fluid connector). In some variations, the sterilant source may include or be coupled to a pump configured to circulate the sterilant through the sterilant port 1650. In some variations, the sterilant port 1650 may be coupled to one or more of a fluid source, such as a sterilant source and a heated air source. For example, the first sterilant port may be configured to couple to a first sterilant source, the second sterilant port may be configured to couple to a second sterilant source, and the third sterilant source may be configured to couple to an air source.

[0338] The separate portions of the fluid connector 1600 may be brought together and mated. The method 2700 may include coupling a first port of a first connector to a second port of a second connector 2704. FIG. 16C is a schematic illustration of the fluid connector 1600 with the first port 216 and the second port 226 in a mated configuration (e.g., a docked position) forming a first seal. In some variations, the first connector 1610 and the second connector 1620 may be axially and / or rotationally aligned, and one or more of the connectors 1610, 1620 may be translated to couple the connectors 1610, 1620 together. In FIG. 16C, the first port 1616 and the second port 1626 are each in a closed configuration, and the lumens of the respective first connector 1610 and second connector 1620 are sealed from the external environment, maintaining sterility of the lumens of the fluid connector 1600. Additionally, the first valve 1618 and the second valve 1628 are each in a closed configuration, sealing the proximal and distal ends of the connector from one another. For example, the first valve 1618 in the closed configuration forms a seal (e.g., a barrier) between the first proximal end 1612 and the first distal end 1614. Similarly, the second valve 1628 in the closed configuration forms a seal between the second proximal end 1622 and the second distal end 1624. In this manner, even if one portion of the connector (e.g., the first distal end 1614) becomes contaminated, other portions of the fluid connector 1600 (e.g., the first proximal end 1612, the second connector 1620) can remain sterile by one or more of the port seals and valve seals.

[0339] The ports can be transitioned to an open configuration so that the distal ends of the connectors can be in fluid communication. The method 2700 can include transitioning the ports to the open configuration 2706. Figure 16D is a schematic illustration of the fluid connector 1600 in which the first port 1616 and the second port 1626 are transitioned to an open port configuration to create a shared volume between the valves 1618, 1628 that is isolated from the external environment. In Figure 16D, the first valve 1618 and the second valve 1628 are in a closed configuration such that the chamber 1615 defines a volume (e.g., a cavity) of the fluid connector 1600 between the first valve 1618 and the second valve 1628. That is, the first distal end 1614 is in fluid communication with the second distal end 1624. The ports 1616, 1627 can be received and / or retained within their respective housings 1617, 1627 in the closed configuration. In some variations, the robot may be configured to transition the ports 1616, 1626 between an open configuration and a closed configuration, as described in more detail herein. Additionally or alternatively, the first port 1616 and the second port 1626 may automatically transition (e.g., mechanically actuated) from the closed configuration to the open configuration upon mating of the first port 1616 with the second port 1626.

[0340] In some variations, a fluid may be flowed into the fluid connector to aid in sterilization. The method 2700 may include flowing a fluid (e.g., liquid, gas) into the fluid connector through a sterilant port 2708. FIG. 16E is a schematic diagram of a fluid connector 1600 in which a first chamber 1615 receives a fluid, such as air, at a predetermined temperature, pressure, and / or humidity. In some variations, one or more portions of the fluid connector 1600 may be dehumidified. For example, pressurized hot air may optionally be circulated within the chamber 1615 to remove residual fluid, moisture, and increase the temperature of the interior surfaces of the chamber 1615. The circulating fluid may flow through the housings 1617, 1627 and over the interior and / or exterior surfaces of the ports 1616, 1626.

[0341] Generally, sterilization of a fluid connector may include one or more steps of dehumidification, conditioning, decontamination, and aeration (e.g., ventilation). Dehumidification may include removing moisture from the fluid connector. Conditioning may include heating the surface of the fluid connector to be decontaminated to prevent condensation and aid in sterilization. Decontamination may include circulating a sterilant through the fluid connector at a predetermined concentration, rate, and exposure time. Aeration may include removing the sterilant from the fluid connector by circulating a gas (e.g., sterile air) through the fluid connector.

[0342] A sterilant can be flowed into the fluid connector to sterilize one or more portions of the fluid connector. As described in more detail herein, the sterilant can be, for example, vaporized hydrogen peroxide (VHP) and / or ionized hydrogen peroxide (IHP). The method 2700 can include flowing the sterilant into the fluid connector through a sterilant port 2710. FIG. 16F is a schematic diagram of a fluid connector 1600 in which a first chamber 1615 receives a sterilant for a predetermined time (e.g., a dwell time). For example, the sterilant can be circulated within the chamber 1615 to sterilize the chamber 1615 of the fluid connector 1600 and any contents disposed therein (e.g., other fluids, biological material). In some variations, the dwell time can be up to about 10 minutes, from about 1 minute to about 10 minutes, including all ranges and subvalues ​​therebetween. In some variations, the vaporized hydrogen peroxide may comprise a concentration of about 50% to about 70%, including all ranges and subvalues ​​the...

Claims

1. 1. An automated system for cell processing, comprising: a plurality of cartridge modules including a bioreactor module, a counterflow centrifugation elutriation module, and at least one of an electroporation module, a magnetic-activated cell selection module, a fluorescence-activated cell selection module, or a spinoculation module; at least one sterile fluid transfer port; and a cartridge including a liquid transfer bus fluidly coupled to each cartridge module; an automated system comprising: a docking station comprising a plurality of docking station modules corresponding to the plurality of cartridge modules, each of the plurality of docking station modules being independently configured to cooperate with a respective cartridge module to perform one or more cell processing operations on the cartridge, the docking station being sized and shaped to receive a single cartridge.

2. 10. The system of claim 1, wherein the liquid transfer bus is fluidly coupled to each cartridge module by a fluid conduit disposed between a port on the liquid transfer bus and a respective port on each cartridge module.

3. The system of claim 2 , wherein the fluid conduit comprises a tube or channeling.

4. The system of claim 1 , further comprising a sterile fluid transfer device configured to facilitate the transfer of fluid between the cartridge and a closed volume fluidic device.

5. The system of claim 4 , wherein the closed volume fluidic device is a fluid container.

6. The system of claim 4 , wherein the sterile liquid transfer device comprises at least one actuator.

7. The system of claim 6 , wherein the actuator actuates one of the at least one sterile liquid transfer port of the cartridge and a corresponding sterile liquid transfer port of a closed volume fluidic device.

8. The system of claim 7 , further comprising a fluid reservoir disposed within a housing of the system, the fluid reservoir being accessible by a user for inserting and / or removing one or more closed volume fluidic devices.

9. The system of claim 1 , wherein each of the one or more cell processing operations performed on the cartridge is performed on cells within the cartridge.

10. The system of claim 1 , wherein the plurality of cartridge modules comprises the electroporation module.

11. The system of claim 1 , wherein the plurality of cartridge modules comprises the magnetically activated cell selection module.

12. The system of claim 1 , wherein the plurality of cartridge modules comprises the fluorescence-activated cell selection module.

13. The system of claim 1 , wherein the plurality of cartridge modules comprises the spinoculation module.

14. The system of claim 1 , wherein the plurality of docking station modules comprises a corresponding bioreactor module.

15. The system of claim 1 , wherein the plurality of docking station modules comprises a corresponding magnetically activated cell selection module.

16. The system of claim 1 , wherein the plurality of docking station modules comprises a corresponding fluorescence-activated cell selection module.

17. The system of claim 1 , wherein the plurality of docking station modules comprises a corresponding electroporation module.

18. The system of claim 1 , wherein the plurality of docking station modules include corresponding countercurrent centrifugal elutriation modules.

19. The system of claim 1 , wherein the plurality of docking station modules comprises a corresponding spinoculation module.

20. The system of claim 1 , wherein the cartridge comprises a pump fluidly coupled to the liquid transfer bus.

21. 21. The system of claim 20, further comprising a pump actuator configured to interface with the pump.

22. The system of claim 1 further comprising a waste reservoir for capturing waste generated by the cartridge.

23. The system of claim 4 , wherein the transfer of liquid between the cartridge and the closed volume fluidic device comprises extraction of at least a portion of a cell processing sample from the cartridge.

24. 1. An automated cell processing method comprising: performing at least two cell processing operations in a cartridge positioned in a docking station, the cartridge comprising a plurality of cartridge modules, at least one sterile liquid transfer port, and a liquid transfer bus fluidly coupled to each cartridge module, the docking station comprising a plurality of docking station modules corresponding to the plurality of cartridge modules and sized and shaped to receive a single cartridge; the plurality of cartridge modules comprising a bioreactor module, a counterflow centrifugation elutriation module, and at least one of an electroporation module, a magnetic-activated cell selection module, a fluorescence-activated cell selection module, or a spinoculation module; An automated cell processing method, wherein said cell processing operations are automatic upon execution of a received set of instructions.

25. 25. The method of claim 24, further comprising activating one of the at least one sterile liquid transfer port of the cartridge and a corresponding sterile liquid transfer port of a closed volume fluidic device to facilitate liquid transfer therebetween.

26. 26. The method of claim 25, wherein facilitating the transfer of liquid between the cartridge and the closed volume fluidic device comprises activating the one of the at least one sterile liquid transfer port and the corresponding sterile liquid transfer port.

27. 26. The method of claim 25, wherein the transfer of liquid between the cartridge and the closed volume fluidic device comprises extraction of at least a portion of a cell processing sample from the cartridge.

28. 25. The method of claim 24, wherein the plurality of cartridge modules comprises the electroporation module.

29. 25. The method of claim 24, wherein the plurality of cartridge modules comprises the magnetically activated cell selection module.

30. 25. The method of claim 24, wherein the plurality of cartridge modules comprises the fluorescence-activated cell selection module.

31. The method of claim 24, wherein the plurality of cartridge modules comprises the spinoculation module.

32. 25. The method of claim 24, wherein the plurality of docking station modules comprises a corresponding bioreactor module.

33. 25. The method of claim 24, wherein the plurality of docking station modules comprises corresponding counter-flow centrifugal elutriation modules.

34. 25. The method of claim 24, wherein the plurality of docking station modules comprises a corresponding electroporation module.

35. 25. The method of claim 24, wherein the plurality of docking station modules comprises a corresponding magnetically activated cell selection module.

36. 25. The method of claim 24, wherein the plurality of docking station modules comprises a corresponding fluorescence-activated cell selection module.

37. The method of claim 24 , wherein the plurality of docking station modules includes a corresponding spinoculation module.

38. 25. The method of claim 24, wherein the two or more cell treatments performed in the cartridge are performed on cells within the cartridge.

Images