Systems, devices, and methods for cell processing

Abstract

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

Description

[0001] Incorporated by reference

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 427,720, filed on November 23, 2022, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The devices, systems, and methods herein relate to the use of automated systems to manufacture cellular products for biomedical applications. Background Art

[0004] Typically, cell therapies based on hematopoietic stem cells (HSC), chimeric antigen receptor (CAR) T cells, NK cells, tumor infiltrating lymphocytes (TIL), T cell receptors (TCR), regulatory T cells (Treg), γδT cells, etc. rely on the manufacture of cell products. Cell product manufacturing generally involves multiple cell processing steps. Conventional solutions for cell product manufacturing may rely on cumbersome manual operations performed in expensive biosafety cabinets and / or clean rooms. However, skilled laboratory technicians, sufficiently sterile housings (such as clean room facilities), and the design and use of relevant protocols and procedures for regulated (GMP) manufacturing may be expensive. Many current manufacturing processes can also adopt many manual reagent preparations and instrument manipulation steps during the manufacturing protocol, and these processes may take several days or even weeks. However, even platforms described as automated cell processing in closed systems may generally rely on pre-configured instruments and pipeline groups, which limit operational flexibility and cannot reliably prevent process failures caused by unexpected operator / human error. Furthermore, most efforts to automate cell product manufacturing are directed toward automating individual processing steps of 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 to automate cell processing are addressed in the various embodiments disclosed herein. Summary of the Invention

[0005] According to one embodiment, the present disclosure generally relates to methods and systems for processing cellular products.

[0006] In some variations, the present disclosure further relates to an automated system for cell processing, the system comprising: a cartridge comprising a plurality of cartridge modules, the plurality of cartridge modules comprising a bioreactor module, a countercurrent centrifugal elutriation module, and at least one of an electroporation module, a magnetically activated cell selection module, a fluorescence-activated cell selection module, or a spin inoculation 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 being independently configured to cooperate with the corresponding cartridge module to perform a procedure on the cartridge. One or more cell processing operations, wherein the docking station is sized and shaped to receive a single cartridge, wherein the liquid transfer bus is fluidly coupled to each cartridge module via a fluid conduit disposed between a port on the liquid transfer bus and a corresponding port of each cartridge module, wherein the fluid conduit comprises a tubing or flow channel, wherein the system further comprises a sterile liquid transfer device configured to facilitate liquid transfer between the cartridge and a closed-volume fluidic device, wherein the closed-volume fluidic device is a fluid reservoir, wherein the sterile liquid transfer device comprises at least one actuator, wherein the actuator actuates one of the at least one sterile liquid transfer port of the cartridge to transfer liquid to the sterile liquid transfer port. and corresponding sterile liquid transfer ports of the closed-volume fluidic devices, wherein the system further comprises a fluid reservoir disposed within the housing of the system, the fluid reservoir being accessible by a user for inserting and / or removing one or more closed-volume fluidic devices, wherein each of the one or more cell processing operations performed on the cartridge is performed on cells within the cartridge, wherein the plurality of cartridge modules comprises the electroporation module, wherein the plurality of cartridge modules comprises the magnetically activated cell selection module, wherein the plurality of cartridge modules comprises the fluorescence activated cell selection module, wherein the plurality of cartridge modules comprises the spinoculation module, wherein the plurality of docking station modules comprises corresponding bioreactor modules, wherein the plurality of The docking module includes a corresponding magnetically activated cell selection module, wherein the multiple docking modules include a corresponding fluorescence-activated cell selection module, wherein the multiple docking modules include a corresponding electroporation module, wherein the multiple docking modules include a corresponding countercurrent centrifugal elutriation module, wherein the multiple docking modules include a corresponding rotary inoculation module, wherein the box includes a pump fluidically connected to the liquid transfer bus, wherein the system also includes a pump actuator configured to interface with the pump, wherein the system also includes a waste liquid reservoir for capturing waste liquid generated by the box, and / or wherein the liquid transfer between the box and the closed volume fluid device includes extracting at least a portion of the cell processing sample from the box.

[0007] In some variations, the present disclosure further relates to an automated cell processing method, the method comprising: performing at least two cell processing operations within a cartridge positioned in a docking station, wherein the cartridge comprises a plurality of cartridge modules, at least one sterile liquid transfer port, and a liquid transfer bus fluidically coupled to each cartridge module, and wherein the docking station comprises a plurality of docking station modules corresponding to the plurality of cartridge modules, and the docking station is sized and shaped to receive a single cartridge, wherein the plurality of cartridge modules comprises a bioreactor module, a countercurrent centrifugal elutriation module, and at least one of an electroporation module, a magnetically activated cell selection module, a fluorescence activated cell selection module, or a spinoculation module, and wherein the cell processing operations are automated upon execution of a set of received instructions, wherein the method further comprises: actuating 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 between the sterile liquid transfer port and the corresponding sterile liquid transfer port, wherein facilitating the transfer of liquid between the cartridge and the corresponding sterile liquid transfer port. The liquid transfer between the closed volume fluid device includes actuating the one sterile liquid transfer port of the at least one sterile liquid transfer port and the corresponding sterile liquid transfer port, wherein the liquid transfer between the box and the closed volume fluid device includes extracting at least a portion of the cell processing sample from the box, wherein the multiple box modules include the electroporation module, wherein the multiple box modules include the magnetically activated cell selection module, wherein the multiple box modules include the fluorescence-activated cell selection module, wherein the multiple box modules include the spin inoculation module, wherein the multiple docking station modules include corresponding bioreactor modules, wherein the multiple docking station modules include corresponding countercurrent centrifugal elutriation modules, wherein the multiple docking station modules include corresponding electroporation modules, wherein the multiple docking station modules include corresponding magnetically activated cell selection modules, wherein the multiple docking station modules include corresponding fluorescence-activated cell selection modules, wherein the multiple docking station modules include corresponding spin inoculation modules, and wherein the two or more cell processing performed within the box is performed on cells within the box.

[0008] Additional variations, features, and advantages of the invention will become apparent from the following detailed description and by practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] This patent or patent application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0010] Figure 1A is a block diagram of an illustrative variation of a cell processing and manufacturing system. Figure 1B is a block diagram of an illustrative variation of the cartridge.

[0011] Figure 2Ais a block diagram of an illustrative variation of a cell processing and manufacturing system. Figure 2B is a perspective view of an illustrative variation of a cell processing station of a cell processing and manufacturing system. Figure 2C is a side view of an illustrative variation of a cell processing station and cartridge of a cell processing and manufacturing system. Figure 2D is a perspective view of an illustrative variation of a cell processing and manufacturing system. Figure 2E is an interior diagram of an illustrative variation of a cell processing and manufacturing system. Figure 2F is a block diagram of an illustrative variation of a cell processing and manufacturing system. Figure 2G is a block diagram of another illustrative variation of a cell processing and manufacturing system.

[0012] Figure 3 is a block diagram of another illustrative variation of a cell processing and manufacturing system.

[0013] Figure 4A is a perspective view of another illustrative variation of a docking station and cartridge for a cell processing and manufacturing system. Figure 4B and Figure 4C are additional perspective views of another illustrative variation of a docking station and cartridge for a cell processing and manufacturing system.

[0014] Figure 5A to Figure 5V Additional perspective views of aspects of another illustrative variation of at least one of a docking station, a cartridge, and a sterile liquid transfer instrument of a cell processing and manufacturing system are provided.

[0015] Figure 6 is a schematic diagram of an exemplary variation of the box.

[0016] Figure 7 is a schematic diagram of another exemplary variation of the box.

[0017] Figure 8A is a side view of an exemplary variation of the cartridge. Figure 8B is a top view of an exemplary variation of the cartridge. Figure 8C is a side view of an exemplary variation of the cartridge. Figure 8D is a perspective view of an exemplary variation of the cartridge.

[0018] Figure 9 A cross-sectional side view of an exemplary variation of a cartridge is shown.

[0019] Figure 10A Illustrative variations of rotary valves and actuators are shown. Figure 10B An illustrative variation of a rotary valve interfacing with an actuator is shown.

[0020] Figure 11A is a perspective view of an illustrative variation of a cassette including a CCE module in an extended configuration. Figure 11Bis a cross-sectional side view of an illustrative variation of a CCE module in a retracted configuration. Figure 11C is a cross-sectional side view of an illustrative variation of a CCE module in an extended configuration.

[0021] Figure 12A is a perspective view of an illustrative variation of a docking station magnetically activated cell sorting (MACS) module including magnets in an ON configuration. Figure 12B is a perspective view of an illustrative variation of a docking station MACS module including magnets in an OFF configuration.

[0022] Figure 13 is a perspective view of an illustrative variation of a cassette and docking station bioreactor module. Figure 14 is a perspective view of an illustrative variation of a cassette coupled to a docking station bioreactor module.

[0023] Figure 15 is a block diagram of an illustrative variation of a fluid connector system.

[0024] Figure 16A is a schematic diagram of an illustrative variation of a fluid connector. Figure 16B yes Figure 16A Detailed schematic diagram of the fluidic connector depicted. Figure 16C is in a connected configuration Figure 16A Schematic diagram of the fluidic connector depicted. Figure 16D Is in open port configuration Figure 16A Schematic diagram of the fluidic connector depicted. Figure 16E It receives gas Figure 16A Schematic diagram of the fluidic connector depicted. Figure 16F Receives sterilant Figure 16A Schematic diagram of the fluidic connector depicted. Figure 16G Is in open valve configuration Figure 16A Schematic diagram of the fluidic connector depicted. Figure 16H It is used to transfer fluid between fluid devices connected to the fluid connector. Figure 16A Schematic diagram of the fluidic connector depicted. Figure 16I Is in closed valve configuration Figure 16A Schematic diagram of the fluidic connector depicted. Figure 16J Is in closed port configuration Figure 16A Schematic diagram of the fluidic connector depicted. Figure 16K is in the decoupled configuration Figure 16A Schematic diagram of the fluidic connector depicted. Figure 16L Is disconnected from the sterilant source Figure 16A Schematic diagram of the fluidic connector depicted.

[0025] Figure 17Ais a front perspective view of the fluid connector in a closed port configuration. Figure 17B Is in closed port configuration Figure 17A A rear perspective view of the depicted fluid connector. Figure 17C Is in closed port configuration Figure 17B A rear view of the fluid connector is depicted. Figure 17D is a front perspective view of the fluid connector in an open port configuration. Figure 17E Is in open port configuration Figure 17D A rear perspective view of the depicted fluid connector. Figure 17F Is in open port configuration Figure 17E A rear view of the fluid connector is depicted.

[0026] Figure 18A is a side view of the fluid connector in an uncoupled configuration. Figure 18B is a cross-sectional side view of the fluid connector in an uncoupled configuration. Figure 18C is a side view of the fluid connector in a coupled configuration. Figure 18D is a cross-sectional side view of the fluid connector in a coupled configuration. Figure 18E is a side view of the fluid connector in an open port configuration. Figure 18F is a cross-sectional side view of a fluid connector in an open port configuration. Figure 18G is a side view of the fluid connector in an open valve configuration. Figure 18H is a cross-sectional side view of a fluid connector in an open valve configuration.

[0027] Figure 19 is a schematic diagram of an illustrative variation of a fluid connector system.

[0028] Figure 20A is a schematic diagram of an illustrative variation of a fluid connector system. Figure 20B and Figure 20C is a schematic diagram of an illustrative variation of a fluid connector connection process.

[0029] Figure 21 is a block diagram of an illustrative variation of a fluid connector system.

[0030] Figure 22 is a block diagram of an illustrative variation of a fluid connector system.

[0031] Figure 23 is a block diagram of an illustrative variation of a fluid connector system.

[0032] Figure 24A is a block diagram of an illustrative variation of a fluid connector system. Figure 24B is a schematic diagram of an illustrative variation of a fluid connector connection process. Figure 24Cis a schematic diagram of an exemplary variation of a valve.

[0033] Figure 25A is a block diagram of an illustrative variation of a fluid connector system. Figure 25B is a schematic diagram of an illustrative variation of a fluid connector connection process. Figure 25C is a schematic diagram of an exemplary variation of a valve.

[0034] Figure 26A is a side view of an illustrative variation of a pump actuator and pump. Figure 26B is a side view of an illustrative variation of a pump actuator coupled to a pump.

[0035] Figure 27 is a flow chart of an illustrative variation of a method of transferring fluid using a fluid connector.

[0036] Figure 28 is a flow chart of an exemplary variation of a cell processing method.

[0037] Figure 29 is a flow chart of an exemplary variation of a cell processing method.

[0038] Figure 30A Flowchart of exemplary variations of the cell processing method for autologous CART cells or engineered TCR cells. Figure 30B Flowchart of exemplary variations of the cell processing method for allogeneic CART cells or engineered TCR cells.

[0039] Figure 31 is a flow chart of an exemplary variation of a cell processing method for HSC cells.

[0040] Figure 32 is a flow chart of an exemplary variation of a cell processing method for TIL cells.

[0041] Figure 33 is a flow chart of an exemplary variation of a cell processing method for NK-CAR cells.

[0042] Figures 34A to 34C Is used for T reg Flowchart of exemplary variations of cell processing methods for cells.

[0043] Figure 35 is a flow chart of an exemplary variation of a cell processing method.

[0044] Figure 36 is a flowchart of an illustrative variation of a method of performing a transformation model.

[0045] Figure 37 is an illustrative variation of a graphical user interface associated with the initial process design interface.

[0046] Figure 38 are illustrative variations of graphical user interfaces associated with the creation process.

[0047] Figure 39 is an illustrative variation of a graphical user interface associated with a null process.

[0048] Figure 40 are illustrative variations of a graphical user interface associated with adding reagents and consumable containers.

[0049] Figure 41 are illustrative variations of graphical user interfaces associated with process parameters.

[0050] Figure 42 are illustrative variations of graphical user interfaces associated with patient weight process parameters.

[0051] Figure 43 are illustrative variations of graphical user interfaces associated with pre-processing analysis.

[0052] Figure 44 is an illustrative variation of a graphical user interface associated with a leukocyte count pre-processing analysis.

[0053] Figure 45 are illustrative variations of graphical user interfaces associated with process parameter calculations.

[0054] Figure 46 are illustrative variations of graphical user interfaces associated with completed process setup.

[0055] Figure 47 are illustrative variations of graphical user interfaces associated with process operation activation settings.

[0056] Figure 48 are illustrative variations of graphical user interfaces associated with populating process operation activation settings.

[0057] Figure 49 are illustrative variations of graphical user interfaces associated with initial process operations.

[0058] Figure 50 is an illustrative variation of a graphical user interface related to dragging in process operations.

[0059] Figure 51 is another illustrative variation of a graphical user interface related to dragging in process operations.

[0060] Figure 52 are illustrative variations of graphical user interfaces associated with the process operations of filling.

[0061] Figure 53are illustrative variations of graphical user interfaces related to product monitoring.

[0062] Figure 54 is another illustrative variation of a graphical user interface related to product monitoring.

[0063] Figure 55 is a block diagram of an illustrative variation of a cell processing and manufacturing system.

[0064] Figure 56 is a cross-sectional side view of an illustrative variation of a countercurrent centrifugal elutriation (CCE) module.

[0065] Figure 57 is a cross-sectional side view of an exemplary variation of a magnetic-activated cell selection (MACS) module.

[0066] Figure 58 is a perspective view of an illustrative variation of a CCE system.

[0067] Figures 59A to 59B is a perspective view of an illustrative variation of a CCE system. Figure 59C is a side cross-sectional view of an illustrative variation of a CCE system. Figures 59D to 59F is a side cross-sectional view of an illustrative variation of a rotor of a CCE module.

[0068] Figure 60A is a plan view of an exemplary variation of a rotor of a CCE module. Figure 60B and Figure 60C is a perspective view of an illustrative variation of a rotor of a CCE module. Figure 60D is a side view of an illustrative variation of a rotor of a CCE module. Figure 60E is a perspective view of an illustrative variation of a rotor in a housing. Figure 60F and Figure 60G is a schematic plan view of an exemplary variation of a rotor of a CCE module. Figure 60H is a side view of an illustrative variation of a rotor of a CCE module. Figure 60I is a perspective view of another illustrative variation of a rotor of a CCE module. Figure 60J is a perspective view of yet another illustrative variation of a rotor of a CCE module. Figure 60K is a schematic plan view of another exemplary variation of the rotor size of the CCE module. Figure 60L are images of a set of illustrative variations of the rotor of a CCE module.

[0069] Figures 61A to 61C is a schematic diagram of an exemplary variation of a cell separation process.

[0070] Figure 62A is a perspective view of an illustrative variation of the MACS system in a first configuration. Figure 62Bis a perspective view of an illustrative variation of the MACS system in a second configuration. Figure 62C is a cross-sectional side view of an exemplary variation of the MACS system. Figure 62D is a perspective view of an illustrative variation of the MACS system in a second configuration. Figure 62E is a plan view of an exemplary variation of a flow cell and magnet array for a MACS system. Figure 62F is a plan view of an exemplary variation of a flow cell for a MACS system. Figure 62G is a schematic diagram of an exemplary variation of a flow cell and magnet array.

[0071] Figures 63A to 63E is a perspective view of an illustrative variation of a magnet array.

[0072] Figure 64A is a perspective view of an exemplary variation of a flow cell. Figure 64B is a cross-sectional side view of an exemplary variation of a flow cell. Figure 64C is a schematic diagram of an exemplary variation of the MACS system.

[0073] Figures 65A to 65C is a schematic diagram of an exemplary variation of a flow cell.

[0074] Figures 66A to 66C is a schematic diagram of an exemplary variation of a cell separation process.

[0075] Figures 67A to 67B is a schematic diagram of an exemplary variation of a cell processing and manufacturing system.

[0076] Figure 68A is a cross-sectional perspective view of an illustrative variation of a bioreactor. Figure 68B is a cross-sectional side view of an exemplary variation of a bioreactor. Figure 68C is a perspective view of an illustrative variation of a bioreactor housing. Figure 68D is a plan view of an exemplary variation of a bioreactor housing.

[0077] Figure 68E is a perspective view of an exemplary variation of a membrane for a bioreactor. Figure 68F is a side view of an exemplary variation of a membrane for a bioreactor. Figure 68G is a perspective view of an exemplary variation of a membrane for a bioreactor. Figure 68H is a bottom view of an exemplary variation of a membrane for a bioreactor.

[0078] Figure 69A is a cross-sectional side view of an illustrative variation of a bioreactor housing. Figure 69B is a cross-sectional perspective view of an illustrative variation of a bioreactor housing.

[0079] Figure 70is an exploded perspective view of an exemplary variation of a bioreactor.

[0080] Figure 71A is a plan view of an exemplary variation of a bioreactor. Figure 71B is a cross-sectional side view of an exemplary variation of a bioreactor.

[0081] Figure 72 is a schematic diagram of an exemplary variation of an electroporation system.

[0082] Figure 73 is an exploded perspective view of an exemplary variation of an electroporation module.

[0083] Figures 74A to 74B is a schematic diagram of an exemplary variation of the electroporation procedure.

[0084] Figure 75 is a circuit diagram of an exemplary variation of the electroporation process.

[0085] Figures 76A to 76D is a diagram of an exemplary variation of the electroporation procedure.

[0086] Figure 77A is a flow chart of an exemplary variation of a method of isolating cells. Figure 77B is a flow chart of an exemplary variation of a method for concentrating cells. Figure 77C is a flow chart of an exemplary variation of the buffer exchange method.

[0087] Figure 78 is a flow chart of another exemplary variation of the method of isolating cells.

[0088] Figure 79A is a flow chart of an exemplary variation of a closed-loop method for isolating cells 7900. Figure 79B is a flow chart of an illustrative variation of a closed-loop method for elutriating cells 7910. Figure 79C is a flow chart of an exemplary variation of a closed-loop method for harvesting cells 7920.

[0089] Figure 80A is a flow chart of an exemplary variation of a method of isolating cells. Figure 80B is a flow chart of an exemplary variation of a method for selecting cells.

[0090] Figure 81 is a flow chart of another exemplary variation of the method of isolating cells.

[0091] Figure 82A is a flow chart of an exemplary variation of a method of preparing a bioreactor. Figure 82B is a flow chart of an exemplary variation of a method of loading a bioreactor. Figure 82C is a flow chart of an exemplary variation of a method of preparing a bioreactor. Figure 82D is a flow chart of an exemplary variation of a calibration method for a bioreactor. Figure 82E is a flow chart of an exemplary variation of a method for mixing reagents. Figure 82F is a flow chart of an exemplary variation of a method for mixing reagents. Figure 82G is a flow chart of exemplary variations of a method of culturing cells. Figure 82H is a flow chart of an exemplary variation of a method for cryopreserving cells. Figure 82I is a flow chart of an illustrative variation of the sampling method. Figure 82J is a flow chart of exemplary variations of a method of culturing cells. Figure 82K is a flow chart of an exemplary variation of the medium exchange method. Figure 82L is a flow chart of an illustrative variation of a method of controlling gas. Figure 82M is a flow chart of an exemplary variation of a method for controlling pH.

[0092] Figure 83 is a flow chart of an exemplary variation of a method of electroporating cells.

[0093] Figure 84 is a flow chart of another exemplary variation of a method for electroporating cells.

[0094] Figure 85 is a schematic diagram of an illustrative variation of a fluid connector.

[0095] Figure 86 is a schematic diagram of an illustrative variation of a fluid connector port.

[0096] Figure 87 is a schematic diagram of an illustrative variation of a fluid connector connection process.

[0097] Figure 88 is a schematic diagram of an illustrative variation of a fluid connector connection process.

[0098] Figure 89 is a schematic diagram of an illustrative variation of a fluid connector.

[0099] Figure 90A is a side view of an illustrative variation of a fluid connector. Figure 90B yes Figure 90A A perspective view of the depicted fluid connector. Figure 90C yes Figure 90A A cross-sectional side view of the fluid connector depicted.

[0100] Figure 91A is a side view of an illustrative variation of a fluid connector. Figure 91B yes Figure 91A A perspective view of the depicted fluid connector. Figure 91C yes Figure 91A A cross-sectional side view of the fluid connector depicted.

[0101] Figure 91D is a side view of an illustrative variation of a fluid connector. Figure 91E yes Figure 91D A perspective view of the depicted fluid connector. Figure 91F yes Figure 91D A cross-sectional side view of the fluid connector depicted.

[0102] Figure 92A is a side view of an illustrative variation of a fluid connector. Figure 92B yes Figure 92A A transparent side view of the depicted fluidic connector. Figure 92C yes Figure 92A A perspective view of the depicted fluid connector. Figure 92D yes Figure 92A A cross-sectional side view of the fluid connector depicted.

[0103] Figure 93A is a perspective view of an illustrative variation of a fluid connector. Figure 93B yes Figure 93A A transparent perspective view of the depicted fluid connector.

[0104] Figure 94A is a perspective view of an illustrative variation of a fluid connector. Figure 94B yes Figure 94A A transparent perspective view of the depicted fluid connector.

[0105] Figure 95A is a perspective view of an illustrative variation of a fluid connector. Figure 95B yes Figure 95A A transparent perspective view of the depicted fluid connector. Figure 95C is a detailed side view of the port in the open port configuration. Figure 95D is a detailed side view of the port in the closed port configuration.

[0106] Figure 96A is a plan view of an exemplary variation of the fluidic device. Figure 96B is a side view of an illustrative variation of a fluid device coupled to a robot. Figure 96C is a perspective view of an illustrative variation of a fluidic device held by a robot.

[0107] Figure 97A is a perspective view of an illustrative variation of a MACS module. Figure 97B is a cross-sectional perspective view of an exemplary variation of a MACS module. Figure 97C is a cross-sectional side view of an exemplary variation of a MACS module.

[0108] Figure 98 is a flow chart of an exemplary variation of a cell processing method.

[0109] Figure 99 is a flow chart of an exemplary variation of a cell processing method.

[0110] Figure 100 is a flow chart of an exemplary variation of a cell processing method.

[0111] Figure 101 is a flow chart of an exemplary variation of a cell processing method.

[0112] Figure 102 is a schematic diagram of an exemplary variation of a cell processing and manufacturing system.

[0113] Figure 103A and Figure 103B is a perspective view of an illustrative variation of a sterile fluid transfer device. DETAILED DESCRIPTION

[0114] definition

[0115] The terms "a" or "an" refer to one or more of the entity, i.e., may refer to plural referents. Thus, the terms "a," "an," "one or more," and "at least one" are used interchangeably herein. Furthermore, 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 the presence of one and only one of the element.

[0116] Throughout this application, the term "about" is used to indicate that a value includes the inherent error variation of the equipment or method used to determine the value, or the variation present in the sample being measured. Unless otherwise stated or otherwise obvious from the context, the term "about" means within 10% above or below the reported value (unless the value would exceed 100% or be below 0% of the possible value). When used in conjunction with a range or series of values, the term "about" applies to the endpoints of the range or each value listed in the series, unless otherwise stated. As used in this application, the terms "about" and "approximately" are used as equivalents.

[0117] As used herein, the term "sterile" is used as a non-limiting description of some variations, optional features that provide advantages in the operation of certain systems and methods of the present disclosure. Maintaining sterility is generally desirable for cell processing, but can be achieved in various ways, including but not limited to providing sterile reagents, culture media, cells, and other solutions; sterilizing the cassette and / or cassette components after loading (to protect the cell product from damage); and / or operating the system in a sterile housing, environment, building, room, etc. Such user- or system-performed sterilization steps can sterilize the cassette or cassette components and / or maintain the sterility of the cassette or cassette components.

[0118] As used herein, the term "and" is used interchangeably with "or" unless expressly stated otherwise.

[0119] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprise," "comprising," and the like are to be construed in an inclusive sense and not in an exclusive or exhaustive sense; that is, in the sense of "including but not limited to." Words using the singular or plural number also include the plural and singular number, respectively. Additionally, when used in this application, the words "herein," "above," "hereunder," and words of similar import shall refer to this application as a whole and not to any particular portions of this application.

[0120] Cell processing and manufacturing systems

[0121] Described herein are systems and methods for processing and manufacturing cell products for biomedical applications. In variations, these systems may include a cell processing and manufacturing system (CPMS) comprising a box and a cell processing station, the box comprising the cell product, the cell processing station comprising at least one controller and at least one novel docking station configured to receive the box. In some variations, the CPMS may perform automated manufacturing of the cell product. In short, 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 box. The controller of the cell processing station may include a processor, memory, display, input device, and communication device for performing the methods described herein. The box of the CPMS may include a liquid transfer bus, a sensor (e.g., at least one sensor), a fluid connector, and one or more modules (e.g., at least one box module or multiple box modules). The one or more cartridge modules may include a countercurrent centrifugal elutriation module, a bioreactor, an electroporation module, a spin inoculation module, a fluorescence-activated cell selection module, and a cell separation module, which may be a magnetically 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. In addition, 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 a cell processing step on the cell product, such that the CPMS performs the cell processing step on the cell product. In some variations, multiple cell processing steps may be performed within a single cartridge.

[0122] Furthermore, methods for processing and manufacturing cell products for biomedical applications may include methods for processing a solution containing the cell product. For example, the method may include the following cell processing steps: digesting tissue using an enzymatic reagent to release a selected cell population into a solution, enriching cells using a docking station CCE module, washing cells using a docking station CCE module, selecting cells in the solution using a docking station selection module, sorting cells in the solution using a docking station sorting module, differentiating or expanding cells in a cartridge bioreactor module, activating cells using an activation reagent, electroporating the cells, transducing the cells using a vector, and arranging the cell product.

[0123] Advantageously, in some variations, multiple cell processing steps may be performed on a cartridge positioned within a docking station of a cell processing and manufacturing system without having to remove or transport the cartridge out of the docking station.

[0124] Cell Processing Station (CPS)

[0125] The CPMS described above may generally include a cell processing station having a docking station, the docking station including a plurality of docking station modules, each docking station module being independently configured to perform one or more cell processing operations on a cartridge within 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 magnetically activated cell selection module), a sorting module (e.g., a fluorescence activated cell sorting (FACS) module), an electroporation module, a countercurrent centrifugal elutriation (CCE) module, a reagent reservoir, and the like. In a variation, the docking station module of the CPS may be configured to interface with a corresponding cartridge module of the cartridge to perform cell processing on the cell product within the cartridge.

[0126] The CPS may advantageously include a docking station configured to integrate the functionality of a variety of cell processing tasks into a single structure, thereby allowing the generation of cell products in an end-to-end automated manner without user intervention to change settings, transfer cell products, etc. In particular, the single structure can be a box that is configured to undergo complete end-to-end automation of multiple processes (e.g., washing, enrichment, elutriation, magnetic cell separation, sorting, activation, genetic modification, electroporation, rotational inoculation, formulation, amplification, harvesting / formulation, and cryopreservation) when docked in the docking station to generate cell products. In some variations, the box may include two or more bioreactor modules for generating two or more cell products via a split process. In combination with the box of the present disclosure, cell processing tasks can be performed on the cells in the box in coordination with the corresponding modules of the box according to a predetermined processing schedule to generate processed cell products.

[0127] In some variations, the cell processing station of the present disclosure can be a stand-alone unit (featuring a standard power option) that can be effortlessly moved throughout a laboratory space (e.g., on rollers or casters) 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 a cartridge housed in the docking station) can be compact and easy to implement within any laboratory space because no expensive installation is required. Thus, the cell processing and manufacturing system of the present disclosure can enable users to perform process development studies and scale up before making a larger investment in production. In addition, because the stand-alone unit may not require the cartridge to be moved between modules and / or instruments during cell processing, the design risk and development risk for cell processing can be reduced.

[0128] box

[0129] The box can include multiple box modules (also referred to herein as boxes, consumable boxes, consumables, and cell processing equipment), such as bioreactors, countercurrent centrifugal elutriation (CCE) modules, magnetic cell sorters (e.g., magnetic activated cell selection modules (MACS)), electroporation devices (e.g., electroporation modules (EP)), sorting modules (e.g., fluorescence activated cell sorting (FACS) modules), acoustic flow cell modules, centrifugation modules, microfluidic enrichment modules, combinations thereof, etc. The cell processing and manufacturing systems described herein can reduce operator intervention and increase throughput by automating box (and cell product) processing. For example, the automated cell processing and manufacturing system can facilitate sterile liquid transfer between one or more boxes and cell processing stations (e.g., one or more docking station modules). Additionally or alternatively, the automated cell processing and manufacturing system can facilitate sterile liquid transfer between one or more boxes and other components of the system, such as fluid connectors (e.g., sterile liquid transfer ports), reagent reservoirs, sampling reservoirs (e.g., sterile liquid transfer equipment and combinations thereof, as described in detail herein).

[0130] The cassette can be configured to be portable and facilitate automated and sterile cell processing. For example, the cassette can be configured to be moved by a user from a location outside the CPS to a receiving position in a docking station within the cell processing station.

[0131] In some variations, the cassette 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.

[0132] In some variations, the box may optionally provide a self-contained device capable of performing one or more cell processing steps via one or more modules. The modules may be integrated into a fixed configuration within the box. Additionally or alternatively, the box modules may be configurable or movable within the box, thereby allowing various boxes to be assembled from shared modules. Similarly stated, the box may be a single closed unit, each box module having fixed components; alternatively, the box may contain configurable modules connected by configurable fluid connections, mechanical connections, optical connections, and electrical connections. In some variations, one or more sub-boxes (each containing a set of modules) may be configured to be assembled to perform various cell processing workflows. In addition, the box modules may each be arranged in a different housing, or may be integrated into a box or sub-box together with other modules. Although for simplicity, modules may generally be described herein as different groups of components, modules (and their corresponding components) may be arranged in any suitable configuration. For example, components of different modules may be interspersed with each other within the CPS, so that each module is defined by a set of connected components that perform a predetermined function together. However, the components of each box module may or may not be physically grouped within the box. In some variations, multiple cartridges may be used to process a single cell product by transferring the cell product from one cartridge to another cartridge of the same or different type and / or by splitting the cell product into more cartridges and / or pooling multiple cell products into fewer cartridges.

[0133] Typically, each docking station module in the system can interface with one or more modules of the docking station module on the cassette. For example, the electroporation module on the cassette (if present) can interface with the docking station electroporation module to perform the electroporation step on the cell product, and can also interface with common components, such as components of the fluid bus circuit (e.g., pumps, valves, sensors, etc.). The advantage of such a split module design is that expensive components (e.g., motors, sensors, heaters, lasers, etc.) can be retained in the docking station module of the system. In such a variation, the use of a disposable cassette can eliminate the need to sterilize the cassette between uses.

[0134] Various materials can be used to construct the box and box housing, including metal, plastic, rubber and / or glass, or a combination thereof. The box, its components and its housing can be molded, machined, extruded, 3D printed, or any combination thereof. The box can contain commercially available components (e.g., tubing, valves, fittings); these components can be attached or integrated with custom components or equipment. The housing of the box can constitute an additional shell layer to further protect the sterility of the cell product. In some variations, the box can be designed to be single-use or disposable.

[0135] Typically, an operator can load or unload the box in an ISO-5 or better environment, thereby utilizing aseptic technique to ensure that the sterility of the contents of the box is maintained when the box is opened. In some variations, an operator can use manual aseptic connection (e.g., sterile tubing welding) to perform loading or unloading of the box.

[0136] Reagent Library

[0137] The system can generally include one or more reagent depots for storing reagents therein. Non-limiting examples of one or more reagents can include cell culture media, buffers, cytokines, proteins, enzymes, polynucleotides, transfection reagents, non-viral vectors, viral vectors, antibiotics, nutrients, cryoprotectants, solvents, cell materials and pharmaceutically acceptable excipients. Additionally or alternatively, waste liquid can be stored in a reagent depot. In some variations, samples can be stored in a reagent depot during extraction from one or more boxes. The reagent depot can include one or more temperature-controlled compartments (e.g., at, for example, a refrigerator, cooler, water bath, heating chamber or other at about -80°C, about -20°C, about 4°C, about 25°C, about 30°C, about 37°C and about 42°C). In the cell manufacturing process, the temperature in these compartments can change to heat or cool reagents. In variations of the method disclosed herein, a robot (or manually by an operator) can engage each of the box and the reagent depot. The reagent depot can interface with one or more sterile liquid transfer ports on the box so that reagents or materials can be dispensed into the box. In some embodiments, the present invention provides a kind of aseptic liquid transmission instrument, and this aseptic liquid transmission instrument is configured to be similarly transferred to the box or to transmit out from the box in an automated, manual or semi-automatic manner.The operator can manually be aseptic liquid transmission instrument reserve reagent.The reagent storehouse can have automatic door to allow aseptic liquid transmission equipment and / or other reagent receptacles to enter, and alternatively, each automatic door is all under the closed-loop temperature control independently.Equipment and receptacle can be configured to pick up and place mobile by robot.In some embodiments, the reagent storehouse can comprise one or more sample picking-up areas.For example, the robot can be configured to move one or more reagents to and from one or more sample picking-up areas in the sample picking-up area.

[0138] In some variations, the size of the reagent reservoir can be designed to accommodate multiple sterile liquid transfer devices required to support a single patient process. The sterile liquid transfer devices can be manually provided to the reagent reservoir and / or replaced within the reagent reservoir on a predetermined schedule (e.g., every 3 days or as needed for cell processing tasks).

[0139] Cell selection system

[0140] The CPMS described herein may include a cell selection system configured to separate cells based on predetermined criteria. For example, cells can be separated based on physical properties (such as size and / or density) using, for example, countercurrent centrifugal elutriation. Cells can also be separated based on the presence of predetermined antigens on the cells using, for example, magnetically activated cell selection. In some variations, a CPMS with modules for these separation methods (e.g., box modules, docking station modules) can 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 system described herein can increase throughput and cell yield output in a compact and portable structure. For example, before cells are subjected to magnetic separation, a cell suspension can be mixed with an excess or a magnetic reagent at a predetermined concentration (e.g., cells / mL). Similarly, after cells are subjected to magnetic separation, the cells can be washed in a solution (e.g., a suitable buffer solution).

[0141] In some variations, a cell separation system (or cell separator) may include: a rotor configured for counterflow centrifugal elutriation of cells in a fluid; a first magnet configured to magnetically rotate the rotor and separate the cells from the fluid in the rotor; a flow cell in fluid communication with the rotor and configured to receive the cells from the rotor; and a second magnet configured to magnetically separate the cells in the flow cell.

[0142] In some variations, the CCE module may be integrated into the box 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 and a magnet, the rotor being configured to separate cells from a fluid (e.g., separating cells of different sizes and / or densities from cells retained in the fluid), the magnet being configured to magnetically rotate the rotor. The housing may be configured to move relative to the magnet, or vice versa (e.g., move the magnet relative to the housing). The CCE modules described herein can provide cell separation within a compact and portable housing, wherein the magnet can be disposed outside the housing (e.g., the magnet is disposed within a corresponding CCE module of a docking station).

[0143] In some variations, a compact rotor that can facilitate cartridge integration may include input and output fluid conduits extending from the rotor toward opposite sides of a 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, wherein the second side is opposite the first side. An elutriation chamber (e.g., a cone) may be coupled between the first and second fluid conduits.

[0144] In some variations, a method of separating cells from a fluid may comprise: moving a rotor toward a magnet, the rotor defining an axis of rotation; flowing a fluid through the rotor; using the magnet to rotate the rotor (e.g., magnetically) about the axis of rotation while flowing the fluid through the rotor; and moving the rotor away from the magnet.

[0145] In some variations, a method of separating cells from a fluid can include flowing a fluid containing cells into a flow cell. A population of cells can be labeled with magnetic particles. The population of cells can be magnetically attracted toward a magnet array for a residence time, and the population of cells can flow out of the flow cell after the residence time.

[0146] In some variations, a flow cell can include an elongated cavity having a cavity height and a magnet array comprising a plurality of magnets, each of the magnets being separated by a spacing distance. A predetermined ratio between the cavity height and the spacing distance can optimize magnetic separation of cells in the flow cell.

[0147] electroporation

[0148] In some variations, an electroporation module (EP) or electroporator as described herein may be configured to facilitate one or more of transduction and transfection of cells. As described in more detail herein, the EP may 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 may also be configured to apply an electroporation signal (e.g., a voltage pulse, a waveform) to each discrete batch of fluid individually to improve electroporation efficiency and thereby increase flux. In some variations, active electric field compensation may similarly improve electroporation efficiency and flux.

[0149] In some variations, a 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 comprising a processor and a 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 the third fluid; and generate an electroporation signal to electroporate the cells in the fluid conduit using the set of electrodes.

[0150] In some variations, a method of electroporating cells may comprise receiving a first fluid comprising cells in a fluid conduit; receiving a second fluid comprising 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.

[0151] 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.

[0152] bioreactor

[0153] In some variations, a bioreactor may include a housing including a base and sidewalls, and a gas-permeable membrane coupled to one or more of the base and sidewalls of the housing. The gas-permeable membrane may facilitate cell culture. In some variations, a 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 an orbital motion.

[0154] Fluid connectors

[0155] It is noteworthy that conventional cell processing systems lack automated multi-purpose sterile fluid connector solutions for cell therapy production, in which a group of sterile fluid connectors are capable of performing multiple connection and disconnection cycles with a given system. For example, conventional sterile fluid connectors can typically be disposable devices, and therefore can be expensive and labor-intensive in manufacturing and performing cell processing procedures. Typically, the fluid connectors described herein may include multiple sealed housings between a sterile portion (e.g., a fluid connector lumen or cavity) and an external (e.g., non-sterile) surrounding environment, thereby promoting sterile control of the fluid connector and the equipment coupled thereto. The fluid connectors described herein can be durable components that can be reused for multiple cycles while maintaining sterility and / or bioburden control. For example, a sterilant can be used to sterilize the fluid connector without damaging cell products or other biomaterials.

[0156] In some variations, aseptic manufacturing systems as described herein can utilize one or more sterile fluid connectors and have a configuration suitable for being manipulated by a robot (such as a robot arm, etc.). Aseptic fluid connectors as described herein can enable fluid to be transmitted in an automated, sterile and quantitative manner to automate cell therapy manufacturing. Automation of cell therapy manufacturing can then provide lower per-patient manufacturing costs, lower process failure risks, and the ability to meet the needs of commercial-scale patients for cell therapy. In some variations, sterile fluid connectors can improve one or more of sterility, efficiency, and speed by removing human operators from the manufacturing process. As described herein, automated and integrated sterilization processes can be applied to fluid connectors to maintain the sterility of the system. For example, a fluid connector can maintain sterility by multiple connection / disconnection cycles between separate sterile closed volume fluid equipment (e.g., housings, containers, reservoirs, boxes, docking station modules, box modules, bioreactors, encapsulated reservoirs, sealed chambers). Therefore, systems, devices, and methods described herein can reduce the complexity of sterilization processes, reduce energy usage, and improve sterilization efficiency.

[0157] 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 respective proximal ends of the connectors may be configured to connect (e.g., be fluidically connected, form a fluid pathway) with respective fluid devices to transmit one or more of a fluid (e.g., liquid and / or gas) and a biological material (e.g., a cell product) between the fluid devices. The distal end of the connector may include a port configured to mate with each other. The fluid connector may also include a sterilant port configured to facilitate sterilization of the chamber within the distal ends of the first connector and the second connector. The fluid connector may be sterilized before or after connection as needed to ensure sterility. In this way, the fluid connector can be reused for multiple connection and disconnection cycles.

[0158] In some variations, a cell processing and manufacturing system utilizing the fluidic connectors described herein may include a robot configured to operate the fluidic connector, and a controller configured to control the robot to manipulate (e.g., move, connect, open, close, disconnect) a first connector and a second connector together while maintaining sterility of the fluidic connector and a plurality of fluidic devices (without human interaction), 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, and the like.

[0159] Cell processing control

[0160] This article describes a system and method for manufacturing cell products for biomedical applications using an automated system. Conventional semi-automatic solutions for cell processing may not allow users to define bioprocesses. Instead, users can select from a limited set of predefined machine processes and process control parameters. In addition, conventional semi-automatic solutions may not provide scalable manufacturing solutions for cell therapy production. For example, cell therapy manufacturing can traditionally be performed in batches (that is, a product will be manufactured in a single room / suite, and the required processing tools are located inside). This can be guided by technicians following standard operating procedures (SOPs), or in some cases, processing tools (e.g., Miltenyi Prodigy, Lonza Cocoon) can perform a series of processing steps for a single patient product on a single multifunctional processing tool. However, existing solutions (e.g., Miltenyi Prodigy) may not allow users to define bioprocesses. In addition, the manual work required for conventional solutions may increase the risk of product contamination and human error.

[0161] In some variations of the present disclosure, a group of cell therapy biomanufacturing processes can be transformed into a group of machine instructions suitable for the automated execution of the system described herein. For example, a method for transforming a user-defined cell treatment operation into a cell treatment step to be executed by a processor of an automated cell treatment and manufacturing system can include receiving an ordered input list of cell treatment operations, and executing a transformation model to create an ordered output list of cell treatment steps that can be executed by the system on the ordered input list. As used herein, a transformation model can refer to an algorithm, process or transformation configured to transform a group of cell treatment steps into a group of machine instructions or hardware instructions for a system. Advantageously, in some variations, a plurality of cell treatment steps can be performed on a box positioned in a docking station for cell treatment and manufacturing systems without having to remove or transport the box out of the docking station. In some variations, the docking station module can be controlled to perform a cell treatment step on each cell product. In this way, systems and methods enable biologists to define manufacturing processes in biological terms, and enable the system to transform the biological model (e.g., process definition) into an instruction for a group of machine execution.

[0162] The end-to-end closed system automation described herein can reduce process failure rates and costs. For example, compared with conventional manual methods, end-to-end automation can reduce manufacturing time (e.g., residence time) and increase throughput. That is, multiple processes (e.g., 10 or more processes) can be performed simultaneously. The methods described herein can further reduce the chance of contamination and user error. Therefore, the systems, devices, and methods described herein can improve one or more of cell processing automation, repeatability, reliability, process flexibility, instrument throughput, process scalability, and reduce one or both of labor costs and process duration.

[0163] I. System

[0164] Described herein are systems and devices configured to perform cell processing steps to manufacture cell products (e.g., cell therapy products). 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. The use of a controller may facilitate one or more of automation, efficiency, and sterility of the cell processing and manufacturing system.

[0165] In some variations, a cell processing and manufacturing system may include a cell processing station comprising a housing.

[0166] Figure 1A 1 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 docking station 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 docking station 125 may include a plurality of docking station modules 112 and a docking station 115 configured to receive a cartridge 114, wherein each docking station module in the plurality of docking station modules 112 may correspond to a cartridge module of the cartridge 114.

[0167] In some variations, the CPS may include a fully or at least partially encapsulated 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 a housing that can be configured for use in a clean room, a biosafety cabinet, or other aseptic location. In some variations, the CPS may be configured to perform aseptic liquid transfers that enter and leave the box in a fully or partially automated process. For example, one or more fluids may be stored in a sterile liquid transfer device 142. In some variations, the sterile liquid transfer device 142 may be a portable consumable that can be moved within the CPMS 100. The sterile liquid transfer device and fluid connector described herein can transfer fluids in an automated, sterile, and quantitative manner to automate cell therapy manufacturing. In some variations, the housing of the CPS may be configured to meet International Organization for Standardization (ISO) standards ISO 7 or better (e.g., ISO 6 or ISO 5). The 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 fully filtered air space). Alternatively, the facility may be an ISO 8 or ISO 9 facility. In some variations, the CPS may include less than about 800 m 3 , less than about 700m 3 , less than about 600m 3 , less than about 500m 3 , less than about 300m 3 , less than about 250m 3 , less than about 200m 3 , less than about 150m 3 , less than about 100m 3 , less than about 50m 3 , less than about 25m 3 , less than about 10m 3 and less than about 5m 3 The volume of (including all ranges and subvalues ​​therebetween).

[0168] In some variations, the cassette 114 may receive cell products from different donors or contain cell products intended for different recipients. If sufficient product needs to be generated for therapeutic use, or when a donor provides cell products for several recipients (e.g., for allogeneic transplantation), the cell product from a single donor may be shunted between multiple cassettes 114. In some variations, multiple cassettes 114 may be placed in a docking station 115 in sequence (e.g., a first cassette may be processed in a docking station, and then a second cassette may be processed in a docking station). In yet other variations, a first cassette may be placed in a docking station 115 of a first cell processing station 110, and a second cassette may be placed in a docking station 115 of a second cell processing station 110. If desired, the cell product for a single recipient may advantageously be shunted between multiple cassettes 114 and processed in separate CPSs to generate sufficient product for therapeutic use for the recipient. Similarly, if desired, a cell product for a single recipient can be split between multiple cassettes 114 and processed within separate CPSs to generate several cell products with unique genetic modifications, which can then be recombined at certain ratios for therapeutic use with the recipient. In other variations, a cell product from a single donor can be split between two separate bioreactors within the same cassette, whereby these cell products undergo separate processes. These cell products can then be combined and used as part of a therapeutic treatment.

[0169] like Figure 1B As illustrated, the box 114 may include a plurality of box modules, which include one or more of a bioreactor 150, a cell separator 152, an electroporator 160, a rotary inoculator 153, a cell sorter 155, a countercurrent centrifugal elutriator 151 and other modules 168, as appropriate and as described in more detail herein. It should also be understood that one or more of the box modules in the plurality of box modules can use shared equipment to complete the tasks of the module. The box 114 may also include a liquid transport 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. The cell separator 152 may include one or more of a rotor 154, a flow cell 156 and a magnet 158. In some variations, the 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 the cells in the flow cell 156.

[0170] Cell Processing Station (CPS)

[0171] In some variations, CPS110 may include an at least partially enclosed housing (e.g., a shell) in which one or more automated cell processing steps are performed. For example, CPS110 may be configured to transfer sterile liquid into and out of box 114 in a fully or partially automated process. In some variations, CPS110 may not have a housing and be configured for use in a clean room, a biosafety cabinet, or other suitable clean or sterile location. In some variations, CPS110 may be part of CPMS100, which includes a feed-through biosafety cabinet, quality control instruments, pumps, consumables (e.g., fluid equipment), fluid connectors, consumable feed-throughs, and a sterilization system (e.g., a sterilant source and / or generator, a fluid source, a heater / dryer, a ventilator).

[0172] Figure 2A 2 is a block diagram of a cell processing and manufacturing system 200 including a cell processing station (CPS) 210 and a box 214. The CPS 210 may include a housing 211 having four walls, a base, and a top. The CPS 210 may be divided into an interior area having a feedthrough access port 213 and optionally a quality control (QC) instrument. 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 area. This air filtration may maintain sterile cell processing in an ISO 8 or ISO 9 manufacturing environment. The CPS 210 may also include an air filter on the air outlet to maintain the ISO grade of the room. In some variations, the CPS 210 may also include a novel docking station 215 having multiple docking station modules, such as a bioreactor, a pool selector (e.g., MACS), an electroporator (EP), a countercurrent centrifugal elutriator (CCE), a cell sorter (e.g., fluorescence activated cell sorting (FACS)), a rotary inoculator, etc. In a variation, the CPS 210 may also include a sterile liquid transfer instrument, a reagent reservoir, and a sterilization system. The reagent reservoir may be accessed by the user through the sample pickup port 219 .

[0173] In some variations of the method according to the present disclosure, a human operator can load a box 214 into the feed-through port 213, which can be empty or include any combination of modules described herein. The box 214 can be pre-sterilized, or after passing through the feed-through port 213 and entering the docking station 215, it can be sterilized using ultraviolet radiation (UV) or a chemical sterilant provided as steam, spray or washing. In some variations, the box 214 can be pre-loaded with an input cell product by the user. After the sterilized, loaded box 214 is positioned in the docking station 215 via the feed-through port 213, the user can use a computer processor in a computer server rack (e.g., a controller 220) to initiate automated cell processing. Advantageously, in some variations, the box can undergo an automated end-to-end process to generate a desired cell product while remaining in the docking position in the docking station 215. At the end of cell processing, the box 214 now having the processed cell product can be retrieved from the docking station 215 by the user via the feed-through port 213. In some variations, an exterior surface of the housing 202 may include an input / output device 208 (eg, a display, a touch screen) that is communicatively coupled to the controller 220 .

[0174] Figure 2B yes Figure 2BIn some variations, the height of the CPS 210 may be greater than about one meter, such as between about 1 m and about 1.1 m, between about 1 m and about 1.2 m, between about 1 m and about 1.3 m, between about 1 m and about 1.4 m, between about 1 m and about 1.5 m, between about 1 m and about 1.6 m, between about 1 m and about 1.7 m, between about 1 m and about 1.8 m, between about 1 m and about 1.9 m, between about 1 m and about 2 m, between about 1 m and about 2.5 m, between about 1 m and about 3 m, between about 1 m and about 5 m, between about 3 m and about 10 m, between about 5 m and about 20 m, between about 10 m and about 30 m, between about 20 m and 100 m, and greater than about 100 m, including all values ​​and ranges therebetween. For example, in some variations, the height of the CPS may be approximately 1.9 m. In some variations, one or both of the length and width of the CPS 210 may be greater than approximately 1 meter, such as between approximately 1 m and approximately 5 m, between approximately 3 m and approximately 10 m, between approximately 5 m and approximately 20 m, between approximately 10 m and approximately 30 m, between approximately 20 m and 100 m, and greater than approximately 100 m, including all values ​​and ranges therebetween. Conversely, in some variations, one or both of the length and width of the CPS 210 may be less than approximately 1 meter, such as between approximately 0.2 m and approximately 0.9 m, between approximately 0.3 m and approximately 0.9 m, between approximately 0.4 m and approximately 0.5 m, between approximately 0.5 m and approximately 0.9 m, between approximately 0.6 m and approximately 0.9 m, between approximately 0.7 m and approximately 0.9 m, or between approximately 0.8 m and approximately 0.9 m. For example, in some variations, one or both of the length and width of the CPS may be approximately 0.9 m.

[0175] As in Figure 2B In the embodiment of the present invention, CPMS200 may include a box 214 and a CPS210 with a housing 211. The box 214 can be fed through the feed-through port 213 of the housing to be arranged in the docking station 215 of the CPS210. The CPS210 may also include a sample pickup port 219, which may also be a reagent library 218 with one or more sterile liquid transfer devices 242. In a variation, the CPS210 may also include a rack module 221 for accommodating one or more of a controller 220, a power distribution unit (PDU), and a refrigerator, wherein the controller 220 may be controlled by at least a user interface of the input / output device 208. In some variations, the CPS210 may be mobile. For example, the CPS210 may be equipped with wheels or casters for repositioning and repositioning. Since the CPS210 can be easily transportable, if necessary, it can be transported to any room in a facility (e.g., a hospital), or two or more CPSs can be transported to a single room.

[0176] Figure 2C is a side view of a cell processing and manufacturing system 200 depicting a cartridge 214 (e.g., any of the cartridges described herein) being introduced into a CPS 210 (e.g., any of the CPSs described herein). Each inserted cartridge 214 may undergo one or more cell processing operations. Figure 2C As shown, the cartridge 214 can be positioned on a docking station drawer 216 that can be moved through a feed-through port 214 of the housing 211 of the CPS 210 to position the cartridge 214 within a docking station 215 of the CPS 210. The CPS 210 can also include a built-in reagent reservoir 218 configured to store one or more sterile liquid transfer devices 242 that can be inserted, removed, and / or replaced (e.g., by an operator or by a robot of the CPS) via a sample pickup port 219. In some variations, the docking station 215 can also 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. The sterile liquid transfer instrument 243 (which may optionally be detachable from but releasably coupled to the cassette 214) may be configured to transfer fluid from and / or to the sterile liquid transfer devices 242 of the reagent reservoir 218 (or other fluid reservoirs similarly configured with fluid connectors, as will be described later, so that they can be controlled by the sterile liquid transfer instrument 243 to exchange fluids with the cassette 214). In some variations, the sterile liquid transfer instrument 243 may include a robot that can be controlled to interact with each of the sterile liquid transfer devices 242 of the reagent reservoir 218 (these sterile liquid transfer devices may generally be referred to herein as fluid transfer devices 242) and with the cassette 214 to supply and remove fluids from the modules of the cassette 214 as required by the cell processing operation.

[0177] Similarly, Figure 2D is a perspective view of the CPMS 200 depicting a cassette 214 positioned on a docking station drawer 216 of the CPS 210. As shown, the drawer 216 can include a telescoping feature that can extend from the docking station 215 (e.g., extending outside the CPS to receive the cassette 214) and retract into the docking station 215 (e.g., upon receiving the cassette 214) such that the entire drawer 216 can be enclosed within the docking station 215. Figure 2DAlso depicted is a sample pickup port 219 and a door 270 with an input / output device 208. In some variations, the door 270 can be movable relative to the sample pickup port 219. In particular, the door 270 can be configured to be repositioned so that it forbids or allows access to the sample pickup port 219. The door 270 can be repositionable (e.g., by an operator and / or by controller 220) manually and / or automatically. For example, in some variations, the door 270 can be slidable (e.g., translatable along a mounting member) toward the sample pickup port 219 (and in front of the sample pickup port) and away from the sample pickup port. Therefore, the door 270 can provide temporary access to the aseptic liquid transfer device and / or other reagent receptacles at the sample pickup port 219. In addition, in some variations, the door 270 can support the input / output device 208, or can be the input / output device. For example, at least a portion of door 270 may include an input / output device 208 configured to directly receive user input (eg, via a touch screen and / or button controls).

[0178] As described above, the CPS 210 may generally include at least one docking station module (e.g., Figure 1A ), the at least one docking station module being configured to engage the cassette 214 and / or interface with a corresponding module of the cassette 214. Figure 2EDepicted is an internal view of a CPS 210 with several exemplary docking station modules. For example, in some variations, the CPS 210 may include a nesting control module for ensuring that the box is correctly oriented in the docking station 215 and / or that the box 214 is connected to the CPS 210. The nesting control module may include one or both of an electrical interface 272 and a box presence sensor 274. The electrical interface 272 may be, for example, a PCB with spring pins that are configured to physically and electrically interface with the corresponding electrical portion of the box 214. In addition, the presence sensor 274 may include one or both of a box in-place sensor and a box presence sensor. The box in-place sensor may be a diffuse reflection sensor that is configured to guide a light beam through the box 214 and detect any light reflected at the sensor. When the light beam is reflected, the box may be incorrectly positioned. In some embodiments, the box 214 is positioned in the CPS 210 and is positioned in the CPS 210. The box 214 is positioned in the CPS 210 and is positioned in the CPS 210. The box 214 is positioned in the CPS 210 and is positioned in the CPS 210. The box 214 is positioned in the CPS 210 and is positioned in the CPS 210. The box 214 is positioned in the CPS 210 and is positioned in the CPS 210. The box 214 is positioned in the CPS 210 and is positioned in the CPS 210. The box 214 is positioned in the CPS 210 and is positioned in the CPS 210. The box 214 is positioned in the CPS 210 and is positioned in the CPS 210. The box 214 is positioned in the CPS 210 and is positioned in the CPS 210. The box 214 is positioned in the CPS 210 and is positioned in the CPS 210. The box 214 is positioned in the CPS 210 and is positioned in the CPS 210.

[0179] Furthermore, 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 a fluid bus (e.g., to and from the cartridge 214, as described herein) by regulating the amount of pressure applied to the valve via a pneumatic cylinder. The sensor 278 may detect bubbles in one or more fluid pathways of the fluid bus. For example, the sensor 278 may be a bubble sensor that uses optical fibers to visualize changes in the amplitude of the sensor output, which may indicate whether air, liquid, or both air and liquid (i.e., bubbles) are flowing through the fluid 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 that enable the one or more pump rollers to act as a peristaltic pump to move fluid through the cartridge 214. The pump mechanism 280 can be coupled to the cassette 214 via tubing to form one or more fluid pathways. In some variations, the pump mechanism 280 can be considered a separate module (i.e., a pump module) configured to couple with multiple cassette modules. That is, any module requiring gas or fluid flow (e.g., gas and / or liquid flow) can be coupled with the pump module.

[0180] Additionally or alternatively, the CPS may include a bioreactor module for culturing various cell types (described herein). Within the docking station 215, the bioreactor module may include one or both of a port 281 and an observation window 282 (also referred to herein as a fluid window). The port 281 may be a supply support (e.g., a gas supply port, a fluid supply port, or a solid supply port) configured to feed nutrients (e.g., CO2, N2, and / or air) into the bioreactor so that the cells can be properly cultured. The observation window 282 may, for example, allow a camera to detect the liquid level of one or more containers of the bioreactor.

[0181] Brief reference Figure 2F In some variations, the bioreactor module may include a manifold 283 located external to the CPS 210. The manifold 283 may be configured to supply gases to the CPS 210 via a gas mixer 284 that receives external sources for clean dry air (CDA), nitrogen (N2), carbon dioxide (CO2), etc. and provides the gases to the CPS 210. Thus, the bioreactor module may formulate gases for supply to the bioreactors 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 (in Figure 2F). The one or more mass flow meters can be considered a first flow meter that is configured to combine the gases and provide the resulting gas to a second mass flow meter before 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). The mass flow meter 285 can be configured to provide information about the mass flow rate of the gases (e.g., the volume per second of each gas) and ensure that an appropriate cell growth environment is provided. That is, cells require a specific gas mixture (e.g., CO2, O2) for growth, and the required gas mixture may change depending on the growth state of the cells.

[0182] Reference again Figure 2E , the CPS may include a magnetically activated cell selection module (MACS) for magnetically selecting cells having a predetermined property or component (such as an antigen) (described throughout this document). Accordingly, the MACS may include magnets 286 that can be engaged with the actuator and the cartridge 214 to perform magnetic selection of cells. Additionally, in some variations, the MACS may include an observation window 287 (also referred to herein as a fluid window) that can allow a camera (e.g., an external camera) to observe the volume of liquid and / or the extent of cell separation that occurs during magnetic selection.

[0183] 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) (described throughout this document). 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 having a magnet coupled thereto, wherein the magnet is configured to mate with a mating magnet within 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 CCE module. Next, the camera 290 may be configured to observe the elution chamber of the CEE within the cartridge 214 (e.g., a double cone or funnel in which cell separation occurs). The tachometer 291 may be configured to calculate the RPM of the CCE as the CCE is rotating. In particular, the tachometer 291 may include a sensor configured to direct a light beam to the rotor of the CEE within the cartridge 214, and the rotor may be reflective (e.g., may include a reflective tape or coating) such that the tachometer 291 can calculate the RPM of the CEE relative to the rotor. In some variations, the tachometer 291 may be paired with a camera 290 so that images can be captured to visualize the cells in the CEE within the cartridge 214. Additionally, the sensor 292 may be a leak detection sensor configured to detect leaks in the CEE rotor using optical fibers. The sensor 292 may be configured to shut down the CEE process when a leak occurs. In some variations, the sensor 292 may be electrically coupled to the controller and / or input / output device 208 to indicate to an operator (e.g., visually, audibly, tactilely, etc.) whether a leak is occurring.

[0184] As another example, the CPS can include an electroporation (EP) module for facilitating intracellular delivery of macromolecules (described throughout this document) (i.e., transfection via electroporation). The EP module can include one or both of a sensor 294 and an actuator 296. The sensor 294 can be a liquid volume sensor configured to detect the volume of fluid within the flow cell of the EP module using optical fibers. Additionally, the actuator 296 can be a high-voltage actuator configured to form a high-voltage coupling between the cartridge 214 and the CPS 210.

[0185] In some variations, the above description of Figure 2E One or more components of the described modules may be shared between modules. For example, the electrical interface 272 described with respect to the nested control module may additionally or alternatively be a component of one or more docking station modules described above (e.g., a fluid control module, a bioreactor module, a magnetic cell selection module, a CEE module, and an EP module) and / or one or more docking station modules described in more detail below (e.g., an electronics and control module).

[0186] Figure 2G is a block diagram of the CPMS 200 described throughout this document. Specifically, Figure 2G The block diagram shows the 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 Figure 2E and Figure 2F As described. For example, the CPS 210 may include one or more of an electronics and control module, a nesting control module, a fluid control module, a gas control module, a bioreactor sensor, a bioreactor agitation module, a temperature control module, a pulse generator 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, an observation window, a magnetic coupling (e.g., for a CEE and / or bioreactor module), a gas gasket, a bioreactor sensor interface, an electroporator (EP) high voltage (HV) connection, and a CCE magnetic coupling may enable the docking station 215 of the CPS 210 to interface with the box 214. In variations, the CPS 210 may also include or be operatively coupled to a manifold (e.g., a gas manifold) that may be integrated or separated with the CPS 210, a power supply, a network box, a cooling source, a gas source, and software (e.g., SW interface, orchestration) for automating cell processing operations within the box 24. In some variations, the electronics and control module may include one or more of a programmable logic controller (PLC) and / or an inter-process communication (IPC), as well as one or more of the controller 220, the input / output device 208, and the electrical interface 272. Thus, the electronics and control module may allow a user to interact with the CPS 210 and may thereby coordinate at least a subset of the operations and processes of the CPMS 200.

[0187] also, Figure 2F The dashed lines in FIG. 1 depict how the components of CPS 210 may be grouped to form docking station modules that are configured to interface with cartridge 214 and / or corresponding modules of cartridge 214, as described above with respect to FIG. Figure 2E For example, a fluid control module, which may include a valve actuator, a pump, and / or a bubble sensor, may interface and / or engage with a fluid bus, an exterior of the cartridge 214, a fluid connector of the cartridge 214, and / or a fluid window of the cartridge 214. As another example, a nested control module may engage with an electrical portion of the cartridge 214 and / or one or more exterior portions of the cartridge 214 (e.g., to clamp the cartridge 214 within the docking station 215).

[0188] As yet another example, the bioreactor module of cartridge 214 may be controllable via a magnetic coupling, a gas gasket, and / or a bioreactor sensor interface. In some variations, the control may be performed by, or in conjunction with, one or more of a temperature control module, a bioreactor stirring 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 substrate temperature sensor, and the like. In variations, the bioreactor stirring module may include one or more of a stirring motor and an encoder, a magnetic coupler, and the like. In variations, the bioreactor sensor may include one or more of a thermistor reader, a dissolved oxygen (DO) reader, a pH reader, and the like. 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, and the like (as described with respect to Figure 2G ). Similarly, the electroporator module of box 214 can be controllable by a pulse generator control module via an EP HV connection, wherein in some variations, the pulse generator control module can include one or more of a pulse generator, an HV connection actuator, a voltage and / or current reader, etc. Similarly, the CCE module and / or magnetic cell separation module of box 214 can be controllable by a CCE control module and an MS control module (etc.) via a CCE magnetic coupling, respectively. In some variations, the CCE control module can include one or more of a motor and encoder, a magnetic coupler, a strobe light, a camera, an optical trigger, and a CCE leak sensor. In some variations, the MS control module can include one or more of a magnetic engagement actuator, a magnetic engagement sensor, etc.

[0189] Figure 3 A two-dimensional (2D) schematic diagram of the docking station 315 of the CPS according to the cartridge modules, modules, instruments, and docking station modules described above is provided. In particular, Figure 3 The 2D schematic diagram shows a flat version of the docking station 315 of the CPS, so that the relative positions of the interfaces between the corresponding modules are shown, and it is assumed that the front of the docking station 315 is open to receive the cartridge therein. In other words, assuming that the docking station 315 is a rectangular prism, Figure 3 The panels are labeled “Bottom,” “Back,” “Top,” “Left,” and “Right,” and modules (e.g., box modules and / or docking station modules) and instruments are identified based on their positioning on the docking station 315. Figure 3 The 2D schematic illustrates how the CPS can advantageously perform multiple functions on a cartridge docked within a docking station 315 by carefully utilizing panels to perform different cell processing steps in a partially or fully enclosed system, something that other systems cannot do. Figure 3References to Sterile Liquid Transfer Instruments (SLTIs), Bioreactors (BRs), and Magnetic Separators (MS) (such as MACS) are included.

[0190] Docking station 315 is provided with the support of the bioreactor module of the invention.For example, (with the nested control module interface) magnetic separator (MS) and bar code or bar code scanner are interfaced on the left wall of docking station 315. The top wall of docking station 315 comprises electroporator (EP) box and (fluid control module) bubble sensor reader. The right wall of docking station 315 comprises electroporator (EP) high voltage (HV) interface and fluid bus, and this fluid bus and pump module and valve and fluid window interface, to each box module supply fluid in the box module in box and remove fluid from this each box module. The back wall of docking station 315 comprises strobe light, CCE motor and camera, and they are included in the CCE control module with the CCE module interface of box. The bottom wall of docking station 315 comprises bioreactor groove, gas mixer and gas sensor (they are configured to the bioreactor module interface with box), and is configured to box is fixed on the clamping plate on the drawer of docking station 315.

[0191] According to the above drawings, the following will refer to Figure 4A to Figure 5V Describe in more detail Figure 3 2D schematic diagram of .

[0192] Figure 4A A perspective view of a rendering of a docking station 415, a sterile liquid transfer instrument 443 coupled to the docking station, and a cartridge 414 (eg, consumables) disposed on a docking station drawer 416 is provided. Figure 4A As shown, the cartridge 414 has not yet been moved into the chamber of the docking station 415 via the feedthrough 413. In a variation, the sterile liquid transfer instrument 443 includes a first robot 444 and a second robot 445 that are configured to provide a two-axis range of motion to the sterile liquid transfer instrument 443. In a variation, and as Figure 4A As shown, the sterile liquid transfer device 442 can be coupled to the sterile liquid transfer instrument 443 at a location so as to be proximate to the cassette 414 when the cassette 414 is located within the docking station 415. Thus, the sterile liquid transfer instrument 443 can be advantageously used to supply culture medium (e.g., fluids and liquids) to or extract culture medium from the cassette when the cassette is docked within the docking station 415. In a variation, the docking station drawer 416 is a telescoping drawer, as will be shown in FIG. Figure 4B was used in.

[0193] like Figure 4B As shown, the cassette 414 can be retracted onto the docking station drawer 416 and into the docking station 415 via the feed-through port 413. The cassette can then be retracted into the docking station 415 via the elevator 417 ( Figure 4C415 , and the cassette 414 can be positioned in contact with, and in a variation, clamped to, the top interior surface of the docking station 415. In a variation, contacting the cassette 414 with the top interior surface of the docking station 415 allows the cassette 414 to be engaged by the sterile liquid transfer instrument 443. For example, after engagement between the cassette 414 and the sterile liquid transfer instrument 443, the sterile liquid transfer device 442 can supply fluid to or extract fluid from the cassette 414 via the sterile liquid transfer port 466 of the sterile fluid transfer device 442.

[0194] Figure 5A to Figure 5V Various aspects of the CPMS and the arrangement of components relative to a docking station of the CPS are illustrated, including a docking station module and, in variations, a cartridge module. According to some variations, Figure 5A to Figure 5V The panels and associated features shown may be Figures 4A to 4C Part of the same assembly shown.

[0195] Figure 5A and Figure 5B Aspects of the electroporation assembly of the CPMS of the present disclosure are illustrated. Figure 5A and Figure 5B Each of the figures depicts a cartridge 514 within a docking station 515, wherein the docking station 515 is coupled to a sterile liquid transfer instrument 543 on its top surface. Figure 5A As shown, docking station 515 includes a docking station electroporation module, of which electroporator actuator 561 is shown. Figure 5B Aspect 571 of the corresponding cartridge electroporation module is shown, where it can be appreciated that the electroporation actuator 561 of the docking station electroporation module interfaces with aspect 571 of the cartridge electroporation module.

[0196] Figure 5C and Figure 5D Aspects of the magnetic cell sorting assembly of the CPMS of the present disclosure are illustrated. Figure 5C and Figure 5D Each depicts a cartridge 514 within a docking station 515, wherein the docking station 515 is coupled on its top surface to a sterile liquid transfer instrument 543. In a variation, the docking station 515 includes a docking station magnetically activated cell selection (MACS) module and the cartridge 514 includes a cartridge MACS module. Figure 5C The docking station MACS module 559 is shown with its actuator in a retracted position. Figure 5D Aspect 579 of the MACS assembly 559 is shown with the actuator of the docking station MACS module 559 in an extended position and configured to supply magnetism to a corresponding cartridge MACS module of the cartridge 514 . Figures 5E to 5GAdditional details are provided 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 so that a reflector 574 is deployed to allow viewing of the corresponding cartridge MACS module of the cartridge 514 through the viewing window 575.

[0197] Figures 5H to 5J A liquid transfer bus actuator 563 of a docking station 515 is illustrated having a sterile liquid transfer instrument 543 thereon, wherein the liquid transfer bus actuator 563 is configured to interface with a liquid transfer bus of a cassette disposed within the docking station 515 to direct fluid within the cassette. Figure 5I Provides a closer look at the liquid transfer bus actuator 563, and Figure 5J A closer look at a single actuator 562 of these liquid transfer bus actuators is provided. In a variation, an actuator in the liquid transfer bus actuator 563 can be used to compress a corresponding pinch valve on the cassette to allow flow to be directed to different places within the cassette.

[0198] Figure 5K Illustrated with Figures 5H to 5J The liquid transfer bus actuators work in conjunction with bubble sensors 533 that are disposed on an inner surface of the docking station 515 and are configured to detect any bubbles that may be present within the liquid transfer bus.

[0199] Fluid may be pumped throughout the cassette by motors 534, which are attached to pump rollers disposed on the back wall of the docking station 515. Together, this permits the pump rollers to act as a peristaltic pump to move fluid through the cassette.

[0200] Figure 5M and Figure 5N The embodiment illustrates the engagement between a bioreactor substrate 549 of a docking station 515 and a cartridge 514 disposed therein. In a variation, the bioreactor substrate 549 may be integrally formed with an elevator (described previously), which enables the cartridge 514 to be lifted from a docking station drawer and into contact with the top interior surface of the docking station 515. In a variation, the bioreactor substrate 549 is an aspect of a docking station bioreactor module that is configured to interact with the cartridge bioreactor modules of the cartridge 514. To this end, the docking station bioreactor module interacts with the bioreactors of the cartridge bioreactor modules via motors within the docking station bioreactor module to, for example, drive impellers within the bioreactors to agitate the fluids and suspend the liquids in the bioreactors. Additionally, premixed gas may be provided from the docking station bioreactor module to the bioreactors of the cartridge bioreactor modules via the bioreactor substrate 549 to support cell growth.

[0201] Figure 5O Illustrate various aspects of the docking station bioreactor module of the present disclosure. Figure 5O As shown, the docking station bioreactor module may include a camera 548, a viewing window 546, and a reflector 547. The camera 548 is capable of monitoring the liquid level within the bioreactor of the cartridge bioreactor module through the viewing window 546. In a variation, the camera 548 is positioned so that the reflector 547 is deployed to allow viewing of the corresponding cartridge bioreactor module through the viewing window 546.

[0202] Similarly, Figure 5P Illustrated are aspects of a docking station waste module configured to assess the waste volume level within a cartridge. To this end, the docking station waste module may include a camera 577, an observation window 578, and optionally a reflector (not shown). The camera 577 is capable of monitoring the waste level within the cartridge through the observation window 578. In a variation, the camera 548 is positioned so that the reflector is deployed to allow observation of the waste level within the cartridge through the observation window 546. In a variation, the cartridge may be directly plumbed to a waste tank that may be emptied, cleaned, and / or replaced during each procedure.

[0203] Figures 5Q to 5S The sterile liquid transfer instrument 543 is illustrated coupled to the top surface of the docking station 515. The sterile liquid transfer instrument 543 may include a first robot 544 and a second robot 545 that provide at least two axes of movement relative to the docking station 515 when actuated using the stage 541. As previously described, the sterile liquid transfer device 542 may be coupled to the stage 541 of the sterile liquid transfer instrument 543 so as to be positioned so that engagement with the cartridge 514 is possible. To this end, as shown in FIG. Figure 5S As shown, engagement may include supplying or withdrawing fluid from the cartridge 514 via the sterile fluid transfer port 566 of the sterile fluid transfer apparatus 542 , wherein such supply and withdrawal is performed by the motor 537 in conjunction with the pump roller 534 , thereby providing peristaltic fluid flow into and out of the sterile fluid transfer apparatus 542 .

[0204] Figure 5T to Figure 5V Various aspects of the docking station CCE module are illustrated. Figure 5T As shown, the docking station CCE module can be disposed in the rear (i.e., back) wall of the docking station 515 and can include at least one magnet 558 actuated by a motor 572 and configured to magnetically couple to the cartridge CCE module to rotate the CCE compartment of the cartridge CCE module. As the CCE compartment rotates, the CCE compartment can be imaged by imaging components of the docking station CCE module, including a camera light 576, a viewing window 575, and a camera 573.

[0205] Figure 66 is a schematic diagram of a box 600, which can be a consumable product produced at a cost that makes recycling or limited use feasible. Box 600 may include a liquid transfer bus 624 fluidically coupled to 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 countercurrent centrifugal elutriation (CCE) module 622. In some variations, the cell selection module 616 may be a magnetically activated cell selection (MACS) module. The cell sorting module 618 may include a fluorescence activated cell sorting (FACS) module. Box 600 may include a housing 602 that makes the box self-contained and optionally protects contents from contamination. Sterile liquid transfer ports (SLTP) 606a-606k may be fluidically coupled to reservoirs 607a-607k, and each reservoir may be a flexible bag or rigid container independently. In some variations, the flexible bag may be configured to hold a large volume and permit fluid to be transferred without replacing the transferred fluid with liquid or gas to maintain pressure in the reservoir, as the bag may collapse as fluid is transferred out and expand as fluid is transferred in.

[0206] In some variations, the liquid transfer bus 624 may include valves V1 to V28 and corresponding pipes that fluidically connect the valves to each other and to each module in the module. The valve shown coupled to four fluid lines is a 4 / 2 (4-port 2-position) valve, and the valve shown coupled to three fluid lines is a 3 / 2 (3-port 2-position) valve. The internal flow paths of the valves are indicated in the legend. The cartridge may also include a first pump 632a and a second pump 632b, each of which exposes a pipe to the outside of the housing 602 to allow each pump to interface with a pump actuator (e.g., a rotor) in some docking station modules in the CPS. The liquid transfer bus 624 may be fluidically coupled to a reservoir 607d and a product bag that is fluidically coupled to the SLTP 606d and fluidically coupled to product input lines 627a-627b. An operator can input a cell product into reservoir 607d by connecting product input line 627a or 627b to an external cell source (e.g., a bag of cells collected from a donor). SLTP 606d can be configured to enable a system (e.g., a CPS) according to the present disclosure to automatically add fluid to reservoir 607d. For example, one or more fluid-carrying containers (such as reservoirs 607a-607k, bags, etc.) can receive fluid via the SLTP. Additionally or alternatively, the SLTP can be configured to periodically sample one or more of the fluid-carrying containers. The cartridge can also include collection bags 626a-626c, which are fluidically coupled to liquid transfer bus 624 via valves V17-V19. The cartridge 600 can be configured to permit an operator to remove collection bags 626a-626c after cell processing by the system is complete.

[0207] Figure 7 700 is a schematic diagram of another variation of cassette 700. For example, cassette 700 may include a reduced feature set compared to cassette 600. Cassette 700 may include a liquid transfer bus 724 fluidly coupled to a bioreactor module 714, a countercurrent centrifugal elutriation (CCE) module 722, and at least one module 716 selected from a cell selection module, a cell sorting module, an electroporation module, a spin seeding module, or any other cell processing module. Cassette 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 be a flexible bag or a rigid container. SLTP 706g is fluidly coupled to bioreactor module 714 to permit direct access to the bioreactor by a system or operator. Reservoir 707c may be fluidly coupled to SLTP 706c and product input line 727. In some variations, the liquid transfer bus 724 may include 14 valves V1-V3, V9, V11-V12, V17-V23, and V28 and tubing that fluidly couples the valves to each other and / or to each of the modules. The cassette may also include collection bags 726a-726c that are fluidly coupled to the liquid transfer bus 724 via valves V17-V19. The cassette may also include a pump 732 that exposes tubing outside of the housing 702 to permit each pump to interface with a pump actuator in the system (e.g., a cell processing station).

[0208] corresponding Figure 8A and Figure 8B Shown is a side view and a top view of another modification of box. In some modifications, box 800 may include bioreactor 814, pump 816 and countercurrent centrifugal elutriation (CCE) module 822. Box 800 may include a blank 818, 819 and 820 configured to accommodate additional modules, such as cell selection module, cell sorting module, electroporation module, small bioreactor module, rotary inoculation module, etc. In some modifications, the blank 818, 819 and 820 can be configured to accommodate additional modules. The blank 818, 819 and 820 can limit the empty volume of the box to accommodate the module at another time. In some modifications, box 800 may include two or more additional bioreactors and / or reservoirs in the blank 818, 819, 820. Along the near surface of box 800, it may be the sterile liquid transfer port 806a-806j that is fluidically connected to reservoir 807a-807f. Reservoir 807b and 807e may include fluid (for example, buffer or culture medium). Along the top surface are product input lines 827a-827d, which may be fluidly connected to reservoirs 807a, 807b, 807e, and 807f, respectively.A liquid transfer bus 824 may fluidly connect the SLTP, reservoirs, and product input lines to the modules via tubing.

[0209] In some variations, the outer dimensions of the housing 802 may be approximately 225 mm by approximately 280 mm by 385 mm, approximately 225 mm by approximately 295 mm by 385 mm, and approximately 450 mm by approximately 300 mm by approximately 250 mm, including all values ​​and subranges therebetween. In some variations, the volume of the cartridge 800 may be approximately 10%, approximately 20%, approximately 30%, or less, including all ranges and subranges therebetween. In some variations, the volume of the cartridge 800 may be approximately 10%, approximately 20%, approximately 30%, approximately 50%, approximately 100%, approximately 200%, or more, including all ranges and subranges therebetween. Of course, the outer dimensions of the cartridge 800 are partially dependent on the inner dimensions of the docking station of the CPS, as the cartridge 800 must be sized to be received within the docking station and to interface with the corresponding docking station module disposed adjacent to the inner wall of the docking station.

[0210] In some variations, such as Figure 8C Side view and Figure 8D The cassette 800 shown in the perspective view of FIG can include a MACS module 818. For example, the bioreactor module 814 can 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 behind a connector, and a coolant input line 815e and an output line 815f (for heat exchange) from the docking station bioreactor module interface when the docking station bioreactor module interface is interfaced with the cassette bioreactor module 814. For example, the gas input line 815c can be configured to transfer gas to the fluid (e.g., via a headspace gas control or gas permeable membrane).

[0211] Figure 9 A cross-sectional side view of a cartridge 900 is shown. In some variations, the cartridge 900 may comprise a housing (e.g., a shell), 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, the cell selection module 616 may be a magnetically activated cell selection (MACS) module 917. The cartridge may also include a collection bag 926. The cartridge 900 may optionally include a reserved position configured to accommodate additional modules, such as a cell selection module, a cell sorting module, an electroporation module 918, and the like. In some variations, the cartridge 900 may include one or more bioreactors and / or reservoirs in the reserved position.

[0212] In some variations, the cartridge may include one or more valves. In some variations, the valve 1000 on the cartridge may be configured to receive a docking station module (e.g., a docking station for a CPS) Figure 10AWhen 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). Figure 10B 1000 from one position to another. In some variations, the valve can be configured to clamp a section of flexible tubing. The pinch valve can include a closed configuration, and an external actuator can be configured to interface with the pinch valve (e.g., using a solenoid with linear motion) to open or close the valve. The valve itself can be configured to be disposable, while the actuator can be integrated into a docking station module configured to repeatedly process cartridges.

[0213] Countercurrent centrifugal elutriation

[0214] Countercurrent centrifugal elutriation (CCE) is a technique for separating cells based on properties such as size and / or density. Countercurrent centrifugal elutriation combines centrifugation with countercurrent elutriation, wherein centrifugation corresponds to a sedimentation process under the influence of a centrifugal field, and countercurrent elutriation corresponds to a separation process by washing. Separation occurs in a cone (e.g., biconical, funnel) shaped elutriation chamber. The particles (e.g., cells) transported into the elutriation chamber in a fluid are subjected to two opposing forces: centrifugal force, which drives the fluid away from the axis of rotation; and fluid velocity, which drives the fluid toward the axis of rotation (e.g., countercurrent). By changing the flow rate and centrifugal force, separation of particles (e.g., cells) can be achieved. For example, as described in more detail herein, particles can be separated based on properties such as size and density.

[0215] Countercurrent centrifugal elutriation can be performed and can be used for multiple operations of cell therapy manufacturing workflow, including but not limited to cell washing, cell concentration, culture medium / buffer exchange, transduction, and the separation of white blood cells and other blood components (for example, platelets and red blood cells). In some variations, the fluid source (for example, a single collection bag) for cell separation process can include a suspension of white blood cells, red blood cells, platelets and plasma. In order to separate the immune cells of interest, white blood cells can be isolated, and then these white blood cells are magnetically labeled for magnetic separation. The white blood cell separation step can be performed in the CCE module to separate cells based on size and density, and magnetic separation can be performed in the MACS module. In some variations, the CCE module can be integrated into the box so that cell processing and manufacturing system can separate cells based on one or more of the following: cell cycle progression (for example, G1 / M phase cells are greater than G0 phase cells, S phase cells or G2 phase cells) and cell type (for example, separation of white blood cells and red blood cells and / or platelets).

[0216] Typically, the rotor configured to rotate can include an elutriation chamber (e.g., cone, bicone). The fluid comprising a cell suspension can be pumped into the rotor under continuous flow. When cells are introduced into a cone (e.g., bicone), the cells migrate to the location in the gradient according to their sedimentation rate, and at these locations, the influence of two forces on these cells is balanced. Smaller cells (e.g., platelets) with a low sedimentation rate can be quickly washed toward the axis of rotation with an increased flow rate. Such smaller cells can be the output of the cone (e.g., washed out from the cone). Relatively large (or denser) cells (e.g., red blood cells) flow through the cone relatively slowly and reach equilibrium at the elutriation boundary where centrifugal force and drag balance are balanced, and because the cone has widened, the fluid velocity is relatively low. The largest or densest cells (e.g., leukocytes) remain near the chamber inlet where centrifugal force and fluid velocity are high. By gradually increasing the flow velocity, increasingly larger or increasingly denser cell (e.g., platelets → red blood cells → leukocytes) components can be output from the rotor one after another. Continued increases in fluid flow rate will eventually elute all cells from the cone.

[0217] Now back to the attached figure, Figure 55 is a block diagram of a cell separation system 5600 comprising 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) counterflow centrifugal elutriation (CCE) module 5632 (e.g., a first magnet), a DS magnetically activated cell selection (MACS) module 5642 (e.g., a magnet array, a second magnet), a fluid transfer connection 5652 (which may be a sterile liquid transfer port or a connection to a liquid transfer bus of a cartridge), a pump 5654, an imaging system comprising an optical sensor 5660 and an illumination source 5662, a sensor 5664, and a processor 5670. In some variations, a 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 connection 5650, which may be a sterile liquid transfer port or liquid transfer bus of the cartridge and which may correspond to the fluid transfer connection 5652 of the CPS 5610. For example, a cartridge for cell processing may include a liquid transfer bus and a plurality of modules, each module being fluidly connected to the liquid transfer bus.

[0218] In some variations, an 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 a fluid flow through a rotor of the cartridge CCE module 5630 may be analyzed and used to control the flow rate of the fluid and / or the rotation rate 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 approximately 100 μm, a working distance between approximately 40 mm and approximately 100 mm, and a focal length of less than approximately 8 mm. The optical sensor 5660 may be configured to operate synchronously with the illumination source 5662. In some variations, the optical sensor 5660 may include one or more of a colorimeter, a turbidity sensor, and an optical density sensor. In some variations, illumination source 5662 may operate as a strobe light configured to output light pulses synchronized with the rotation rate of the rotor of CCE module 5630 .

[0219] In some embodiments, sensor 5664 may include one or more of the following: an optical density sensor configured to measure fluid intensity, 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 a fluid line, a bubble sensor configured to detect the presence of bubbles in a fluid conduit, a colorimetric sensor, a vibration sensor, etc.

[0220] In some variations, the fluid transfer connection 5652 may include one or more valves configured to control the flow of fluid 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 connection 5652 (e.g., a valve), the optical sensor 5660, the illumination source 5662, and the sensor 5664.

[0221] In some variations, a CPMS 5600 for cell processing may include a cartridge 5600 comprising a rotor configured for counterflow centrifugal elutriation of cells in a fluid, a CCE module 5630. A first magnet of the docking station CCE module 5632 may be configured to magnetically rotate the rotor and separate the cells from the fluid in the rotor. The cartridge may also 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 the cells in the flow cell.

[0222] In some variations, illumination source 5662 may be configured to illuminate cells. Optical sensor 5660 may be configured to generate image data corresponding to the cells. In some variations, CPMS 5600 may include one or more of an oxygen depletion sensor, a leak sensor, an inertial sensor, a pressure sensor, and a bubble sensor. In some variations, CPMS 5600 may include one or more valves and pumps.

[0223] Figure 56 is a cross-sectional side view of a counter-flow centrifugal elutriation (CCE) module 5700, which includes a housing 5710 (e.g., a shell), a rotor 5720 configured to rotate within and relative to the housing 5710, and one or more fluid ports 5730 (e.g., a fluid inlet, a fluid outlet).

[0224] Figure 57 5 is a cross-sectional side view of a magnetically activated cell selection (MACS) module comprising a housing 5810 (e.g., a casing), a first fluid port 5820 (e.g., a fluid inlet), a second fluid port 5830 (e.g., a fluid outlet), and a flow cell 5810 coupled between the first and second fluid ports 5820, 5830. As described in more detail herein, the flow cell 5810 may comprise a cavity (e.g., a chamber) comprising one or more flow channels (e.g., linear flow channels, laminar flow channels). In some variations, the cavity of the flow cell 5810 may be substantially empty. For example, the flow cell 5810 may not contain a screen, microbeads, a tortuous flow channel, etc. In some variations, the flow cell 5810 may have a longitudinal axis aligned perpendicular to the ground. That is, the flow cell 5810 may be vertically oriented, with the first fluid port 5820 disposed at a higher elevation than the second fluid port 5830, such that gravity can assist fluid flow through the flow cell 5810.

[0225] Figure 58 is a semi-transparent perspective view of the CPMS 5900 including the CCE system for cell processing. Figure 59A A relatively opaque perspective view of the CPMS 5900 including the CCE system is provided. Figure 58 and Figure 59AAs shown, 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.

[0226] In some variations, a CPMS 5900 for cell processing may include a cartridge 5930 comprising a housing 5931 and a rotor 5910 in the housing that is configured to separate cells from a fluid. A corresponding docking station module 5932 comprising a magnet may be configured to interface with the cartridge 5930 to magnetically rotate the rotor 5910. The cartridge 5930 may be configured to move cell products between a plurality of 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 holes 5937 that are configured to facilitate visualization (e.g., imaging) of the rotor 5910. Figure 58 and Figure 59A Magnet 5932 is depicted near but not attached to housing 5931. Figure 59B is a perspective view of the rotor 5910 and housing 5931 without the magnet 5932, optical sensor 5960, and illumination source 5962.

[0227] In some variations, the cartridge 5930 (e.g., housing 5931, 5910) may include consumable components, such as disposable components, limited use components, single use components, etc. In some variations, the magnet 5932 may include a durable component that can 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 movable relative to the magnet 5932 to facilitate magnetic coupling between the magnet 5932 and the plurality of cartridges 5930. Additionally or alternatively, the magnet 5932 may be configured to move relative to the housing 5931.

[0228] Figure 59Cis a side cross-sectional view of a cartridge CCE module 5930. In some variations, the housing 5931 of the rotor 5910 may include 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, with the second side 5935 being opposite the first side 5933. The rotor 5910 (including a cone or a double 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 counterflow centrifugal elutriation. As a result, the rotor 5910 may have low alignment sensitivity with the magnet 5932, as well as low vibration between the rotor 5910 and the magnet 5932. Furthermore, the space between the rotor 5910 and the magnets 5932 enables the second fluid port 5914 to extend toward the second side 5935 of the housing 5931 , thereby allowing fluid to flow on each side of the rotor 5910 .

[0229] In some variations, counterflow centrifugal elutriation can be performed by the CPMS 5900 by moving the magnet 5932 toward the rotor 5910 (or vice versa). The rotor can define an axis of rotation (e.g., coaxial with the first fluid port 5912 and the second fluid port 5914). Fluid can flow through the rotor via the first fluid port 5912 and the second fluid port 5914. The magnet 5932 can magnetically rotate the rotor about the axis of rotation while the fluid flows through the rotor 5910. The rotor can be moved away from the magnet. For example, moving the rotor 5910 can include advancing and retracting the rotor 5910 relative to the magnet 5932.

[0230] In some variations, fluid may flow through first fluid port 5912 along first side 5933 of rotor 5910 and into rotor 5910. Following counterflow centrifugal elutriation through rotor 5910, fluid may exit rotor 5910 through second fluid port 5914 along second side 5935 of rotor 5910.

[0231] In some variations, counterflow centrifugal elutriation can be visualized by an optical sensor 5960 and an illumination source 5962 so as to monitor and modify 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 can be configured to image any portion of the rotor through which the fluid flows (e.g., the first fluid conduit, the second fluid conduit, the third fluid conduit, the first double cone, the second double cone). For example, the optical sensor 5960 can be used to generate image data of one or more of the fluid and cells in the rotor 5910. In some variations, the illumination source 5962 can be used to illuminate one or more of the fluid and cells. For example, the output of the cone can be imaged by the optical sensor to identify non-target cells being elutriated.

[0232] In some variations, one or more of the rotor's rotational speed and the fluid's flow rate may be selected based at least in part on the image data. For example, the rotor may have a rotational speed of up to 6,000 RPM. For example, while the rotor is rotating, the fluid may have a flow rate of up to approximately 150 ml / min. 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.

[0233] Figure 59D 59 is a side cross-sectional view of a rotor 5910 including a first fluid port 5912 (e.g., a fluid conduit, an inlet) and a second fluid port 5914 (e.g., a fluid conduit, an outlet). In some variations, the first fluid port 5912 and the second fluid port 5914 may extend parallel to each other and / or extend parallel to the axis of rotation of the rotor 5910. In some variations, the first fluid port 5912 and the second fluid port 5914 may be disposed on opposite sides of the rotor 5910, which may simplify fluid path design (routing), box design, and also reduce manufacturing costs. For example, fluid seals may be simplified because they each contain only a single lumen. Traditionally, a complex fluid flow path (including an inlet and an outlet) is 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. Figure 59E and Figure 59F is a cross-sectional side view of the rotor 5910 disposed within the housing 5931.

[0234] Figure 60Ais a plan view of a rotor 6000 that may be used with any of the CCE systems, CCE modules, cassettes, housings, combinations thereof, and the like described herein. The rotor 6000 may include a first fluid conduit 6010, a cone 6020 (e.g., a double cone), a second fluid conduit 6030, a magnetic portion 6040 (e.g., a magnet), and a housing 6050. The 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 include 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., about Figure 60A Entering and leaving the page). In some variations, at least a portion of the first fluid conduit 6010 and at least a portion of the second fluid conduit 6030 can be coaxial.

[0235] In some variations, cone 6020 may comprise a double cone having a first cone with a first base and a second cone with a second base, such that the first base faces the second base. In some variations, the double 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 cone of the rotor may comprise a single cone.

[0236] 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 fluids (e.g., cells) within rotor 6000. For example, cone 6020 and portions of first and second fluid conduits 6010, 6030 may be transparent.

[0237] In some variations, the cone may comprise a volume between about 10 ml and about 40 ml. In some variations, the cone may comprise a cone angle between about 40 degrees and about 60 degrees.

[0238] In some variations, the cone may include a first cone (e.g., a distal cone) and a second cone (e.g., a proximal cone), wherein the first cone is larger than the second cone. In some variations, the first cone may have a length between approximately 60 mm and approximately 90 mm. In some variations, the proximal cone may have a length between approximately 15 mm and approximately 40 mm. In some variations, the cone diameter (e.g., the maximum diameter of the cone) may be between approximately 15 mm and approximately 40 mm.

[0239] In some variations, rotor 6000 may comprise an asymmetric shape.In some variations, a first portion (eg, the first end) of rotor 6000 may comprise a cone 6020 and a second portion (eg, the second end) may comprise a paddle shape.

[0240] In some variations, the cone may comprise a length of at least about 4 cm (e.g., between about 9 cm and about 12 cm), a cone diameter of about 5 cm or less (e.g., between about 3 cm and about 5 cm), a fluid flow rate of up to about 100 ml / min (e.g., between about 60 ml / min and about 100 ml / min), and a rotation rate of less than about 3000 RPM. The shape of the first and second cones may be generally linear (rather than convex or concave).

[0241] Figure 60B and Figure 60C is a perspective view, and Figure 60D is a side view of rotor 6002 , which includes first fluid conduit 6012 , cone 6022 , second fluid conduit 6032 , and housing 6052 . Figure 60E is a perspective view of the rotor 6002 disposed in the housing 6090.

[0242] Figure 60F FIG6 is a plan view of a rotor 6004 having two cones (e.g., two double cones) configured to elute cells (e.g., red blood cells, leukapheresis product) in the second cone to recirculate buffer for reuse. The rotor 6004 may include 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 conduit 6023, and a third fluid conduit 6032 coupled to the second cone 6024. The first cone 6022 may include a first volume, and the second cone 6024 may include a second volume greater than the first volume. In some variations, the ratio of the second volume to the first volume may be between approximately 2:1 and approximately 5:1. Fluid may flow sequentially through the first fluid conduit 6012, the first cone 6022, the second fluid conduit 6023, the second cone 6024, and the third fluid conduit 6032. In some variations, rotor 6004 may include a magnetic portion 6042 .

[0243] In some variations, the first cone 6022 may comprise a first double cone, and the second cone 6024 may comprise a second double cone. In some variations, the first double cone may comprise a third cone and a fourth cone, the third cone comprising a first base, the fourth cone comprising a second base, such that the first base faces the second base. In some variations, the second double cone may comprise a fifth cone and a sixth cone, the fifth cone comprising a third base, the sixth cone comprising a fourth base, such that the third base faces the fourth base.

[0244] 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.

[0245] 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 away 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), thereby reducing the force on the RBCs 6030 within the second cone 6024. By recirculating the fluid (e.g., buffer), less fluid (e.g., buffer) may be used to elutriate a higher concentration of RBCs. In some variations, white blood cells 6040 may be harvested from the first cone 6022. The optical sensor may be configured to image the first cone 6022 to generate imaging data identifying the boundary between the white blood cells 6040 and the RBCs 6030. In some variations, the recirculated fluid may be passed through a filter to remove small particles (eg, platelets) using less fluid (eg, buffer).

[0246] Figure 60G is a plan view of a rotor 6005 having two cones (e.g., two double cones), and Figure 60H 6006 is a side view of the rotor configured to elute cells (e.g., red blood cells) in the second cone. A rotor having two cones can facilitate recirculation of buffer for reuse. The rotor 6006 can 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.

[0247] Figure 60I is a perspective view of rotor 6006 including cone 6024 and housing 6054. Figure 60J is a perspective view of the rotor 6007 including the cone 6026 and the 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 different sizes.

[0248] Figures 11A to 11C Another variation of a counter-flow centrifugal elutriation (CCE) module 1100 is depicted. Figure 11A is a perspective view of a cassette 1110 including a CCE module 1100 in an extended configuration, the cassette being configured to receive a docking station (DS) CCE module. Figure 11B and Figure 11C 1 is a cross-sectional side view of a cartridge CCE module 1100 in respective retracted and extended configurations. In some variations, the cartridge CCE module may include a tapered element having an inner surface and an outer surface that is fixedly attached to a distal end of a linear member having an inner surface and an outer surface. The proximal end of the linear member is rotatably attached to a fulcrum to enable extension, retraction, and / or rotation of the linear member. For example, Figure 11C A linear member is depicted that extends outside the housing of the cartridge and then rotates to generate a centrifugal force. A cell product can be conveyed between the inner and outer surfaces of the linear member (optionally in a conduit) to a tapered element and fed into an opening at the distal end of the inner surface of the tapered element such that the flow of the cell product can run opposite to the centrifugal force generated by the rotation of the linear member. Due to the countercurrent of the solution and the sedimentation of the cells under the action of the centrifugal force, the cells in the cell product can be separated based on the ratio of the hydrodynamic cross-section of the cells to the mass of the cells. The flow rate can then be increased and / or the rotation of the linear member can be reduced to permit the cells to selectively pass through the voids in the inner surface of the linear member back to the proximal end of the linear member. Selected cells can be directed into a tube that returns the selected cells to the cartridge. After the enrichment / washing steps are performed, the linear member can be retracted into the housing to a position such as Figure 11B Retracted configuration shown.

[0249] Magnetic cell selection

[0250] Typically, the systems and methods described herein can select cells based on magnetically labeled cells corresponding to cells with predetermined antigens. For example, a cell suspension of interest can be immunologically labeled with magnetic particles (e.g., magnetic beads) that are configured to selectively bind to the surface of the cell of interest. When the cell suspension flows through the flow cell, the labeled cells can generate a large magnetic moment. The flow cell can be arranged near a magnet array (e.g., a permanent magnet, an electromagnet) that generates a magnetic field with a gradient across the flow cell to attract labeled cells for separation, capture, recovery, and / or purification. The magnet array can be configured to generate a non-uniform magnetic field at the edge and interface of each magnet to cover the entire volume of the flow cell so that the magnetophoretic force is equal to the drag applied by the fluid flowing through the flow cell.

[0251] Figures 61A to 61C61 is a schematic diagram of a magnetic cell separation (e.g., magnetic activated cell selection) system and process. The magnetic cell separation system may include a flow cell 6110 (the flow cell includes 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 group 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, a group of cells 6160 may be labeled with a magnetic activated cell selection (MACS) reagent. A MACS reagent may be incubated with the group of cells to label (e.g., attach, couple) the cells to the MACS reagent. As described in more detail herein, the magnet array 6120 can be arranged outside the flow cell 6110 so that the magnet array 6120 can be movable relative to the flow cell 6110. For example, the magnet array 6120 can be moved away from the flow cell 6110 to facilitate causing a group of cells 6160 to flow out of the flow cell 6110. Conventional flow cells include tortuous paths that include screens and / or microbeads for capturing cells. However, it is difficult to reclaim labeled cells from a conventional flow cell configuration. In contrast, the flow cell 6110 described herein can lack tortuous paths, such as microbeads, screens, etc., thereby enabling positive selection or negative selection to be used to effectively perform continuous separation. In some variations, the flow cell can typically include a laminar flow channel, as described in more detail herein.

[0252] At step 6102, the magnet array 6120 may magnetically attract a group of cells 6160 toward the magnet array 6120 for a predetermined residence time and / or based on a measured amount of magnetically separated cells. In some variations, the residence time may be at least one minute (e.g., at least two minutes, at least three minutes, at least five 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 (e.g., cells 6160, 6170) within the flow cell may be held statically within the flow cell 6110 for a residence 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 so that gravity assists the flow of fluid through the flow cell 6110. At step 6104 , the magnetic coupling between the magnet array 6120 and the cells 6160 can be released after the dwell time, and the cells 6160 can flow into the third reservoir 6150 .

[0253] In some variations, even after removing the magnet array 6120, static friction also can make cells remain attached to the surface of the flow cell. Therefore, gas can flow through the flow cell 6110 to assist in collecting cells into the third reservoir 6150. The air flow through the flow cell can provide a more improved cell recovery than the liquid flushing through the flow cell. The interface produced by the gas (e.g., bubbles, air gaps) can be maintained by gravity, thereby enabling a relatively wide flow cell, which further improves cell recovery relative to a horizontally oriented flow cell. The MACS module described herein can be configured to perform positive selection and / or negative selection by modifying the order of steps.

[0254] Additionally or alternatively, the optical sensor can be configured to image the flow cell to generate imaging data identifying the amount of cells magnetically attracted to the magnet array. When a predetermined amount of cells has been measured by the optical sensor, the fluid containing the labeled cells can flow out of the flow cell.

[0255] Figure 62A 6200 is a perspective view of a cartridge MACS module 6200 in a first configuration. The cartridge MACS module 6200 can be a component of any of the cartridges described herein. For example, a cartridge for cell processing can include a liquid transfer bus and a plurality of modules, each module fluidly connected to the liquid transfer bus. The cartridge MACS module 6200 can include a flow cell 6210 including an elongated cavity having a cavity height, an inlet 6230, and an outlet 6232. The MACS module 6200 can also include a magnet array 6220 including a plurality of magnets. Each of the magnets can be spaced apart by a spacing distance, such as Figure 62G 、 Figure 63D and Figure 63E As illustrated, but Figures 62A to 62E A magnet array 6220 is illustrated where magnets are in contact with adjacent magnets.

[0256] Figure 62G 6204. In some variations, the flow cell 6210 may include a cavity height 6202 and a cavity width 6204. The 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 magnet including a corresponding width 6222. In some variations, adjacent magnets may be separated by a predetermined spacing distance 6224. Each magnet pair may have the same or different spacing distances 6224. Figure 62GAs shown, the orientation (e.g., magnetic poles) of the magnets in the magnet array 6220 may comprise a predetermined pattern. In some variations, the ratio of the cavity height 6202 to the separation 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, rotary) may be configured to move the magnet array 6220 relative to the flow cell 6210. In some variations, the orientation (e.g., magnetic poles) of the magnets in the magnet array 6220 may comprise a predetermined pattern (e.g., a Halbach array).

[0257] In some variations, the magnet array 6220 may be movable relative to the flow cell 6210, or vice versa. Figure 62A The cartridge MACS module 6200 is illustrated in an open configuration, and Figure 62B The cartridge MACS module 6200 is illustrated in a closed configuration. Figure 62B is a perspective view of the cartridge MACS module 6200 in a second configuration, in which labeled cells can be magnetically attracted toward the magnet array 6220. In the second configuration, the magnetic field lines generated by the magnet array traverse the flow channel, thereby exerting a magnetophoretic force on the magnetically labeled cells injected into the flow channel. Figure 62C is a cross-sectional side view of a cartridge MACS module 6200 including a magnet array 6220. Figure 62D is a perspective view of the cartridge MACS module 6200 in a second configuration. Figure 62E 62 is a plan view of the flow cell 6210 and magnet array 6220 of the MACS system. Figure 62F FIG. 6 is a plan view of the flow cell 6210 of the MACS module.

[0258] Figures 63A to 63E 6300, 6310, 6320, 6330, 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 magnetic permeability material configured to enhance or reduce the field strength and field gradient within the flow cell. The material can be positioned between the magnets and the flow cell. Additionally or alternatively, the material can be positioned within the flow cell and / or on one or more sides of the flow cell.

[0259] Figure 64A and Figure 64B6410 and a magnet array 6420. The flow cell 6410 may include a set of linear flow channels 6412, 6414, 6416, the set of linear flow channels including a first flow channel 6412 parallel to a second flow channel 6414, and a third flow channel 6416 in fluid communication with each of the first flow channel 6412 and the second flow channel 6416. Figure 64B As shown, the third flow channel 6416 can be disposed between the first flow channel 6412 and the second flow channel 6416 and define a volume where fluids from the first flow channel 6412 and the second flow channel 6416 interact (e.g., mix). In some variations, the flow cell 6410 can include a first inlet 6430 coupled to the first flow channel 6412 and configured to receive a first fluid 6460 (e.g., cells). A second inlet 6431 can be coupled to the second flow channel 6414 and configured to receive a second fluid 6470 (e.g., buffer). The flow cell 6410 can include a first outlet 6432 coupled to the first flow channel 6412 and a second outlet 6433 coupled to the second flow channel 6414.

[0260] The magnet array 6420 can be disposed externally to the flow cell 6400 and can be moved relative to the flow cell 6400 as described herein. In some variations, the longitudinal axis of the flow cell 6410 can be perpendicular to the ground, such that fluid flows in a generally vertical direction.

[0261] In some variations, the first flow channel 6412 may have different dimensions than the second flow channel 6414. For example, the first cavity height of the first flow channel 6412 may be greater than the second cavity height of the second flow channel 6414. For example, the ratio of the first cavity height to the second cavity height may be between about 1:1 and about 3:7, between about 1:1 and about 2:3, and between about 2:3 and about 3:7, including all values ​​and subranges therebetween. Due to the larger cavity height of the first flow channel 6412 relative to the second flow channel 6414, the fluid flowing through the first flow channel 6412 may have a slower flow rate relative to the second flow channel 6414. In some variations, the third flow channel 6416 may include a ratio of the length of the third flow channel 6416 to the diameter of the third flow channel 6416 that is between about 2:1 and about 6:1, between about 2:1 and about 3:1, between about 3:1 and about 4:1, between about 4:1 and about 5:1, between about 5:1 and about 6:1, and between about 3:1 and about 5:1, including all values ​​and subranges therebetween.

[0262] like Figure 64BAs shown, a first fluid 6462 can flow through the flow cell 6410 generally in a first direction. When the magnetic attraction generated by the magnet array 6420 pulls magnetically labeled cells 6416 away from the first flow channel 6412 and toward the second flow channel 6414 (e.g., toward the magnet array 6420), the cells 6416 within the first fluid 6462 can be separated from the remaining cells of the first fluid 6462 within the third flow channel 6416. Similarly, a second fluid 6470 (e.g., a buffer) can flow through the second flow channel 6414. When the cells 6416 flow toward the magnet array 6420, they displace the second fluid 6470 flowing through the third flow channel 6416, allowing a portion of the second fluid 6470 to flow into the first flow channel 6412. In this way, the magnetically labeled cells 6416 can be magnetically separated from the first fluid 6462, and the second fluid 6470 can assist in removing the first fluid 642 that does not include the cells 6416.

[0263] In some variations, a set of fluidic circuits may be coupled to the flow cell to enable multiple cell separation cycles. Figure 64C 64 is a schematic diagram of a cartridge MACS module comprising a flow cell 6410, 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 the population of cells from the outlet 6432 of the flow cell 6410 for recovery 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 fluid such as a buffer and unrecovered magnetically labeled cells. The second fluid conduit 6490 can be configured to receive fluid from the flow cell 6410 without the population of cells. Based on the number of cycles performed, higher purity labeled cells can be recovered. For example, a single cell separation cycle may produce about 80% cell purity, a second cell separation cycle may produce about 96% cell purity, a third cell separation cycle may produce about 99.2% cell purity, and a fourth cell separation cycle may produce about 99.84% cell purity.

[0264] In some variations, applying centrifugal force to the magnetic cell separation process can further attract labeled cells toward the magnetic array independently of fluid flow rate in order to maintain throughput. Figures 65A to 65C is a schematic diagram of a cartridge MACS module 6500 that utilizes centrifugal force to assist in the cell separation process. Figure 65A Depicted is a flat flow cell 6510 configured to be wrapped to form a generally cylindrical shape 6512. The flow cell 6510 can include a tortuous flow path 6520.

[0265] Figure 65BA cylindrical flow cell 6510 is illustrated as being concentrically surrounded by (e.g., nested within) a cylindrical magnet array 6530. Figure 65B , only a cross-section of the magnet array 6530 is shown for clarity. The flow cell 6510 can be spaced apart from the magnet array 6530 by a predetermined spacing distance. Thus, the flow cell 6510 can be configured to rotate 6550 about a longitudinal axis to generate a centrifugal force on the fluid 6540 within the flow path 6520 in an outward direction toward the magnet array 6530. During the cell separation process, the fluid can be subjected to Figure 65C The depicted set of forces includes a bulk fluid force 6560 in the axial (e.g., bulk flow) direction, a centrifugal force 6570 in the radially outward direction from the center of rotation (e.g., proportional to the net particle system buoyancy), and a magnetic force 6580 extending radially outward from the center of rotation (e.g., proportional to the net particle system magnetic attraction 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 improving the specificity and efficiency of cell separation.

[0266] Figures 66A to 66C 66 is a schematic diagram of a cell separation system and process. The magnetic cell separation system may include a flow cell 6610 and a magnet array 6630, the flow cell including a flow path 6620 (schematically shown as flat for clarity). As shown in step 6600, a group 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, a group of cells 6640 may be labeled with a magnetically activated cell selection (MACS) reagent. The magnet array 6630 may be disposed outside the flow cell 6610 so 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 a group of cells 6640 out of the flow cell 6610.

[0267] At step 6602, the flow cell 6650 may be rotated to generate centrifugal force to push 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 so that gravity assists the flow of fluid through the flow cell 6610. At step 6604, the magnet array 6630 may magnetically attract a group of cells 6640 toward the magnet array 6630 for a predetermined residence 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 reservoir. In some variations, the fluid (e.g., cells 6160, 6170) within the flow cell may be held statically within the flow cell 6110 for a predetermined residence 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 can be released after a dwell time, and the cells 6640 can be recovered.

[0268] Figure 12A and Figure 12B The magnets of the docking station MACS module 1200 and the cartridge MACS module 1210 are illustrated. Figure 12A is shown in the ON configuration and in Figure 12B Shown in the OFF configuration.

[0269] bioreactor

[0270] The bioreactor described herein may include a reservoir configured to culture mammalian cells. Typically, cell and gene therapy products can be grown in a bioreactor to produce a clinical dose, which can then be administered to a patient. A variety of biological and environmental factors can be controlled to optimize the proliferation rate and success rate of cell growth. The bioreactor module described herein can enable one or more of the monitoring, regulation, and / or control of cell growth (e.g., to promote consistent and effective cell proliferation).

[0271] Figure 67A67 is a schematic diagram of a bioreactor system 6700 that includes one or more of the following: a cartridge bioreactor module 6750, a cartridge bioreactor module 6750 including 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 sensors 6720 may be configured to monitor (e.g., measure, sense, determine) one or more characteristics of the cartridge bioreactor module, including one or more characteristics of cells in the bioreactor 6710. For example, the sensors 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, and the like, which may be used to detect the environment within the bioreactor 6710. One or more of these sensors may be non-invasive optical sensors.

[0272] Figure 67B 67 is a schematic diagram of a cell processing and manufacturing system including a CPS 6760 and a bioreactor system 6700, wherein the bioreactor system 6700 includes a docking station bioreactor module 6712, a cassette 6714, and a cassette bioreactor module 6750. The docking station bioreactor module 6712 may include an agitator 6730 as well as a temperature regulator, a sensor, and a gas regulator (not shown). The CPS 6760 may also include a fluid transfer connector 6780. In some variations, the cassette 6714 for cell processing may include a liquid transfer bus and, in addition to the cassette bioreactor module 6750, include multiple modules (e.g., CCE modules, MACS modules, EP modules). Each module may be fluidically connected to the liquid transfer bus. In variations, the cassette bioreactor module may include at least one bioreactor.

[0273] like Figure 67B As shown, the docking station bioreactor module 6712 can be configured to interface with the cartridge bioreactor module 6750 of the cartridge 6770, wherein the cartridge bioreactor module 6750 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 the cell culture medium containing cells within the bioreactor. In some variations, a fluid transfer connection 6780 can be configured to couple the docking station bioreactor module 6700 to the cartridge bioreactor module 6750.

[0274] like Figure 67BAs shown, the cassette 6714 is disposed within the CPS 6760. Once the fluid transfer connection 6780 couples the docking station bioreactor module 6712 of the CPS 6760 to the cassette bioreactor module 6750 of the cassette 6714 (e.g., to create a sterile flow path), the cell culture medium within the bioreactor of the cassette bioreactor module 6750 can be agitated by the agitator 6730 of the docking station bioreactor module 6712. In some variations, the fluid transfer connection 6780 can include a set of foldable side walls (e.g., like an accordion) that are configured to receive and dissipate agitation from the agitator 6730 without transmitting such motion to the CPS 6760. That is, the fluid transfer connection 6780 can act as a bellows to maintain the connection between the docking station bioreactor module 6712 of the CPS 6760 and the cassette bioreactor module 6780 without agitating the CPS 6760.

[0275] In some variations, the agitator can be configured to generate motion (e.g., orbital, rotational, linear) to the bioreactor of the cartridge bioreactor module 6750 to mix the culture if desired to facilitate interaction with reagents and cells. For example, orbital motion can be used to generate a uniform culture volume so that a smaller sample taken from the culture can represent the entire culture. In some variations, the agitator 6730 can include one or more impellers. The agitator 6730 can be configured to provide variable intensity mixing at defined cycles during the culture period.

[0276] In some variations, orbital motion can promote increased interactions within cell cultures, such as in annular bioreactors described herein, which include geometries that promote continuous and gentle flow of fluid around the bioreactor, thereby aiding uniform mixing with minimal shear stress imparted to the cells.

[0277] In some variations, a temperature regulator can be configured to control the temperature of a bioreactor and a corresponding process. The temperature regulator can be coupled to the bioreactor. For example, the temperature regulator can control the temperature of a cell culture between about 2°C and about 40°C, thereby ensuring that the culture is heated to physiological conditions and cooled to slow metabolic processes (e.g., to maintain cells in a dormant state) as needed. For example, the thermal regulator can include a circulating coolant coupled to a heat exchanger coupled to a thermal interface (e.g., a heating / cooling plate of the docking station bioreactor module 6712).

[0278] In some variations, a gas regulator may be coupled to a 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, such as Figure 2EAs shown. A 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 contain oxygen, such as pure oxygen. In some variations, the gas regulator may include 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 the flow of gas through the bioreactor. The solenoid may be configured to pulse to control the amount and composition of the gas received by 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 to measure the gas mixture and / or flow rate. Additionally or alternatively, the sensor may be configured to perform closed-loop control of the flow of gas through the gas regulator.

[0279] 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, in response to 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 between about 5.5 and about 8.5. One or more of the CO2 composition, buffer and reagent (e.g., acid, alkali) of the gas in the bioreactor can be used to regulate the pH. In some variations, the dissolved oxygen concentration of the bioreactor can be between about 0% and about 21%. The nitrogen component of the gas in the bioreactor can be used to regulate the dissolved oxygen concentration. For example, the flow rate and composition of the agitator in the bioreactor and the gas contacting the cell culture can be controlled to adjust the dissolved carbon dioxide concentration.

[0280] In some variations, the measured dissolved oxygen from the dissolved oxygen sensor can be used to control the oxygen concentration of 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 produce anoxic conditions.

[0281] Figure 68A and Figure 68B6 is a cross-sectional perspective view of a bioreactor 6800 including a housing 6810, which includes a base 6812, sidewalls 6814, and a top 6816. A gas permeable membrane 6820 may be coupled to one or more of the base 6812 and sidewalls 6814 of the housing 6810. In some variations, the housing 6810 may include a first chamber 6830 having a first volume and a second chamber 6832 having a second volume, the first chamber 6830 being separate from the second chamber 6832, and the first volume being 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 greater overall working volume range (e.g., 100:1). The first chamber 6830 may include a well shape with an angled base surface to facilitate pooling of fluid at the center of the first chamber 6830 during aspiration. In some variations, the base 6812 may be positioned on a thermal regulator (not shown), such as a thermoelectric element. In some variations, the housing 6810 may be constructed from a thermally conductive material such as metal (eg, aluminum).

[0282] In some variations, the bioreactor 6800 may be coupled to a gas regulator (not shown) to facilitate the transport of gases across the gas permeable membrane 6820 (e.g., into and out of the culture). The gas permeable membrane 6820 may be configured to hold the cell culture. Gas may diffuse through the surface of the culture that contacts the gas permeable membrane to enable increased oxygenation of the cell culture and removal of gaseous metabolic byproducts of the cell culture, thereby increasing the potential for metabolic activity. For example, the gas permeable membrane 6820 enables dissolved oxygen to diffuse into the culture immediately adjacent to the cell bed, where it may be consumed. In some variations, the bioreactor may be coupled to both a first gas regulator for facilitating the transport of gases across the gas permeable membrane and a second gas regulator for facilitating control of the gas composition in the headspace.

[0283] In addition to gas delivery, the bioreactors described herein can also be configured to effectively control the temperature of the cell culture using thermally conductive interfaces along both the base and sidewalls of the bioreactor (e.g., gas permeable membrane 6820, housing 6810).

[0284] In some variations, the first chamber 6830 may include a working volume between about 10 ml and about 100 ml. In some variations, the first chamber 6830 may include a total volume between about 10 ml and about 130 ml. In some variations, the second chamber 6832 may include a working volume between about 100 ml and about 1000 ml. In some variations, the second chamber 6832 may include a total volume between about 100 ml and about 1400 ml. In some variations, the first chamber 6830 may include a diameter between about 10 mm and about 100 mm, and a height between about 10 mm and about 100 mm. In some variations, the second chamber 6832 may include a diameter between about 100 mm and about 250 mm, and a height between about 10 mm and about 100 mm.

[0285] like Figure 68B As shown, the base 6822 of the breathable membrane 6820 may include an angle between about 3 degrees and about 10 degrees relative to the base 6812 of the housing 6810. Similarly, Figure 69A and Figure 69B A tilted base is depicted. For example, due to the tilt of base 6822, chambers 6830, 6832 are deeper toward the center of bioreactor 6800. This can encourage cell growth toward the center of bioreactor 6800, which can aid in one or more of cell sampling, cell transport, cell recovery, etc. In some variations, the orbital motion of bioreactor 6800 can encourage cell aggregation toward the center of bioreactor 6800, thereby increasing interactions between cells.

[0286] In some variations, the breathable membrane 680 may include a curved surface. In some variations, the breathable membrane may include a set of patterned curved surfaces. For example, the set of patterned curved surfaces may include a radius of curvature between about 50 mm and about 500 mm.

[0287] In some variations, a bioreactor can be configured to facilitate monitoring (e.g., temperature, pH, dissolved oxygen) and fluid flow between chambers (e.g., gas composition, fluid transfer). Figure 68C As shown, the housing 6810 may include one or more nesting surfaces that are curved about a longitudinal axis (e.g., a center) of the housing 6810. For example, the nesting surfaces may include a set of concentric rings. The housing 6810 may include an annular shape. Figure 68C is a perspective view of a housing 6810 including a set of apertures 6818 (e.g., holes, openings, slits, slots), and Figure 68D6818 is a bottom view of the housing. In some variations, aperture 6818 can enable gas and / or heat transfer between components and chambers of bioreactor 6800. Additionally or alternatively, one or more sensors can be coupled to aperture 6818. For example, aperture 6818 can be coupled to a non-contact sensor (e.g., pH, DO), such as an optical sensor (not shown) configured to detect fluorescent spots disposed on a surface of the bioreactor. In some variations, one or more of a sensor and a fluid connector can be introduced through aperture 6818.

[0288] In some variations, the breathable membrane extends along the base 6812 and sidewalls 6814 of the housing 6810, as shown in FIG. Figure 68B In some variations, the breathable membrane extends only along the base 6812 of the housing 6810. Figure 68E is a perspective view of the breathable membrane 6820 and Figure 68F 68 is a side view of the breathable membrane, wherein the outer surface of the breathable membrane 6820 includes one or more protrusions 6824 (e.g., protrusions, spacers, ribs). The protrusions 6824 are also Figure 68G Perspective drawing and Figure 68H 6820. The protrusion 6824 contacts the housing 6810 and defines a cavity between the housing 6810 and the gas permeable membrane 6820. That is, the protrusion 6824 can be configured to mechanically separate the housing 6810 from a portion of the gas permeable membrane 6820 to facilitate heat transfer from the housing 6810 to the cell culture. In some variations, the gas permeable membrane can comprise polydimethylsiloxane (PDMS) (e.g., silicone), fluorinated ethylene propylene (FEP), polyolefin (PO), polystyrene (PS), ethylene vinyl acetate (EVA), and have a thickness between about 0.1 mm and about 0.4 mm, between about 0.2 mm and about 0.3 mm, and about 0.25 mm, including all ranges and subvalues ​​therebetween.

[0289] Figure 69A is a cross-sectional side view of a housing 6910 of a bioreactor including a first chamber 6912 , a second chamber 6914 , and a post 6916 extending along a longitudinal axis of the housing 6910 . Figure 69B is a cross-sectional perspective view of the housing 6910 showing the nested curves of the housing 6910. The column 6916 can be configured to facilitate cell culture in conjunction with agitation such as orbital motion.

[0290] Figure 70 is an exploded perspective view of a bioreactor 7000, which includes a housing 7010, a gas permeable membrane 7020, and a top 7030. The top 7030 can be constructed of a material such as polyethylene.

[0291] Figure 71Ais a plan view of a bioreactor 7100 , which includes a first chamber 7110 and a second chamber 7120 . Figure 71B is a cross-sectional side view of bioreactor 7100.

[0292] Figure 13 and Figure 14 is a perspective view of the cartridge 1300 and the docking station bioreactor module interface 1310. Figure 14 In FIG, the docking station bioreactor module interface 1310 is coupled to the cartridge 1300 .

[0293] Electroporation module

[0294] In some variations, the electroporation module can be configured to promote 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 groups of electrodes for applying direct current or alternating current to the chamber. The discharge from one or more capacitors or current sources can generate enough current in the chamber to promote the transport of polynucleotides, proteins, nucleoprotein complexes or other macromolecules into cells in a cell product. As with other modules described herein, one or more components for a process step (here, electroporation) can be provided on the box or in a docking station to which the box is interfaced. For example, a capacitor and / or a battery can be provided in a docking station module on the box module. In some variations, the electroporation module can be configured to apply an electric field to a cell suspension under a continuous flow in a microfluidic device, for example, as described in Sci.Rep.6:21238 (2016) by Garcia et al.

[0295] In addition or alternatively, the intracellular delivery of macromolecules can also be achieved by other methods (such as mechanical perforation).It should be understood that throughout the disclosure, the modification including the electroporation module may alternatively or additionally include a mechanical perforation module, or include another module configured to perform any suitable method for delivering macromolecules to cells.Mechanical perforation can be achieved by, for example, applying instantaneous fluid pressure to a solution containing cells or by (for example, via a microneedle) applying physical pressure to cells. The exemplary method of mechanical perforation by passing a cell suspension through a constriction is provided in, for example, International Patent Publication No. WO 2017 / 041051 and No. WO 2017 / 123663, and is incorporated herein by reference.Mechanical perforation can also be achieved by applying eddy current to a cell suspension in a microfluidic device.

[0296] Figure 72is a schematic diagram of an electroporation module 7200 (e.g., an electroporation system) that includes an electroporation chamber 7210 (which may include 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.

[0297] In some variations, fluid conduit 7210 can be configured to receive a first fluid containing cells and a second fluid. A set of electrodes can be coupled to fluid conduit 7210. A pump can be coupled to fluid conduit 7210. Controller 7260 can be configured to: generate a first signal to introduce the first fluid into fluid conduit 7210 using pump 7220; generate a second signal to introduce the second fluid into fluid conduit 7210 such that the second fluid separates the first fluid from the third fluid; and generate an electroporation signal to electroporate cells in fluid conduit 7210 using the set of electrodes.

[0298] In some variations, the second fluid may include gas or oil. In some variations, the controller may be configured to generate a third signal to introduce the 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 a plurality of modules, such as an electroporation module 7200. Each module may be fluidically connected to the liquid transfer bus.

[0299] A set of sensors 7250 can be configured to measure a fluid change in a fluid conduit, such as a change from a first fluid to a second fluid (e.g., liquid to air) in the fluid conduit. The module 7200 can also include a set of valves configured to ensure that the fluid does not flow back into the electroporation chamber 7210 and / or the fluid source 7240. The electroporation chamber 7210 can include 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 can generate square wave pulses, as described in more detail herein.

[0300] In some variations, the electroporation module 7200 (e.g., valve 7234, pump 7220, sensor 7250, and controller 7260) can be configured to control the flow of fluid through the electroporation chamber 7210 in a discontinuous (e.g., batch process) manner. For example, a first batch of cells can undergo electroporation and be physically separated from a second batch of cells by an intermediate fluid (such as air) or fluid (such as oil). Separating the cell batches can reduce mixing of transfected cells with non-transfected cells and further ensure a fixed batch volume. That is, the fluid gap can form a visually verifiable boundary between the cell batches to reduce diffusion and mixing between electroporated cells and non-electroporated cells. Separating the cell batches can reduce the duration of cell exposure to certain cytotoxic agents (e.g., electroporation buffer), thereby improving performance.

[0301] In some variations, a batch of cells can be electroporated when substantially stationary (e.g., substantially without fluid flow). In contrast, conventional continuous flow electroporation has an upper limit on the fluid flow rate associated with transfection efficiency. In the batch processing described herein, a batch of cells can be transferred to and from the electroporation chamber 7210 at a predetermined rate to increase the total flux of the system 7200 without reducing electroporation efficiency. In addition, the electroporation system 7200 does not utilize precisely controlled flow rates / pulse rates, such as those required for continuous flow electroporation systems.

[0302] Figure 73 7 is an exploded perspective view of an electroporation module 7300 of a cartridge, which may include electrodes 7310, a fluid conduit 7320 (e.g., an electroporation chamber), a base plate 7330 (e.g., an alloy busbar), a housing 7340, and fasteners 7350. In some variations, the fluid conduit 7320 may be configured to hold a fluid volume between approximately 0.4 ml and approximately 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 insulating washers. 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 matter (e.g., charged molecules, DNA, proteins) on the electrode surfaces.

[0303] Generally, a method for 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 and separated from the first fluid by the second fluid. In some variations, the first fluid may be substantially static when the electroporation signal is applied.

[0304] Figures 74A to 74B Schematic diagram of a variation of electroporation process 7400, 7402. Method 7400 may include loading cells 7410 into electroporation chamber 7450. For example, at step 7412, a first fluid may be pumped into the electroporation chamber by opening valve v1 and pumping to generate negative pressure (valves v2 and v3 are closed). At step 7414, a second fluid (e.g., gas, oil) may separate the first fluid from the third fluid to produce the first batch of cells to be electroporated. For example, valves v1 and v3 may be closed, valve v2 may be opened, and the pump may generate negative pressure. In some variations, the loading volume may be between about 1 ml and about 3 ml, and the pumping time may be between about 8 seconds and about 15 seconds (at a rate of about 20 ml / min). At step 7420, the cells of the first fluid may be electroporated when each valve in the valve is closed and the pump is closed. At step 7430, cells of the first fluid may flow from the electroporation chamber 7450 to an output, with valves v1 and v2 closed, valve v3 open, and a pump creating positive pressure.

[0305] Figure 74B Describe another configuration, wherein the pump is arranged between the input and the electroporation chamber so that the pump can be configured to pump in a single direction. Method 7402 may include loading cells 7411 into the electroporation chamber 7450. For example, at step 7416, the first fluid can be pumped into the electroporation by opening valves v1 and v4 and pumping to generate positive pressure (valves v2 and v3 are closed). At step 7418, a second fluid (e.g., gas, oil) can separate the first fluid from the third fluid to produce the first batch of cells to be electroporated. For example, valves v1 and v3 can be closed, valves v2 and v4 are opened, and the pump generates positive pressure. At step 7422, the cells of the first fluid can be electroporated when each valve in the valve is closed and the pump is closed. At step 7432, the cells of the first fluid can flow out from the electroporation chamber 7450 to the output, wherein valves v1 and v4 are closed, valves v2 and v3 are opened, and the pump generates positive pressure.

[0306] In some variations, the impedance / resistance across the electrodes of the electroporation system may increase over time due to electrode passivation / degradation caused by charged biological matter (e.g., charged molecules, DNA, proteins) adhering to the electrode surface. Active electric field compensation may be applied to ensure consistent electric field strength applied to cells across multiple batches of cells. This may reduce the need for electrode surface modifications to reduce passivation.

[0307] Figure 75 is a circuit diagram of a resistor divider network for electroporation process 7500. For example, a group of cells can be introduced into electroporation chamber 7510 and a voltage V chip can be applied to the electroporation chamber. bcorresponds to the resistance of the fluid (e.g., cell mixture). Assuming uniform cell distribution, the fluid resistance R b should be consistent, again assuming that the volume of each fluid batch being electroporated is the same. i Corresponding to the resistance between the fluid and the electrodes, this resistance increases over time during the electroporation process. ps is constant. However, due to R i increases with time, so the voltage applied to the fluid will decrease with time, resulting in a lower electric field strength.

[0308] Since the fluid resistance R b The interpolation used to compensate for the reduced electric field strength may not accurately compensate for electrode passivation due to the small number of pulses that can be applied.

[0309] 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, before applying the electroporation signal to the fluid, a second fluid containing a gas may be received in the fluid conduit. The first fluid may be separated from a third fluid by the second fluid.

[0310] Figures 76A to 76D Graphs 7600 , 7602 , 7604 , and 7606 are diagrams of measurement waveforms and electroporation waveforms. Figure 76A A first resistance measurement pulse 7620 is depicted having a low voltage and a wide pulse width. Figure 76B A second resistance measurement pulse 7622 is depicted having a high voltage and a short pulse width. Figure 76C A third resistance measurement pulse 7624 is depicted having a continuous low voltage waveform for continuously monitoring impedance changes over time. Figure 76D A fourth resistance measurement pulse 7626 having a low AC voltage waveform is depicted for continuously monitoring impedance changes over time. Each of the resistance measurement pulses avoids inducing electroporation in the cells by reducing the voltage and / or pulse width. By monitoring the voltage and current of the applied resistance measurement pulses, changes in resistance can be measured, and the electroporation pulse applied to the cell batch can be compensated accordingly.

[0311] In some variations, the electroporation signal may comprise between about 1 and about 50 pulses, a voltage between about 100 V and about 700 V, a pulse width between about 100 μs and about 1 ms, a pulse interval between about 5 seconds and about 30 seconds, a resistive pulse voltage between about 10 V and about 40 V, and a resistive pulse width between about 10 μs and about 50 μs.

[0312] For example, eight electroporation batches may each receive one electroporation pulse. The electric field strength of each electroporation pulse may be between about 0.5 kV / cm and about 2.0 kV / cm. The electric field strength of the resistance measurement pulse applied before each batch may be less than about 0.2 kV / cm so that electroporation is not induced by the resistance measurement pulse.

[0313] Sterile liquid transfer equipment

[0314] Typically, the sterile liquid 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 box, bioreactor, etc.). In some variations, the sterile liquid transfer device may include a portable consumable that is configured to be moved using a robot. For example, a robot can be configured to move the sterile liquid transfer device from a reagent library to an ISO 7 space, and then to a sterile liquid transfer instrument within the cell processing and manufacturing system. The sterile liquid transfer device is capable of transferring fluids in an automated, sterile, and quantitative manner to automate cell therapy manufacturing.

[0315] Figure 103A and Figure 103B is a perspective view of a sterile liquid transfer apparatus 10300 comprising a fluid chamber 10310 (e.g., a container, a reservoir), a fluid connector 10320, and a pump 10330. Fluid stored within the fluid chamber 10310 can be transferred into and out of the sterile liquid transfer apparatus 10300 through the fluid connector 10320 using the pump 10330. In some variations, the sterile liquid transfer apparatus 10300 may comprise an engagement feature 10340 (e.g., a robotic mount) to facilitate robotic arm control.

[0316] Fluid connectors

[0317] Typically, the aseptic fluid connector described herein can form an aseptic fluid pathway between at least two fluid devices to achieve fluid transmission, which can be one or more of aseptic, fully automatic, and precisely quantitative (e.g., accurately controlling the volume of fluid being transmitted). In some variations, a robot can be configured to couple a fluid connector between at least two aseptic liquid transfer devices and a plurality of cartridge modules. In some variations, a robot can be configured to operate a fluid controller to open and close a group of ports and valves of a fluid connector. Using a robot and a controller to operate a fluid connector can promote the automation and sterility of cell processing and manufacturing systems.

[0318] 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 a robotic arm, generate a first valve signal to translate the first valve relative to the second valve using the robotic arm, and generate a second valve signal to transition the first valve and the second valve to an open configuration.

[0319] 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 the fluid through the sterilant port into the chamber. The controller may be configured to generate a second fluid signal to circulate the sterilant through the sterilant port into the chamber to at least sterilize the chamber. The controller may be configured to generate a third fluid signal to remove the sterilant from the chamber.

[0320] In some variations, the controller may be configured to generate a port signal to couple the first port to the second port using a robot, generate a first valve signal to translate the first valve relative to the second valve using a robot, and generate a second valve signal to transition the first valve and the second valve to an open configuration.

[0321] The fluid connector can further allow multiple connection cycles in a sterile system and can be controlled without human intervention. For example, the fluid connector can include one or more engagement features for facilitating robotic control and alignment features for ensuring proper connection between connector components. Figure 15 is a block diagram of an exemplary variation of a fluid connector system 1500 that includes a fluid connector 1510, a first fluid device 1520 (e.g., a box, a box module, a sterile liquid transfer device (SLTD)), a second fluid device 1522 (e.g., a box, a box module, a SLTD), a sterilant source 1530, a fluid source 1532, a robot 1540, and a controller 1550.

[0322] The fluid connector 1510 can be removably coupled (e.g., connected / disconnected, attached / detached) to each of the first fluid device 1520, the second fluid device 1522, the sterilant source 1532, the fluid source 1532, and the robot 1540. In some variations, the fluid device can include one or more of a cassette and a sterile liquid transfer device. For example, the sterile liquid transfer device can be in fluid communication with the cassette via the fluid connector.

[0323] As described in more detail herein, the individual parts (e.g., male connector, female connector) of fluid connector 1510 can be removably coupled to one another. Robot 1540 can be configured to physically manipulate (e.g., removably couple) one or more of fluid connector 1510, first fluid device 1520, second fluid device 1522, sterilant source 1530, and fluid source 1532 in a predetermined manner. For example, robot 1540 can connect fluid connector 1510 between first fluid device 1520 and second fluid device 1522. Robot 1540 can also connect sterilant source 1530 and / or fluid source 1532 to a sterilant port of fluid connector 1510. In some variations, robot 1540 can control one or more valves and / or ports of fluid connector 1510 and thereby initiate a sterilization process for one or more parts of fluid connector 1510 using, for example, a sterilant from sterilant source 1530. A 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 fluid transfer and sterilization.

[0324] Figure 16A 16. It is a schematic diagram of an exemplary variation of fluid connector 1600. Fluid connector 1600 may include an inner cavity extending along its length and be arranged between first fluid equipment 1630 and second fluid equipment 1640 so that fluid can flow through fluid connector 1600. In some variations, first fluid equipment 1630 and second fluid equipment 1640 can be aseptically connected and disconnected using fluid connector 1600. Fluid equipment 1630, 1640 may include closed sterile equipment and may be fluid equipment of the same or different types. For example, fluid equipment 1630, 1640 may include one or more of a sterile liquid transfer device and consumables (e.g., box). In some variations, 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 be coupled to the first fluid equipment 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.

[0325] The fluid connector 1600 may also 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. Figure 16A 16, first connector 1610 includes a sterilant port 1650 configured to be coupled to a sterilant source (not shown). Additionally or alternatively, second connector 1620 can include sterilant port 1650. Sterilant port 1650 can be configured to be in fluid communication with first distal end 1614 and second distal end 1624 when second port 1626 is coupled to first port 1616, as described in greater detail herein.

[0326] In some variations, fluidic devices 1630, 1640 can include a sterilant chamber and a sterilant port configured to receive a sterilant. The sterilant chamber can enclose a fluidic device connector (not shown) configured to couple to the proximal end of first connector 1610 or second connector 1620. Fluidic devices 1630, 1640 can receive a sterilant in a manner similar to fluidic connector 1600.

[0327] Figure 16B 16 is a detailed schematic diagram of a first connector 1610 including a first port housing 1617 and a chamber 1615. The chamber 1615 can be defined by a cavity enclosed by one or more of the distal ends 1614, 1624. For example, Figure 16B The chamber 1615 may comprise a portion of the first connector 1610 (eg, the first distal end 1614) between the first valve 1618 and the first port 1616 in the closed configuration. In some variations, the first chamber 1615 may comprise a portion of the first connector 1610 (eg, the first distal end 1614) between the first valve 1618 and the first port 1616 in the closed configuration. 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 (eg Figure 16D 16), chamber 1616 may comprise a portion of fluid connector 1600 between first valve 1618 and second valve 1628 (e.g., first distal end 1614 and second distal end 1624). Chamber 1615 may comprise an enclosed volume configured to receive a fluid, such as a sterilant, from sterilant port 1650. In some variations, sterilant port 1650 may comprise an inlet 1652 and an outlet 1654. Figures 16C to 16L and Figure 27 Methods of using the fluid connector are described in more detail.

[0328] In some variations, fluid connector 1600 may include one or more alignment features and robotic engagement features configured to facilitate robotic manipulation, as described in more detail herein. In some variations, fluid connector 1600 may be coupled to one or more sensors, pumps, and valves to facilitate fluid transfer and monitoring.

[0329] In some variations, the fluid-contacting components of the fluid connector can be USP Class VI compatible with cell processing and / or GMP applications. In some variations, the components of the fluid connector can be constructed of materials including, but not limited to, one or more of: cyclic olefin copolymer (COC), polytrifluorochloroethylene, polyetherimide, polysulfone, polystyrene, polycarbonate, polypropylene, silicone, polyetheretherketone, polymethyl methacrylate, nylon, acrylic resin, polyvinyl chloride, vinyl resin, phenolic resin, petroleum-based polymers, glass, polyethylene, polyethylene terephthalate, metal, stainless steel, titanium, aluminum, cobalt chromium, chromium, silicates, glass, alloys, ceramics, carbohydrate polymers, mineral substances, and combinations or composites thereof.

[0330] 17A to 18D Depicted are exterior and interior views of variations of fluid connectors. Figure 17A is a front perspective view of fluid connector 1700 in a closed port configuration. Figure 17B is a rear perspective view of fluid connector 1700, and Figure 17C is a rear view of the fluid connector. Typically, a fluid connector may include internal seals to reduce contamination and aid in sterilization, and alignment features to aid in proper registration of the fluid connector assembly.

[0331] Fluid connector 1700 may include an inner cavity extending along its length. In some variations, fluid connector 1700 may include a first connector 1710, which includes a first proximal end 1712 and a first distal end 1714. The first proximal end 1712 may be configured to be coupled to a first fluid device (not shown for clarity). The first proximal end 1712 may include a Luer connector or any other suitable connector. The first distal end 1714 may include a first port 1716 and a first housing 1717. Figure 17A 17. A first housing 1717 is shown in the figure, which holds a first port 1716 in a closed configuration. The first connector 1710 also includes sterilant ports 1750, 1752 configured to be coupled to a sterilant source (not shown for clarity). In some variations, the sterilant port may include 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 entering the sterilant port when the sterilant port is 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 ensures that minor positioning errors due to robotic manipulation do not affect the operation of the fluid connector.

[0332] The fluid connector 1700 may further include a second connector 1720 comprising a second proximal end 1722 and a second distal end 1724. The second proximal end 1722 may be configured to couple to a second fluid 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 comprise a second port 1726 and a second housing 1727. Figure 17A 17. A second housing 1727 is shown in FIG. 17, which holds the second port 1726 in a closed configuration. The second connector 1720 may include 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 may be configured to couple to the first alignment feature 1760 in a predetermined axial and rotational configuration to assist in mating the first connector 1710 and the second connector 1720.

[0333] The first and second ports 1716, 1726 retained within the first and second housings 1717, 1727 of the respective first and second distal ends 1714, 1724 facilitate robotic control because the ports 1716, 1726 cannot be separated from the fluid connector 1700, thereby reducing the risk of failure of automated handling by the robot.

[0334] 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. 17A to 17F Additionally or alternatively, a user may manually actuate the robotic engagement features 1770 , 1772 to actuate the corresponding ports 1716 , 1726 .

[0335] Figure 17D is a front perspective view of fluid connector 1700 in an open port configuration. Figure 17E is a rear perspective view, and Figure 17F 17 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 in Figure 17D Shown in.

[0336] Figure 18Ais a side view of fluid connector 1800 in an uncoupled configuration, and Figure 18B is a cross-sectional side view of the fluid connector. In some variations, fluid connector 1800 may include a first connector 1810 comprising a first housing 1817 including 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 couple to a corresponding alignment feature (not shown) of a second connector 1820. Fluid connector 1800 may include a second connector 1820 comprising a second housing 1827 including a second port 1826. First connector 1810 and second connector 1820 may be axially aligned, and the alignment features may assist in rotational alignment of first connector 1810 with second connector 1820. First valve 1818 may include a first valve stem 1819, and second valve 1828 may include a second valve stem 1829.

[0337] Figure 18C is a side view of fluid connector 1800 in a coupled configuration, and Figure 18D 18 is a cross-sectional side view of the fluid connector with first housing 1817 and second housing 527 brought together, but with first connector 1810 and second connector 1820 not in fluid communication because first port 1816 and second port 1826 are both in a closed configuration. The first alignment feature on each connector 1810, 1820 can be configured to ensure axial and / or rotational alignment between first connector 1810 and second connector 1820.

[0338] Figure 18E is a side view of fluid connector 1800 in an open port configuration, and Figure 18F is a cross-sectional side view of the fluid connector. Each of the first port 1817 and the second port 1827 transitions from a closed configuration to an open configuration. This creates a closed internal volume within the respective distal ends of each connector 1810, 1820. Each of the first valve 1818 and the second valve 1828 is in a closed configuration, inhibiting fluid flow between the first connector 1810 and the second connector 1820. Each half remains restricted due to the self-closing valves on both sides.

[0339] Figure 18G is a side view of the fluid connector 1800 in an open valve configuration (with the first valve 1818 coupled to the second valve 1828), and Figure 18H 18 is a cross-sectional side view of the fluid connector. For example, the second valve 1828 can be translated along the longitudinal axis of the second connector 1820 toward the first valve 1818. Figure 18G and Figure 18HAs shown, second connector 1820 can be axially compressed to translate second valve 1828 toward first valve 1818. First valve 1818 coupled to second valve 1829 can form a radial seal, and first valve stem 1819 and second valve stem 1828 can contact to enable fluid communication between first connector 1810 and second connector 1820.

[0340] Figures 19 to 26B is a schematic diagram of a variation of a fluid connector system for coupling fluidic devices. In some variations, the fluid connector may include a first connector configured to couple to any one of a plurality of second connectors. Figure 19 1 is a schematic diagram of an exemplary variation of a fluid connector system 1900, which includes a first connector 1910, a plurality of second connectors 1920, 1921, 1922, a first fluid device 1930 (e.g., a sterile liquid transfer device), a second fluid device 1940 (e.g., a consumable), and a robot 1960 (e.g., a robotic arm, a 3DOF robot). The first connector 1910 can be coupled in fluid communication with the first fluid device 1930, and the second connectors 1920, 1921, 1922 can be coupled in fluid communication with the second fluid device 1940. The first connector 1910 and the second connectors 1920, 1921, 1922 can 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.

[0341] 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 fluidic devices, fluid connectors, and sterilization systems. For example, Figure 96A is a plan view of a fluidic device 9600 (eg, 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). Figure 96B and Figure 96Care respective side and perspective views of a fluidic device 9600 coupled to a 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.

[0342] In some variations, the fluid connector may include a third connector disposed between the first connector and the second connector. Figure 20A 2021, 2022, a third connector 2070 (e.g., an instrument, a sterile housing), 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 can be coupled in fluid communication with the first fluidic device 2030, and the second connectors 2020, 2021, 2022 can be coupled in fluid communication with the second fluidic device 2040. The third connector 2070 can 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 a sterilant and circulate the 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.

[0343] 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., 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, fluidic connectors, and the sterilization system.

[0344] Figure 20B and Figure 20C This is a schematic diagram of the fluid connector connection process. Figure 20B , at 2002, the third connector 2070 can be coupled to the distal end of the first connector 2010. At 2004, the distal end of the second connector 2020 can be coupled to the third connector 2070. At 2006, the second connector 2020 can be translated through the third connector 2070 to directly couple the second connector 2020 to the first connector 2010.

[0345] exist Figure 20C , at 2002, the third connector 2070 may be coupled to the distal ends of the first connector 2010 and the second connector 2020. At 2005, each of the first connector 2010 and the second connector 2020 may be translated toward each other via the third connector 2070. 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. Figure 20C Further illustrated are a first port 2090 and a second port 2092 transitionable between a closed port configuration and an open port configuration.

[0346] 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. Figure 21 FIG2 is a block diagram of an exemplary variation of a fluid connector system 2100, which includes a first connector 2110, a plurality of second connectors 2120, 2121, 2122, a third connector 2170 (e.g., an instrument, a sterile housing), a first fluid device 2130 (e.g., a sterile liquid transfer device), a second fluid device 2140 (e.g., a consumable), a first robot 2160, and a second robot 2166. The first connector 2110 can be coupled in fluid communication with the first fluid device 2130, and the second connectors 2120, 2121, 2122 can be coupled in fluid communication with the second fluid device 2140. The third connector 2170 can be coupled between the first connector 2110 and one of the second connectors 2120, 2121, 2122. The third connector 2170 may include an internal lumen configured to receive a sterilant and circulate the 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.

[0347] 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 fluidic connectors, and the sterilization system.

[0348] In some variations, a fluid connector may include a sterilant source coupled to a plurality of second connectors. Figure 22 2 is a block diagram of an exemplary variation of a fluid connector system 2200, which includes a first connector 2210, a plurality of second connectors 2220, 2221, 2222, a first fluid device 2230 (e.g., a sterile liquid transfer device), a second fluid 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 can be coupled in fluid communication with the first fluid device 2230, and the second connectors 2220, 2221, 2222 can be coupled in fluid communication with the second fluid device 2240. The robot 2260 can 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 fluidic device 2230 may include one or more fluid ports 2250 configured to be coupled to an end effector 2262. In some variations, a 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 fluidic 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.

[0349] 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 fluid connector system. Figure 232 is a block diagram of an exemplary variation of a fluid connector system 2300, which includes 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 a housing of the second fluidic device 2340, and a sterilant switch 2392. The first connector 2310 can be coupled in fluid communication with the first fluidic device 2330, and the second connectors 2320, 2321, 2322 can be coupled in fluid communication with the second fluidic device 2340. The robot 2360 can 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 fluid device 2330 may include one or more fluid ports 2350 configured to be coupled to an end effector 2362. In some variations, a 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 fluid device, the fluid connector, and the sterilization system. In some variations, a sterilant conduit may be routed from the switch 2392 through the second fluid device 2340 to the respective second connectors 2320, 2321, 2322.

[0350] In some variations, a fluid connector may include a sterilant source coupled to a plurality of second connectors, each second connector having a sterilant port (eg, a sterilant valve) and a sterilant conduit passing through the fluidic device. Figure 24A2 is a block diagram of an exemplary variation of a fluid connector system 2400, which includes a first connector 2410, a plurality of 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 can be coupled in fluid communication with the first fluidic device 2430, and the second connectors 2420, 2421, 2422 can be coupled in fluid communication with the second fluidic device 2440. The robot 2460 can include one or more end effectors 2462, 2464 configured to manipulate and / or couple to one or more of the first fluidic device 2430 and the first connector 2410. For example, the first fluid device 2430 may include one or more fluid ports 2450 configured to be coupled to an end effector 2462 .

[0351] In some variations, each of the second connectors 2420, 2421, 2422 may include a respective sterilant port 2494, 2496, 2498 comprising 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 ports 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.

[0352] Figure 24B 2402, 2404, 2406 of a fluid connector connection process in which a first connector 2410 is coupled to a second connector 2420. For example, when the first connector 2410 and the second connector 2420 are separated and disconnected 2402, the sterilant port 2494 is in a closed valve configuration. Figure 24C is a detailed schematic diagram of sterilant valve 2494. In some variations, at 2404 and 2406, when the first connector 2410 is coupled to the second connector 2420, the valve 2494 may transition to an open valve configuration.

[0353] In some variations, the plurality of second connectors may include one or more pneumatic sterilant valves and a sterilant path through the fluidic device. Figure 25A2 is a block diagram of an exemplary variation of a fluid connector system 2500, which includes a first connector 2510, a plurality of second connectors 2520, 2521, 2522, a first fluid device 2530 (e.g., a sterile liquid transfer device), a second fluid device 2540 (e.g., a cassette), a robot 2560, and a sterilant switch 2592 coupled to a sterilant source (not shown). The first connector 2510 can be coupled in fluid communication with the first fluid device 2530, and the second connectors 2520, 2521, 2522 can be coupled in fluid communication with the second fluid device 2540.

[0354] In some variations, each of the second connectors 2520, 2521, 2522 may include a respective pneumatic sterilant port 2594, 2596, 2598 comprising 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 ports 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.

[0355] 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 configured to couple to the end effector 2562 to pneumatically actuate the sterilant ports 2594, 2596, 2598. Pneumatically actuated sterilant ports may enable sterilant conduits to be formed with fewer check valves between the sterilant ports 2594, 2596, 2598 and the switch 2592.

[0356] Figure 25B 25 is a schematic diagram of a fluid connector connection process 2502 and 2504, wherein a first connector 2510 is coupled to a second connector 2520. For example, when the first connector 2510 and the second connector 2520 are separated and disconnected 2502, the sterilant port 2594 is in a closed valve configuration. Figure 25Cis a detailed schematic diagram of sterilant valve 2594. In some variations, at 2504, when the first connector 2510 is coupled to the second connector 2520 and the valve 2594 is pneumatically actuated, the valve 2594 may transition to an open valve configuration.

[0357] Liquid transfer bus

[0358] Typically, to permit transfer of one or more of a cell product (i.e., a solution containing a cell product), a fluid, and a reagent between modules, the modules of the cartridge may be fluidically coupled to one another, either directly or via one or more liquid transfer buses. In some variations, the liquid transfer bus may comprise a portion of the cartridge configured to control the flow and distribution of the cell product between the modules and the reservoir. The liquid transfer bus may comprise one or more of a fluid manifold, a fluid conduit (e.g., a tubing), and one or more valves (including, but not limited to, a 2 / 2 valve, a 3 / 2 valve, a 3 / 3 valve, a 4 / 2 valve, and a rotary selector valve).

[0359] Transferring cell products, reagents, or fluids within the cassette can be accomplished by any pump or other structure that creates a pressure differential between a fluid in one portion of the cassette and a fluid in another portion of the cassette. For example, the cassette may include one or more pumps; the cassette may be preloaded with a pressurized fluid contained behind a valve; the cassette may be connected to a fluid source or reservoir. The cassette may contain one or more mechanical pumps (e.g., a linear pump, a peristaltic pump, a gear pump, a progressive cavity pump, a plunger pump), or portions of a pump (i.e., a pump may interface with a pump actuator). External pressure may be applied to the cassette, to tubing within the cassette, or to a bag within the cassette (i.e., applying pressure to the liquid in the bag or to the headspace gas in the bag). In some variations, the arrangement of the cassette components may facilitate gravity-based fluid transfer within the cassette (e.g., gravity-fed pumping). Although one advantage of the disclosed variations may be reduced operator intervention, the systems and methods of the present disclosure may be used with manual operation in a designed workflow or as an adjunct to automated operation in situations where automated systems operate imperfectly. For example, process steps may include manual intervention, such as fluid input or output. An operator may intervene in an automated process to correct device operation (e.g., manually compressing a bag to flush excess fluid into the system). Fluids may include liquids and / or gases, as compressed gas supplied externally or provided in a pressurized chamber may be used to generate liquid flow, for example, to transfer a solution containing a cell product from one module to another.

[0360] In some variations, the liquid transfer bus may be configured to deliver the cell product to each module in a series of modules in an order set by the design of the box or in an order determined by the operation of the system by one or more processors. Similarly stated, some variations of the box may have the following advantages: the order of the cell processing steps and the processing parameters for any of the cell therapy processing steps may not be set by the box, but rather controlled by the controller. In some variations, the liquid transfer bus may be controlled to deliver the cell product to the modules in any of a variety of sequences, or to skip one or more modules (e.g., by configuring the state of valves attached to the fluid bus). In some variations, a module may be used more than once in a 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 exemplary method, the method includes culturing a cell product in a first bioreactor module, transferring the cell product to a CCE module for enrichment of a desired cell type, transferring the cell product to a second bioreactor module for a second culturing step, washing the CCE module with a wash solution, and transferring the cell product to a CCE module for a second enrichment step.

[0361] In some variations, one or more liquid transfer buses may be fluidly coupled to a plurality of bags or reservoirs for providing solutions or reagents, storing cellular products, or collecting waste fluids or reagents.

[0362] In some variations, the box may include one or more pumps that are fluidly coupled to a liquid transfer bus and / or one or more modules. The pump may include a motor operatively coupled to a control circuit and a power source (e.g., a battery or an electrical connector for a power source external to the box). In some variations, the pump may be separated into a pump on the box and a pump actuator on one or more docking station modules of the system. The pump may be an opening in the box with tubing arranged around the circumference of the opening and configured to receive a pump actuator (e.g., a peristaltic rotor). By separating the components of the pump that contact the cell product (i.e., the tubing) from the components of the pump that perform operations on the cell product (i.e., the pump actuator, e.g., a peristaltic rotor), the box may be compact and simplified. For example, Figure 26A and Figure 26B The example is in the decoupled configuration ( Figure 13 A) Pump head 2610 and pump 2610 of the cassette in a coupled configuration.

[0363] 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 include one or more of a positive displacement pump (e.g., a peristaltic pump, a diaphragm pump, a syringe pump), a centrifugal pump, combinations thereof, etc. One or more fluid sources may be coupled to the pumps.

[0364] In some variations, the pump may be configured to receive a pump signal (generated by a 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., an inert gas, air) to remove the sterilant.

[0365] In some variations, a discontinuous flow pump (e.g., a peristaltic pump) may generate a pulsating flow when, for example, a tube contracts and relaxes between rollers. In some variations, closed-loop feedback from a flow sensor may be used to compensate for the pulsating flow to produce a substantially continuous flow rate. For example, a flow sensor may be coupled to a 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 that is configured to reduce the "ripple" measured by the flow sensor. Additionally or alternatively, the controller may apply a periodic error correction to the pump signal to reduce periodic errors that may be unique to each pump. For example, a flow sensor may measure and determine the periodic error of the pump. The pump signal including the periodic error correction may correspond to a waveform having an inverse shape of the error. The resulting pump flow rate may be corrected for fluctuations in the flow rate.

[0366] Controller

[0367] 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 enclose 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 remotely provided to the CPS 110 (e.g., the input device 128, the display 130).

[0368] processor

[0369] The processors described herein (e.g., processor 122) can process data and / or other signals to control one or more components of a system (e.g., CPS 110, controller 120). The processors can be configured to receive, process, compile, compute, store, access, read, write, and / or transmit data and / or other signals. Additionally or alternatively, the processors can be configured to control one or more components of a device and / or one or more components of a controller (e.g., a console, a touch screen, a personal computer, a laptop computer, a tablet computer, a server).

[0370] 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 the storage medium (e.g., memory, flash drive, memory card, database). In some variations, the processor may be any suitable processing device configured to run and / or execute a set of instructions or codes, 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 for reducing data rates and / or memory requirements), encryption processors (e.g., for secure wireless data transmission), and / or central processing units (CPUs). The processor may be, for example, a general-purpose processor, a field programmable gate array (FPGA), an application-specific integrated circuit (ASIC), a processor board, and / or the like. 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 company can provide basic device technologies for a variety of component types, for example, 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 (for example, silicon conjugated polymer and metal conjugated polymer-metal structures), mixed analog and digital, etc.

[0371] The systems, devices, and / or methods described herein may be implemented by software (implemented on hardware), hardware, or a combination thereof. The hardware modules may include, for example, a general-purpose processor (or microprocessor or microcontroller), a field programmable gate array (FPGA), and / or an application-specific integrated circuit (ASIC). The software modules (implemented on hardware) may be expressed in a variety of software languages ​​(e.g., computer code), including structured text, typescript, C, C++, C#, Python, Ruby, Visual and / or other object-oriented, procedural, or other programming languages ​​and development tools. Examples of computer code include, but are not limited to, microcode or microinstructions, machine instructions (such as those generated by a compiler), code for generating network services, and files containing high-level instructions that are executed by a computer using an interpreter. Additional examples of computer code include, but are not limited to, control signals, encrypted code, and compressed code.

[0372] Memory

[0373] The cell processing and manufacturing systems and devices described herein may include a memory (e.g., memory 124) configured to store data and / or information. In some variations, the memory may include one or more of a random access memory (RAM), a static RAM (SRAM), a dynamic RAM (DRAM), a memory buffer, an erasable programmable read-only memory (EPROM), an electrically erasable read-only memory (EEPROM), a read-only memory (ROM), a flash memory, a volatile memory, a non-volatile memory, a combination thereof, or the like. In some variations, the memory may store instructions to cause a processor to execute 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 communication. Some variations described herein may involve a computer storage product having a non-transitory computer-readable medium (also referred to as a non-transitory processor-readable medium) having instructions or computer code thereon for performing various computer-implemented operations. Computer-readable media (or processor-readable media) are non-transitory in the sense that they do not inherently include transient propagating signals (e.g., propagating electromagnetic waves that carry information over a transmission medium such as space or a cable). Computer code (also referred to as code or algorithm) may be that which is designed and constructed for a specific purpose or purposes. In some variations, the memory may be configured to store any data received 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.

[0374] Input devices

[0375] In some embodiments, the display may include an input device 128 (e.g., a touch screen) configured to receive input data from a user and / or be operatively coupled to the input device. For example, user input to the input device 128 (e.g., a keyboard, buttons, touch screen) 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 user input. For example, the input device may include a touch surface for the user to provide input corresponding to the user input (e.g., a finger contact to the touch surface). An input device including a touch surface may be configured to detect contact and movement on the touch surface using any of a variety of touch-sensitive technologies, including capacitive technology, resistive technology, infrared technology, optical imaging technology, dispersive signal technology, acoustic pulse recognition technology, and surface acoustic wave technology. In variations of the input device including at least one switch, the switch may have, for example, at least one of a button (e.g., a hard key, a soft key), a touch surface, a keyboard, an analog stick (e.g., a joystick), a directional pad, a mouse, a trackball, a dial, a stepper 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 the optical sensor and classify user gestures as user input. The microphone may receive audio data and recognize user speech as user input.

[0376] In some variations, in addition to a display, the cell processing and manufacturing system may optionally include one or more output devices, such as an audio device and a tactile device. The audio device may audibly output any system data, alarms, and / or notifications. For example, when a fault is detected, the audio device may output an audible alarm. In some variations, the audio device may include at least one of a speaker, a piezoelectric audio device, a magnetostrictive speaker, and / or a digital speaker. In some variations, a user may use the audio device and a communication channel to communicate with other users. For example, a user may establish an audio communication channel (e.g., a VoIP call).

[0377] Additionally or alternatively, the system can include a haptic device configured to provide additional sensory output (e.g., force feedback) to the user. For example, the haptic device can generate a tactile response (e.g., vibration) to confirm user input to an input device (e.g., a touch surface). As another example, tactile feedback can notify the user that the input is overridden by the processor.

[0378] Communication equipment

[0379] 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 via one or more wired networks and / or wireless networks. In some variations, the communication device may include a radio frequency receiver, a transmitter, and / or an optical (e.g., infrared) receiver and transmitter that are configured to communicate with one or more devices and / or networks. The communication device may communicate by wire and / or wirelessly.

[0380] A communication device may include RF circuitry configured to receive and transmit RF signals. The RF circuitry may convert electrical signals into / from electromagnetic signals and communicate with a communication network and other communication devices via the electromagnetic signals. The RF circuitry may include well-known circuitry 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, and the like.

[0381] Wireless communication by any device may use any of a variety of communication standards, protocols, and technologies, including, but not limited to, 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-HSPDA), 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 (WiFi) (e.g., IEEE 802.11a, IEEE 802.11b, IEEE 802.11g, IEEE 802.11b), IEEE 802.11c, IEEE 802.11d ... 802.11n, etc.), Voice over Internet Protocol (VoIP), Wi-MAX, email protocols (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 extended for Instant Messaging and Presence Support (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, the devices herein can communicate directly with each other without transmitting data through a network (e.g., via NFC, Bluetooth, WiFi, RFID, etc.).

[0382] In some variations, the systems, devices, and methods described herein may communicate with other wireless devices via, for example, one or more networks, each of which may be any type of network (e.g., a wired network, a wireless network). Communications may or may not be encrypted. A wireless network may refer to any type of digital network that is not connected by any type of cable. Examples of wireless communications in a wireless network include, but are not limited to, cellular, radio, satellite, and microwave communications. However, a wireless network may be connected to a wired network to connect to the Internet, other carrier 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. There are many different types of wired networks, including wide area networks (WANs), metropolitan area networks (MANs), local area networks (LANs), internetworking networks (IANs), campus networks (MWANs), global area networks (GANs) (such as the Internet), and virtual private networks (VPNs). Hereinafter, a network refers to any combination of wireless, wired, public, and private data networks, typically interconnected via the Internet, to provide a unified networking and information access system.

[0383] Cellular communications can encompass technologies such as GSM, PCS, CDMA or GPRS, W-CDMA, EDGE or CDMA2000, LTE, WiMAX, and 5G network standards. Some wireless network deployments combine networks from multiple cellular networks or use a hybrid of cellular, Wi-Fi, and satellite communications.

[0384] monitor

[0385] 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 comprise 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 electronic paper / electronic ink display, a laser display, and / or a holographic display.

[0386] II. Methods

[0387] Generally, the systems and apparatus described herein can perform one or more cell processing steps to produce a cell product. Figure 282800 is a flow chart of a cell processing method 2800. Method 2800 may include enriching a selected cell population 2802 in a solution (e.g., a fluid). For example, the solution may be transported to a cartridge CCE module via a liquid transport bus. The cartridge may be moved by a user to a docking station of a cell processing station (CPS). The docking station may include a plurality of docking station modules corresponding to one or more modules of the cartridge. For example, the docking station module may include a docking station CCE module that is configured to interface with the cartridge CCE module to perform cell processing on the cells within the cartridge. In this case, the cell process includes enriching a selected cell population via countercurrent centrifugal elutriation.

[0388] Additionally or alternatively, for any of the steps described herein, the cell product can be introduced into and out of the cassette via a sterile liquid transfer port (manually or automatically). In some variations, the cassette can be sterilized in the feed-through port (manually or automatically).

[0389] In some variations, the selected cell population can be washed in a solution 2804. For example, the solution can be delivered to a CCE module of a cartridge within a docking station via a liquid transfer bus. The docking station CCE module can be operated to cause the cartridge CCE module to remove culture medium from the solution, introduce culture medium into the solution, and / or replace culture medium in the solution.

[0390] In some variations, a cell population in a solution can be selected 2806. For example, the solution can be delivered to a selection module of the cartridge via a liquid transfer bus. The docking station selection module can interface with the cartridge selection module, and the docking station selection module can be operated to cause the cartridge selection module to select the selected cell population.

[0391] In some variations, cell populations in a solution can be sorted 2808. For example, the solution can be delivered to a sorting module of a cartridge via a liquid 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 populations.

[0392] In some variations, the solution may be delivered to the bioreactor module of the cartridge via a liquid transfer bus to rest 2810. The 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 cells at a predetermined set of conditions.

[0393] In some variations, cells may be expanded in a solution 2812. For example, the solution may be delivered to the bioreactor module of the cartridge via a liquid transfer bus. The docking station bioreactor module may interface with the cartridge bioreactor module and may be operated to cause the cartridge bioreactor module to expand the cells through cell replication.

[0394] In some variations, tissue may be digested by delivering an enzymatic reagent via a liquid transfer bus to a cartridge module containing a solution containing the tissue, causing the tissue to release a selected cell population into the solution 2814 .

[0395] In some variations, a selected cell population 2816 in solution may be activated by delivering an activation reagent via a fluid transfer bus to a cartridge module containing a solution containing a cellular product.

[0396] In some variations, the solution may be delivered to the electroporation module of the cartridge via the liquid transfer bus and receive an electroporation signal to electroporate the cells in the solution 2818. For example, a docking station electroporation module may interface with the cartridge electroporation module and be operable to cause the cartridge electroporation module to electroporate a selected cell population in the presence of genetic material.

[0397] In some variations, an effective amount of vector may be delivered via a liquid transfer bus to a cartridge module containing a solution containing a cell product, thereby transducing 2820 a selected cell population in the solution.

[0398] In some variations, the formulation solution may be delivered via a liquid transfer bus to a cartridge module containing a cell product to generate a prepared cell product 2822. For example, the prepared cell product may be delivered to one or more product collection bags. In some variations, preparing the cell product may include one or more of the following steps: washing the cells, concentrating the cells, exchanging the cell buffer with a formulation buffer, and dosing the cells in the formulation buffer in predetermined amounts into one or more product collection bags and / or reservoirs.

[0399] In some variations, the cell product may be removed from the cartridge manually or automatically to harvest the cells 2824 .

[0400] In some variations, the cell product may include one or more of immune cell genetically engineered chimeric antigen receptor T cells, genetically engineered T cell receptor (TCR) cells, hematopoietic stem cells (HSC), and tumor infiltrating lymphocytes (TIL). In some variations, the immune cell may include natural killer (NK) cells.

[0401] The cell processing method may include a subset of the cell processing steps in any suitable order. For example, the cell processing method may include, in sequence, an enrichment step 2802, a selection step 2806, an activation step 2816, a transduction step 2820, an expansion step 2812, and a harvesting step 2824. In some variations, the cell processing method may include, in sequence, an enrichment step 2802, a selection step 2806, a resting step 2810, a transduction step 2820, and a harvesting step 2824. In some variations, the cell processing method may include, in sequence, a tissue digestion step 2820, a wash step 2804, an activation step 2816, an expansion step 2812, and a harvesting step 2824. In addition, one or more of the enrichment step 2802, the washing step 2804, the selection step 2806, the sorting step 2808, the resting step 2810, the amplification step 2812, the digestion step 2814, the activation step 2816, the electroporation step 2818, the transduction step 2820, the sorting step 2822 and the removal step 2824 can be performed while the box remains stationary within the docking station.

[0402] In general, the methods described herein can offload the complex steps performed in a cell processing operation to a set of docking station modules, thereby reducing the cost of the cartridge (which can be a consumable). In some variations, the cartridge can contain a cell product (e.g., a solution containing cells) throughout the manufacturing process, with different docking station modules interfaced with the cartridge being engaged at appropriate times to perform one or more cell processing steps. For example, a cell processing step can include delivering cells and reagents to each of the modules within the cartridge. The set of docking station modules interfaced with the cartridge facilitates process flexibility, wherein a CPS and the docking stations within the CPS can be customized for a predetermined cell therapy product using a predetermined set of docking station modules. For example, the order of the cell processing steps can be customized for each cell product, as described herein with respect to Figures 35 to 55 Describe in more detail.

[0403] In some variations, the cell product can be retained in the box throughout the manufacturing process (e.g., workflow). Additionally or alternatively, the cell product can be manually removed from the box by an operator or automatically via a fluid connector or by other access port systems on the box for one or more cell processing steps. The cell product can then be returned to the same box, transferred to another box, or shunted between several boxes. In some variations, one or more cell processing steps can be performed outside the box. In some variations, the processing in the CPS can promote the sterile cell processing in the box.

[0404] Figure 29is a flow chart of a cell processing method and illustrates cell processing steps performed on a cartridge (e.g., consumable) within a CPS that includes 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.

[0405] In some variations, the DS CCE module may include a pump and 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 cartridge bag and the bioreactor. The DS bioreactor module may include one or more sensors, temperature regulators, pumps, agitators, etc., and be configured to interface with the cartridge. In some variations, the cell product may be contained within the cartridge throughout the cell processing.

[0406] Figure 29 The depicted cell processing method may include using a pump to move fluid (e.g., cells in solution) from a product bag to a CCE module (e.g., a rotor) of a cartridge (e.g., a consumable) 2910. In some variations, the cartridge CCE module may be used to enrich a fluid 2912. For example, blood components may be collected in a waste bag 2913. In some variations, the cartridge CCE module may be used to wash a fluid 2914. For example, a buffer may be collected in a waste bag 2915. In some variations, the cartridge CCE module may be used to exchange culture medium 2916. For example, one or more of a buffer (e.g., a formulation buffer) and culture medium may be collected in a waste bag 2917. In some variations, the fluid may be moved to a bioreactor 2918 of the cartridge.

[0407] In some variations, a fluidic connector may fill a bag with reagents 2920. In some variations, reagents (e.g., beads, vectors) may be added to the bioreactor of the cartridge 2922. In some variations, a fluidic connector removes waste from the bag 2924. In some variations, a fluidic connector may optionally remove a sample from the bioreactor.

[0408] 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 medium may be collected in a waste bag 2937. In some variations, the cells may undergo expansion 2938. In some variations, the cells may be harvested 2940 after the medium exchange.

[0409] Figure 30AFlowchart of a cell processing method for autologous CAR T cells or engineered TCR cells. Method 3000 may include the steps of enrichment, selection, activation, genetic modification, expansion, harvesting / formulation, and cryopreservation. Figure 30B Flowchart of a cell processing method for allogeneic CART cells or engineered TCR cells. Method 3010 may include the steps of enrichment, activation, genetic modification (e.g., transduction, transfection), α / β T cell removal, expansion, harvesting / pooling / preparation, and cryopreservation.

[0410] Figure 31 is a flow chart of a cell processing method for hematopoietic stem cell (HSC) cells. Method 3100 may include the steps of enrichment, selection, resting, genetic modification, harvesting / formulation, and cryopreservation.

[0411] Figure 32 Flowchart of a cell processing method for tumor infiltrating lymphocyte (TIL) cells. Method 3200 may include steps of tissue digestion, washing, selection, activation, expansion, harvesting / formulation, and cryopreservation.

[0412] Figure 33 Flowchart of a method for processing natural killer (NK) CAR cells. Method 3300 may include the steps of enrichment, selection, activation, genetic modification, expansion, harvesting / formulation, and cryopreservation.

[0413] Figures 34A to 34C It is used to regulate T(T reg ) cells. Method 3400 may include the steps of enrichment, selection, harvesting / preparation, and cryopreservation. Method 3402 may include the steps of enrichment, selection, activation, genetic modification, amplification, selection (optionally), harvesting / preparation, and cryopreservation. Method 3404 may include the steps of introducing a feeder cell culture for enrichment, selection, activation / amplification, and harvesting / irradiation. Another group of cells may undergo enrichment, selection, co-culture with treated feeder cells, harvesting, and cryopreservation.

[0414] Figures 98 to 101It is a flow chart of a cell processing method for a cell therapy workflow, which includes shunting (e.g., parallel) processing. Method 9800 may include the steps of enrichment, selection, activation, genetic modification, amplification, preparation, and cryopreservation. For example, cell processing method 9800 (e.g., workflow) may include shunting a cell product into two or more parts after the enrichment step. The shunt part can be processed in parallel within a single box. In some variations, one or more shunt parts may be transferred to two or more boxes and processed in parallel. One or more cell processing parameters (e.g., the timing of the process step, the type of reagent added, transfection constructs, etc.) can be independently configured for each shunt part of the cell product. In some variations, the shunt part can be collected after the amplification step.

[0415] Method 9900 may include the steps of enrichment, selection, activation, genetic modification, amplification, preparation and cryopreservation. For example, cell processing method 9900 (e.g., workflow) may include shunting the cell product into two or more parts after the activation step. The shunt portion may be processed in parallel within a single box. One or more cell processing parameters (e.g., the timing of the process step, the type of added reagent, transfection construct, etc.) may be independently configured for each shunt portion of the cell product. In some variations, the shunt portion may be collected after the amplification step and / or genetic modification step.

[0416] Method 10000 can include the steps of enrichment, selection, activation, genetic modification, amplification, preparation and cryopreservation. For example, cell processing method 10000 (e.g., workflow) can include shunting the cell product into two or more parts after the selection step. The shunt part can be processed in parallel within a single box. One or more cell processing parameters (e.g., the timing of the process step, the type of added reagent, transfection construct, etc.) can be independently configured for each shunt part of the cell product. In some variations, the shunt part may not be collected.

[0417] Method 10100 can include the steps of enrichment, selection, activation, genetic modification, amplification, formulation and cryopreservation. For example, cell processing method 10100 (e.g., workflow) can include shunting a cell product into two or more parts as starting material. Individual products can be kept separate and processed in parallel as shunt parts within a single box. One or more cell processing parameters (e.g., timing of process steps, type of added reagents, transfection constructs, etc.) can be independently configured for each shunt part. In some variations, the shunt parts can be collected after the amplification step.

[0418] Figure 102 is a schematic diagram of a cell processing and manufacturing system 10200 configured for split flow processing within a single cartridge. Figures 98 to 101The described methods 9800-10100 may be performed within a cartridge 10210. In some variations, the system 10200 may include a sterile liquid transfer apparatus 10220 including reagents 10222, and a cartridge 10210 including a plurality of 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 reserve module 10280. The cartridge 10210 may also include a reagent storage area 10285, a plurality of product bags 10290, and a liquid transfer bus 10295. The liquid transfer bus 10295 may be configured to couple the components of the cartridge 10210 for fluid communication.

[0419] In some variations, the cell product is loaded into the box and removed from the box and can be performed in the system or outside the system. In some variations, the box is loaded into the bedside of the patient or donor and then delivered to the cell processing and manufacturing system in or near the hospital, or transported to the facility where the cell processing and manufacturing system are installed. Similarly, after processing at the facility or at the expected recipient (patient) closer to the cell product, the cell product can be removed from the box. Optionally, the cell product is frozen before, during or after the method of the present invention (optionally after one or more cryoprotectants are added to the cell product). In some variations, the system includes a refrigerator and / or a liquid nitrogen source. In some variations, the system includes a water bath or a heating chamber containing a controlled temperature gas to allow the controlled thawing of the cell product, for example, a water bath of about 20°C to about 40°C. In some variations, the box is made of a material that resists mechanical damage when freezing.

[0420] Automated cell processing

[0421] This article describes a method for converting user-defined cell processing operations into cell processing steps using the automated cell processing and manufacturing systems and apparatus described herein. In some variations, cell processing operations are received and converted into cell processing steps to be executed by the system given a set of predetermined constraints. For example, a user can input a set of bioprocess steps and corresponding bioprocess parameters to be executed by the cell processing and manufacturing system. Alternatively, process parameters can be customized for each cartridge or multiple groups of cartridges.

[0422] Figure 35 is a flow chart generally describing a variation of an automated cell processing method. Method 3500 may include receiving an ordered input list of cell processing operations 3502. For example, a set of more than one ordered input list 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, Figure 49As shown in GUI 4900 of FIG. 49 and described in more detail herein, one or more bioprocess inputs (e.g., available operations) such as enrichment, MACS selection, activation, transduction, transfection, amplification, and online analysis can be selected as an ordered input list of cell processing operations. In addition, Figure 52 GUI 5200 illustrates a complete ordered input list of cell processing operations (eg, a set of selected operations) 5220 selected by a user.

[0423] 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 at that cell processing step. For example, Figure 40 The GUI 4000 illustrates reagent and container parameters, Figure 42 The GUI 4200 illustrates an example of process parameters, Figure 44 GUI 4400 illustrates an example of a pre-processing analysis, and Figure 48 GUI 4800 illustrates an example set of activation settings.

[0424] In some variations, a transformation model 3506 may be executed on the ordered input list. In some variations, the transformation model may include constraints on the ordered output list determined by a predetermined 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.

[0425] In some variations, the constraints may include one or more of the following: 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 box, the type and / or number of reservoirs on the box, the type and / or number of sterile liquid transfer ports on the box, and the number and positioning of fluid pathways between the modules, reservoirs and sterile liquid transfer ports on the box.

[0426] In some variations, a set of predetermined constraints may be imposed on a set of process control parameters. For example, the volume and / or type of reagents 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 number, cell density, cell viability, dissolved oxygen, glucose levels, volume of onboard reagent storage and waste, combinations thereof, and the like. For example, Figure 40 The GUI 4000 depicts that the reagent has 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. Figure 48GUI 4800 depicts an activation concentration of 12 mg / L, an activation incubation time of 1600 seconds, an activation temperature of 18° C., and a gas mixture comprising 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 a cell product is manufactured.

[0427] In some variations, the order of operations may be constrained based on hardware constraints.

[0428] In some variations, such as Figure 49 As illustrated in GUI 4900 of , the load product operation must be the first operation performed and can be performed once for each process. The fill and sort operation can always be the last operation performed before the product is completed and can be performed once for each process.

[0429] In some variations, the system may prevent a user from performing a set of operations in an order that may not be performed by the system.

[0430] In some variations, a notification (e.g., a warning, an alert) may be output if a user orders a set of operations in a "non-standard" manner. For example, a notification may be output if the same type of operation is repeated in sequence (e.g., enrichment followed by enrichment). Similarly, a notification may be output if an operation (e.g., selection, activation) is used two or more times in a given process, whereas such an operation is typically used only once in a given process.

[0431] 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 executed on multiple sets of ordered input lists to create 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.

[0432] In some variations, the ordered output list is further executed by the system to control one or more of the docking station modules to perform one or more cell processing steps 3512 on one or more cell products of the corresponding cartridge. For example, the computing server rack 210 (e.g., the controller 120) may be configured to control the cartridge electroporation module 220, which is configured to apply a pulsed electric field to the cell suspension of the cartridge 250. In some variations, the ordered output list may include instructions for the docking station module (e.g., a bioreactor) to process the product (e.g., transfer the cell product from the small cartridge bioreactor module to the large cartridge bioreactor module). In addition, the docking station module may be further configured to operate under a set of process parameters (e.g., a 9-hour duration, a pH of 6.7, a temperature between 37.3° C. and 37.8° C., and a mixing mode of 3). As another example, the ordered output list may include instructions for operating the sterile liquid transfer module to perform one or more of the following: remove waste from the cartridge, add culture medium to the cartridge, and add MACS reagent to the cartridge.

[0433] In some variations, one or more electronic batch records 3514 may be generated based on process parameters and data collected from sensors during process execution. Batch records generated by the system may include process parameters, time records, sensor measurements from docking station modules, QC parameters determined by QC instruments, and other records.

[0434] Figure 36 36 is a flow chart generally describing a variation of method 3600 for executing a transformation model. In some variations, one or more bio-functions may be generated and output to a user. For example, a set of configurable bio-function blocks may be displayed on a graphical user interface for user selection. The GUI may enable the user to select and sort the bio-function blocks and define bio-control parameters. If desired, one or more control parameters of the bio-function blocks may be modified by the user. In some variations, one or more bio-function templates may be generated that include a predefined sequence of bio-function blocks. If desired, one or more bio-control parameters of the bio-function templates may be modified by the user.

[0435] In some variations, the cell processing and manufacturing system may be configured to receive and / or store one or more bio-function (e.g., process) inputs from a user 3604. For example, a user may select one or more predefined bio-function templates.

[0436] In some variations, a bioprocess model (e.g., process definition) may be generated based on the bioprocess inputs 3606. In some variations, the bioprocess model may include one or more of enrichment, isolation, MACS selection, FACS selection, activation, genetic modification, gene transfer, transduction, transfection, amplification, formulation (e.g., harvesting, pooling), cryopreservation, T cell depletion, resting, tissue digestion, washing, irradiation, co-cultivation, combinations thereof, and the like.

[0437] In some variations, the bioprocess model may be transformed into a docking station (DS) module execution process model 3608. For example, each biofunction block in the bioprocess 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 bioprocess model. As described herein, the transformation model may include one or more constraints.

[0438] 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 3610 from a user. For example, the user may modify the transformed DS module execution process model if desired. The user may select specific hardware components to perform certain steps, modify timing parameters, etc.

[0439] In some variations, the DS modules may be executed to perform processes to generate cell products 3612. For example, a cell processing and manufacturing system at runtime may process cell products by executing the system defined by the DS module process model.

[0440] In some variations, a DS module execution process 3612 may be executed. In some variations, the DS module execution process model may be transformed back into 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 for the biological process model.

[0441] In some variations, cell product 3616 may be monitored. For example, the corresponding Figure 53 and Figure 54 GUIs 5300 and 5400 illustrate sensor data monitored by the system for a plurality of products. For example, the number of viable cells and the status of the process (e.g., as a function of the percentage of completion) may be graphically illustrated to the user.

[0442] In some variations, electronic records 3618 may be generated based on the monitored data. For example, one or more electronic batch records may be generated in accordance with 21 CFR regulations, for example.

[0443] Graphical User Interface

[0444] In some variations, a graphical user interface (GUI) may be configured for use in the design process and monitoring of the product. Figure 37 3700 includes an initial process design interface. For example, GUI 3700 may be a process design homepage. GUI 3700 may indicate that no process has been selected or loaded. A create icon 3710 (e.g., "Create Process") may be selectable for the user to begin the process design process. In some variations, one or more of the GUIs described herein may include a search bar.

[0445] Figure 38 is a variation of the GUI 3800 associated with the creation process. Figure 37 3800. For example, the GUI 3800 may include a process creation window 3810 that allows the 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.

[0446] Figure 39 3900 is a variation of the GUI 3900 associated with an empty process. After confirming in the GUI 3800 that a process is to be created, the GUI 3900 may be displayed. The GUI 3900 may indicate the process name (e.g., Car T Therapy) and may highlight a process setup icon 3910 and allow the addition of process-specific parameters such as process reagents and containers, process parameters, and pre-processing analyses. The GUI 3900 may also include an add process reagents and containers icon 3920, an add process parameters icon 3930, and an add pre-processing analysis icon 3940. Once the process setup is complete, one or more process elements may be specified.

[0447] In some variations, GUI 3900 may include one or more predefined templates for a set of biological processes (e.g., CAR-T, NK cells, HSCs, TILs, etc.). For example, the templates may assist in process development and be a validated starting point for process development. The templates may be further modified (e.g., customized) based on user requirements.

[0448] Figure 40 A variation of GUI 4000 that is related to adding reagents and consumable containers. Figure 39In the interpolation process reagent and container icon 3920, can show GUI 4000.For example, GUI 4000 can comprise interpolation reagent and container window 4010, and this interpolation reagent and container window enable the user to input and / or select one or more reagents, comprise reagent type, manufacturers, part number, every unit volume, required volume and required reagent input (for example, lot number, expiration date, need container transmission).Interpolation reagent and container window 4010 can comprise one or more in input field, selection box, drop-down selector etc.In addition, interpolation reagent and container window 3810 can enable the user to input and / or select one or more consumable containers, and these one or more consumable containers comprise manufacturers, part number, every unit volume and required container input (for example, lot number, expiration date).In some variations, the user can select from the list of predetermined template.For example, the user can create process and it is saved as template.

[0449] Figure 41 is a variation of the GUI 4100 that is related to process parameters. Figure 39 4100. For example, GUI 4100 may include an add process parameter window 4110 that enables a user to enter and / or select one or more parameters, including a name, parameter identifier, description, data type, unit, and parameter type. Add process parameter window 4110 may include one or more of an input field, a selection box, a drop-down selector, etc. In some variations, a user may select from a list of predefined templates. For example, a user may create parameters and save them as a template. Figure 42 A variation of GUI 4200 related to a patient weight process parameter is shown. For example, GUI 4200 may include an add process parameter window 4110 that has been populated with parameter information, including patient weight, data type (e.g., integer), unit (e.g., kg), and parameter type (e.g., input).

[0450] Figure 43 is a variation of GUI 4300 associated with pre-processing analysis. Figure 39 After selecting the Add Pre-Processing Analysis icon 3940 in the GUI 4300, the GUI 4300 may be displayed. For example, the GUI 4300 may include an Add Pre-Processing Analysis window 4310, which enables the user to enter and / or select one or more parameters, including a name, identifier, description, data type, and display group. The Add Pre-Processing Analysis window 4310 may include one or more of an input field, a selection box, a drop-down selector, etc. In some variations, the user may select from a list of pre-defined templates. For example, the user may create parameters and save them as a template.

[0451] Figure 44 A variation of GUI 4400 related to a white blood cell count pre-processing analysis may be shown. For example, GUI 4400 may include an add pre-processing analysis window 4410 that is already populated with pre-processing 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., floating point), and a display group (e.g., WBC).

[0452] Figure 45 is a variation of GUI 4500 that is related to process parameter calculations. Figure 39 After clicking the Add Pre-Processing Analysis icon 3940 in the GUI and selecting the "Calculation" parameter type, the GUI 4500 may be displayed. For example, the GUI 4500 may include an Add Pre-Processing Analysis window 4510 that enables the user to enter and / or select one or more parameters, including a name, identifier, description, data type, display group, unit, and parameter type. In addition, a calculation builder may enable the user to define a formula (e.g., an algorithm, an equation) to perform a predetermined calculation. For example, the calculation builder may include one or more of a set of available parameters (e.g., patient weight), constant values, equations, and operands.

[0453] Figure 46 A variation of GUI 4600 is shown with respect to a completed process setup. For example, GUI 4600 may include a process setup window 4610 that has been populated with process reagents, containers, process parameters, and pre-processing analysis. Once the process setup is complete, one or more process elements may be specified.

[0454] Figure 47 is a variation of GUI 4700 that is associated with process operation activation settings. Figure 46 4620 in the process element icon, GUI 4700 may be displayed. For example, GUI 4700 may include an activation settings window 4710 that allows the user to enter and / or select one or more of the activation concentration (e.g., mg / L), activation incubation time (e.g., seconds), activation temperature (e.g., ° C.), and gas mixing mode. In some variations, the user may select from a list of pre-defined templates. For example, the user may create a set of activation settings and save it as a template for later selection.

[0455] Figure 48 A variation of GUI 4800 is provided that relates to populated process operation activation settings. For example, GUI 4800 may include activation settings window 4810 that has been populated with activation settings information. In some variations, a set of gases (e.g., O2, N2, CO2) and corresponding concentrations may be specified.

[0456] Figure 49 This is a variation of GUI 4900 related to a process operation interface. GUI 4900 may include an available operation window 4910 and a selected operation window 4920. Available options for selection may include one or more bioprocess inputs as described herein, including but not limited to enrichment, MACS selection, activation, transduction, transfection, amplification, and online analysis. One or more of the operations may be selected and dragged into the selected operation window 4920. The selected operations may be reordered within the selected operation window 4920.

[0457] Figure 50 5000. GUI 5000 may include an available operation window 5010, a selected operation window 5020, and a selected (e.g., dragged) operation 5030 that can be dragged and dropped between available operation window 5010 and selected operation window 5020. Selected operation window 5020 may include multiple selected operations.

[0458] Figure 51 51 is a variation of the GUI 5100 related to the drag process operation. The GUI 5100 may include an available operation window 5110, a selected operation window 5120, and a selected (e.g., dragged) operation 5130 that can be dragged and dropped between the available operation window 5110 and the selected operation window 5120. The selected operation window 5120 may include a plurality of selected operations.

[0459] Figure 52 5200 is a variation of a GUI associated with populated process operations. For example, GUI 5200 may include an available operations window 5210 and a selected operations window 5220 that includes a complete set of selected operations. In some variations, the settings (e.g., parameters) of each operation can be selectively modified by the user by selecting a corresponding icon (e.g., a gear icon).

[0460] Figure 53 and Figure 54 This is a variation of GUIs 5300 and 5400 related to product monitoring. GUIs 5300 and 5400 may include corresponding 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 summaries, process data, online analysis, imaging, process audit logs, process parameters, and process timelines. Monitoring window 5410 may monitor one or more product characteristics of one or more products. For example, product characteristics may include but are not limited to one or more of process name, ID, process ID, progress, estimated completion, current step, and messages.

[0461] Figure 77Ais a flow chart of method 7700 for separating cells using a cartridge CCE module. Figure 77B is a flow chart of method 7710 for concentrating cells using a cartridge CCE module. Figure 77C is a flow chart of a buffer exchange method 7720 using a cartridge CCE module.

[0462] Figure 78 7800 is a flow chart of a method for separating cells. The method 7800 of countercurrent centrifugal elutriation (CCE) may include a step 7802 of moving a rotor toward a magnet. The rotor may define an axis of rotation. In some variations, moving the rotor includes advancing and retracting the magnet relative to the rotor using a robot. The rotor may optionally be moved 7804 toward an illumination source and an optical sensor. A fluid may flow through the rotor 7806. In some variations, flowing the fluid includes a flow rate of up to about 150 ml / min when rotating the rotor. When flowing the fluid through the rotor, a magnet may be used to magnetically rotate the rotor about the axis of rotation 7808. In some variations, rotating the rotor includes a rotation rate of up to 6,000 RPM. An illumination source may be used to optionally illuminate one or more of the fluid and cells 7810. Image data of one or more of the fluid and biological material (e.g., particles, cellular material) in the rotor may be optionally generated 7812 using an optical sensor. One or more of the rotation rate of the rotor and the flow rate of the fluid can be optionally selected based at least in part on the image data 7814. The fluid can flow out of the rotor 7816. The rotor can be moved away from the magnet 7818. The rotor can optionally be moved away from the illumination source and the optical sensor 7820.

[0463] Figure 79A is a flow chart of a closed-loop method 7900 for isolating 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.

[0464] Figure 80A is a flow chart of method 8000 of isolating cells. Figure 80B is a flow chart of method 8010 for selecting cells.

[0465] Figure 818100 is a flow chart of a method for separating cells. The method of magnetic activated cell selection (MACS) may include labeling cells with a reagent 8102. In some variations, a magnetic activated cell selection (MACS) reagent may be incubated with input cells to label a group of cells with the MACS reagent. In some variations, incubating the MACS reagent includes a temperature between about 1° C. and about 10° C. A fluid containing the input cells may flow into a flow cell 8104. A group of cells is labeled with the MACS reagent. In some variations, the magnet array may optionally be moved relative to the flow cell 8106. In some variations, the group of cells may be magnetically attracted toward the magnet array for a certain residence time 8108. In some variations, the residence time may be at least about one 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, microbeads may not be present in the flow cell. In some variations, the magnet array can optionally be moved away from the flow cell to facilitate the flow of the group of cells out of the flow cell 8110. After a residence time, the group of cells can flow out of the flow cell 8112. For example, causing the group of cells to flow out of the flow cell can include flowing a gas through the flow cell. After the residence time, the fluid free of the group of cells can optionally flow out of the flow cell 8114.

[0466] Figure 82A is a flow chart of a method 8200 of preparing a bioreactor. Figure 82B is a flow chart of a method 8210 of loading a bioreactor. Figure 82C is a flow chart of method 8220 of preparing a bioreactor. Figure 82D is a flow chart of calibration method 8230 for a bioreactor. Figure 82E is a flow chart of method 8240 for mixing reagents. Figure 82F is a flow chart of a method 8250 for mixing reagents. Figure 82G is a flow chart of method 8260 of culturing cells. Figure 82H is a flow chart of method 8270 for cryopreserving cells. Figure 82I is a flow chart of sampling method 8270. Figure 82J is a flow chart of method 8280 of culturing cells. Figure 82K This is a flow chart of medium exchange method 8290. Figure 82L It is a flow chart of method 8292 for controlling gas. Figure 82M is a flow chart of method 8294 for controlling pH.

[0467] Figure 83is a flow chart of a method 8300 for electroporating cells using a cartridge electroporation module. In some variations, the cartridge electroporation module may comprise a fluid conduit configured to receive a first fluid comprising cells and a second fluid, a set of electrodes coupled to the fluid conduit, a pump coupled to the fluid conduit, and a controller comprising a processor and a memory.

[0468] A method of electroporating cells may optionally include generating a first signal to introduce a first fluid into a fluid conduit using a pump 8302. A first fluid containing cells may be received in the fluid conduit 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 comprise gas or oil. The second fluid may be received in the fluid conduit to separate the first fluid from the third fluid 8308. 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 the electroporation signal is applied. 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 the second fluid. A third fluid can optionally be received in the fluid conduit, the third fluid being separated from the first fluid by the second fluid 8316 .

[0469] Figure 84 8400 is a flow chart of a method for electroporating cells. The method for 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 8404 using a set of electrodes. The 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.

[0470] Fluid connectors

[0471] Method 2700 for transferring fluid using a fluid connector Figure 27 is described in the flowchart and is Figures 16B to 16L The corresponding steps are schematically illustrated in the depicted diagram. Method 2700 may include a step 2702 of coupling a sterilant source to a fluid connector. For example, Figure 16BAs shown, inlet 1652 and outlet 1654 are coupled to a sterilant source to form a fluid pathway or connection. In some variations, the robot can be configured to use a fluid conduit (such as a tube) to couple the sterilant source to sterilant port 1650 and disconnect the sterilant source from the sterilant port. In some variations, the fluid connector 1600 can include multiple sterilant ports 1650. As described herein, in some variations, the sterilant port can 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 can include or be coupled to a pump configured to circulate the sterilant through the sterilant port 1650. In some variations, the sterilant port 1650 can be coupled to one or more of a sterilant source and a fluid source (such as a heated air source). For example, a first sterilant port may be configured to couple to a first sterilant source, a second sterilant port may be configured to couple to a second sterilant source, and a third sterilant source may be configured to couple to an air source.

[0472] The separate parts of the fluid connector 1600 can be brought together and mated.The method 2700 can include coupling 2704 a first port of a first connector to a second port of a second connector. Figure 16C is a schematic diagram of a fluid connector 1600 in which the first port 216 and the second port 226 are in a coupled configuration (e.g., docked position) that forms 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. Figure 16C In the embodiment of the present invention, the first port 1616 and the second port 1626 are each in a closed configuration, wherein the inner cavity of the corresponding first connector 1610 and the second connector 1620 is sealed with the external environment to maintain the sterility of the inner cavity of the fluid connector 1600. In addition, the first valve 1618 and the second valve 1628 are each in a closed configuration, and the closed configuration makes the proximal end and the distal end of the connector seal each other. For example, the first valve 1618 in the closed configuration forms a seal (for example, 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 way, even if a part of the connector (for example, the first distal end 1614) is contaminated, the other parts of the fluid connector 1600 (for example, the first proximal end 1612, the second connector 1620) can also be kept sterile by means of one or more of the port seals and the valve seals.

[0473] The port can be transitioned to an open configuration such that the distal end of the connector can be in fluid communication. Method 2700 can include transitioning 2706 the port to the open configuration. Figure 16D is a schematic diagram of a fluid connector 1600 in which the first port 1616 and the second port 1626 are transformed into an open port configuration to create a shared volume between valves 1618, 1628 that is isolated from the external environment. Figure 16D 16. In some embodiments, the first and second valves 1618, 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 and second valves 1618, 1628. That is, the first distal end 1614 is in fluid communication with the second distal end 1624. The ports 1616, 1627 may be received and / or retained in the 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, upon mating the first port 1616 to the second port 1626, the first and second ports 1616, 1626 may be automatically transitioned (e.g., mechanically actuated) from the closed configuration to the open configuration.

[0474] In some variations, a fluid may flow into the fluid connector to assist in sterilization. Method 2700 may include flowing a fluid (eg, liquid, gas) into fluid connector 2708 through a sterilant port. Figure 16E 16 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 raise the temperature of the interior surfaces of the chamber 1615. The circulated fluid may flow through the housings 1617, 1627 and over the interior and / or exterior surfaces of the ports 1616, 1626.

[0475] Typically, sterilization of a fluid connector may include one or more steps of dehumidification, conditioning, purification, and ventilation (e.g., ventilation). Dehumidification may include removing moisture from the fluid connector. Conditioning may include heating the surface of the fluid connector to be purified to prevent condensation and assist in sterilization. Purification may include circulating a sterilant through the fluid connector at a predetermined concentration, rate, and exposure time. Ventilation may include removing a sterilant from the fluid connector by circulating a gas (e.g., sterile air) through the fluid connector.

[0476] A sterilant can flow 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). Method 2700 can include flowing the sterilant into the fluid connector 2710 through a sterilant port. Figure 16F is a schematic diagram of fluid connector 1600 in which first chamber 1615 receives a sterilant for a predetermined amount of time (e.g., a dwell time). For example, the sterilant may circulate within chamber 1615 to sterilize chamber 1615 of fluid connector 1600 and any contents disposed therein (e.g., other fluids, biological materials). In some variations, the dwell time may be up to about 10 minutes, and between about 1 minute and about 10 minutes, including all ranges and subvalues ​​therebetween. In some variations, the vaporized hydrogen peroxide may comprise a concentration between about 50% and about 70%, including all ranges and subvalues ​​therebetween. Additionally or alternatively, one or more of first valve 1618 and second valve 1628 may be in an open configuration such that the sterilant may circulate through other portions of fluid connector 1600, such as first proximal end 1612 and second proximal end 1622.

[0477] In some variations, the valves may translate relative to each other.Method 2700 may include translating a first valve 2712 relative to a second valve. Figure 16G 16 is a schematic diagram of a fluid connector 1600 in which a first valve 1618 and a second valve 1628 are coupled to each other (eg, in a transfer position). The first valve 1618 coupled to the second valve 1628 forms a second seal between the first connector 1610 and the second connector 1620 .

[0478] The valve can be transformed into an open configuration so that each end of the fluid connector is in fluid communication. Method 2700 may include transforming the first valve and the second valve from the closed configuration to the open configuration 2714. In some variations, the first valve and the second valve may include a spring-loaded closure member that is configured to actuate to an open configuration, thereby allowing fluid communication between the sterile inner chambers of the first connector 1610 and the second connector 1620. In some variations, each of the first valve 1618 of the first connector 1610 and the second valve 1628 of the second connector 1620 may include an engagement feature, such as a thread, that is configured to promote the coupling between the first valve 1618 and the second valve 1628. For example, once the second valve 1628 translates to contact the first valve 1618, the engagement features of the valves 1618, 1628 can be coupled (e.g., locked) by rotating (e.g., twisting) one of the first valve 1618 and the second valve 1628 so that the corresponding threads of these valves engage with each other. Conversely, one of the first valve 1618 and the second valve 1628 may be rotated in an opposite direction to decouple (eg, unlock) the first valve 1618 from the second valve 1628 .

[0479] In some variations, fluid may flow through fluid connector (2716). Figure 16H It is used to transfer fluid between fluid devices connected to the fluid connector. Figure 16A For example, the contents of first fluidic device 1630 and second fluidic device 1640 (e.g., fluid, biological material) can be transferred through fluidic connector 1600. In some variations, one or more of a pump, gravity feed, etc. can assist in the transfer through fluidic connector 1600.

[0480] In some variations, after the fluid transfer between the first fluidic device and the second fluidic device is completed, another fluid may flow into the fluid connector. Method 2700 may include flowing a fluid (e.g., liquid, gas, sterilant) through a sterilant port into the fluid connector 2708 to remove the fluid and / or biomaterial 2718 from the fluid connector. For example, flowing an inert gas into the fluid connector can reduce droplets formed when the first connector and the second connector are separated. If the sterilant flows through the fluid connector, another fluid (such as an inert gas) may flow to vent the fluid connector and ensure that the sterilant is removed.

[0481] To begin decoupling the fluid connectors, the valves may be translated away from each other. Method 2700 may include decoupling 2720 the first and second connectors. In some variations, the robot may be configured to manipulate the fluid connector 1600 to transition the valves 1618, 1628 to a closed configuration and translate the valves 1618, 1628 away from each other, which may occur simultaneously or independently. The valves 1618, 1628 in the closed con...

Claims

1. An automated system for cell processing, the system comprising: A box comprising A plurality of cartridge modules, the plurality of cartridge modules comprising a bioreactor module, a countercurrent 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 fluid transfer port, and a liquid transfer bus fluidly coupled to each cartridge module; as well as 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 perform one or more cell processing operations on the cartridge in collaboration with the corresponding cartridge module, wherein the docking station is sized and shaped to accommodate a single cartridge.

2. The system of claim 1, wherein the liquid transfer bus is fluidly coupled to each cassette module via a fluid conduit disposed between a port on the liquid transfer bus and a corresponding port of each cassette module.

3. The system of claim 2, wherein the fluid conduit comprises a pipe or a flow channel. 4 . The system of claim 1 , further comprising a sterile liquid transfer device configured to facilitate liquid transfer between the cassette and a closed volume fluidic device.

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

6. The system of claim 4, wherein the sterile fluid 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 cassette 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 the housing of the system, the fluid reservoir being accessible by a user for insertion and / or removal of 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 spin seeding module.

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

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

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

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

18. The system of claim 1, wherein the plurality of docking station modules comprise corresponding counter-flow centrifugal elutriation modules.

19. The system of claim 1, wherein the plurality of docking station modules comprise corresponding rotary seeding modules.

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

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 fluid transfer between the cartridge and the closed volume fluidic device comprises extracting at least a portion of a cell processing sample from the cartridge.

24. An automated cell processing method, comprising: performing at least two cell processing operations within a cartridge positioned in a docking station, wherein the cartridge comprises a plurality of cartridge modules, at least one sterile liquid transfer port, and a liquid transfer bus fluidly coupled to each cartridge module, and wherein the docking station comprises a plurality of docking station modules corresponding to the plurality of cartridge modules, and wherein the docking station is sized and shaped to receive a single cartridge, wherein the plurality of cartridge modules comprises a bioreactor module, a countercurrent 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, and Wherein said cell processing operations are automated in execution of a set of received instructions.

25. The method according to claim 24, further comprising: One of the at least one sterile liquid transfer port of the cassette and a corresponding sterile liquid transfer port of the closed volume fluidic device are actuated to facilitate liquid transfer between the sterile liquid transfer port and the corresponding sterile liquid transfer port.

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

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

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

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

30. 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 spin seeding module.

32. The method of claim 24, wherein the plurality of docking station modules comprise corresponding bioreactor modules.

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

34. The method of claim 24, wherein the plurality of docking station modules comprise corresponding electroporation modules.

35. The method of claim 24, wherein the plurality of docking station modules comprise corresponding magnetically activated cell selection modules.

36. The method of claim 24, wherein the plurality of docking station modules comprise corresponding fluorescence activated cell selection modules.

37. The method of claim 24, wherein the plurality of docking station modules comprise corresponding rotary seeding modules.

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

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