Systems, devices, and methods for cell processing

Abstract

Disclosed herein are cell processing systems, devices, and methods. The system for cell processing can include multiple instruments, each independently configured to perform one or more cell processing operations on a cartridge, and a robot capable of moving the cartridge between each of the multiple instruments.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS)

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 987,745, filed March 10, 2020, and U.S. Provisional Patent Application No. 63 / 093,038, filed October 16, 2020, the contents of each of which are incorporated by reference in their entirety.

[0002] FIELD OF THE INVENTION

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

[0003] Cell therapies based on hematopoietic stem cells (HSCs), chimeric antigen receptor (CAR) T cells, NK cells, tumor infiltrating lymphocytes (TILs), T-cell receptors (TCRs), regulatory T cells (Tregs), gamma delta (γδ) T cells, and others rely on the manufacturing of cell products. The manufacturing of such cell products typically requires multiple cell processing steps. Conventional solutions for manufacturing cell products rely on laborious manual operations performed in expensive biosafety cabinets and / or clean rooms. Skilled laboratory technicians, appropriate sterile enclosures such as clean room facilities, and associated protocols and procedures for regulated (GMP) manufacturing are expensive. Many current manufacturing processes use numerous manual reagent preparation and instrumentation steps during the manufacturing protocol, which can take days or even weeks. Even platforms described as automated cell processing within closed systems typically rely on pre-configured sets of instrumentation and tubing. This limits operational flexibility and does not reliably prevent process failure due to accidental operator / human error.

[0004]

[0004] Most efforts to automate the manufacturing of cellular products have focused on automating individual processing steps in the cell therapy manufacturing workflow. Even systems that automate several steps lack comprehensive process flexibility, process robustness, and process scalability. Various embodiments disclosed herein address these and other limitations in previous attempts to automate cell processing. Summary of the Invention

[0005]

[0005] The present disclosure generally relates to methods and systems for processing cellular products. By processing cellular products in cartridges that are transferred between tools, some variations may achieve one or more advantages over conventional cell manufacturing systems. These advantages include, for example, improved sterility, automation, reduced material costs, reduced labor costs, improved reproducibility, improved reliability, reduced risk of operator error, reduced risk of contamination, increased process flexibility, increased capacity, increased tool throughput, increased process scalability, and reduced process time. Variations of the present disclosure may reduce the cost of providing a cleanroom environment by including sterile enclosures and / or may utilize work cells with smaller footprints than current manufacturing facilities. Additionally, variations disclosed herein may be able to be performed more quickly and with less risk of cellular product loss.

[0006] In some variations, the present disclosure provides a system for cell processing, the system comprising a plurality of instruments, each independently configured to perform one or more cell processing operations on a cartridge, and a robot capable of transferring the cartridge between each of the plurality of instruments.

[0007] In some variations, the system may be housed within a work cell. In some variations, the work cell may be automated. In some variations, the plurality of instruments may be configured to interface with the cartridge to perform cell processing operations on the cartridge. In some variations, the system may include a processor. The processor may be configured to control the robot and the plurality of instruments.

[0008] In some variations, the system may be configured to receive two or more cartridges. In some variations, the system may include the cartridge. In some variations, the cartridge may include multiple modules. In some variations, the cartridge may include a bioreactor module. In some variations, the cartridge may include a cell selection module. In some variations, the cell selection module may include a magnetic-activated cell selection module. In some variations, the cartridge may include a sorting module. In some variations, the sorting module may include a fluorescence activated cell sorting (FACS) module. In some variations, the cartridge may include an electroporation module. In some variations, the cartridge may include a counterflow centrifugal elutriation (CCE) module.

[0009] In some variations, the cartridge may include one or more sterile fluid transfer ports. In some variations, the cartridge may include a fluid transfer bus fluidly coupled to each module. In some variations, the cartridge may include a pump fluidly coupled to the fluid transfer bus.

[0010] In some variations, the system may include a pump actuator configured to interface with the pump. In some variations, the system may include a bioreactor instrument. In some variations, the bioreactor instrument may include a plurality of slots for cartridges. In some variations, the system may include a cell selection instrument. In some variations, the cell selection instrument may include a magnetically activated cell selection instrument.

[0011] In some variations, the system may include a sorting instrument. In some variations, the sorting instrument may include a fluorescence-activated cell sorting (FACS) instrument. In some variations, the system may include an electroporation instrument. In some variations, the system may include a counterflow centrifugal elutriation (CCE) instrument. In some variations, the system may include a reagent reservoir.

[0012] In some variations, the cartridge may include a bioreactor module and a selection module. In some variations, the cartridge may include a bioreactor module and a CCE module. In some variations, the cartridge may include a bioreactor module, a selection module, and a CCE module. In some variations, the cartridge may include a bioreactor module, a selection module, and an electroporation module. In some variations, the cartridge may include a bioreactor module, a selection module, a CCE module, and an electroporation module. In some variations, the cartridge may include a second bioreactor module having an internal volume that is two or more times, five or more times, or ten or more times larger than the internal volume of the first bioreactor.

[0013] In some variations, the system may include an enclosure. In some variations, the enclosure may include an ISO 7 clean room. In some variations, the enclosure may include an ISO 6 clean room. In some variations, the enclosure may include an ISO 5 clean room. In some variations, the enclosure may include a feedthrough. In some variations, the system may perform automated manufacturing of a cellular product.

[0014] In some variations, the present disclosure provides a cartridge for cell processing comprising a fluid transfer bus and a plurality of modules, each module fluidly coupled to the fluid transfer bus.

[0015]

[0015] In some variations, the cartridge may include one or more sterile fluid transfer ports. In some variations, the cartridge may include a bioreactor module. In some variations, the cartridge may include a cell selection module. In some variations, the cell selection module may include a magnetically activated cell selection module. In some variations, the cartridge may include a sorting module. In some variations, the sorting module may include a fluorescence activated cell sorting (FACS) module. In some variations, the cartridge may include an electroporation module. In some variations, the cartridge may include a counterflow centrifugal elutriation (CCE) module.

[0016] In some variations, the cartridge may include a mechanoporation module. In some variations, the cartridge may include a second bioreactor module having an internal volume that is two or more times, five or more times, or ten or more times larger than the internal volume of the first bioreactor. In some variations, the cartridge may include a bioreactor module, a selection module, and a CCE module. In some variations, the cartridge may include a bioreactor module, a selection module, and an electroporation module. In some variations, the cartridge may include a bioreactor module, a selection module, a CCE module, and an electroporation module.

[0017] In some variations, the present disclosure provides a method for processing cells, the method including transferring a cartridge containing a cellular product among a plurality of instruments within an enclosed automated work cell, the instruments being capable of engaging the cartridge to perform cell processing steps on the cellular product.

[0018] In some variations, cell processing steps may be performed on the cell products. In some variations, for each cell product, all cell processing steps in the method are performed in a single cartridge.

[0019] In some variations, the cell product may be divided into multiple cell product portions. In some variations, cell processing steps may be performed in parallel on the multiple cell product portions. In some variations, at least two cell product portions of the multiple cell product portions may be combined.

[0020] In some variations, the work cell may include a robot configured to move the cartridge. In some variations, the work cell may include a processor. The processor may be configured to control the robot and the plurality of tools. In some variations, the work cell may be configured to receive two or more cartridges.

[0021] In some variations, the cartridge may include multiple modules. In some variations, the cartridge may include a bioreactor module. In some variations, the cartridge may include a cell selection module. In some variations, the cell selection module may include a magnetically activated cell selection module.

[0022] In some variations, the cartridge may include a sorting module. In some variations, the sorting module may include a fluorescence-activated cell sorting (FACS) module. In some variations, the cartridge may include an electroporation module. In some variations, the cartridge may include a counterflow centrifugal elutriation (CCE) module. In some variations, the cartridge may include one or more sterile fluid transfer ports. In some variations, the cartridge may include a fluid transfer bus fluidly coupled to each module. In some variations, the cartridge may include a pump fluidly coupled to the fluid transfer bus.

[0023] In some variations, the work cell may include a pump actuator configured to interface with the pump. In some variations, the work cell may include a bioreactor instrument. In some variations, the bioreactor instrument may include multiple slots for cartridges. In some variations, the method may include performing cell processing steps on two or more cartridges in parallel.

[0024] In some variations, the work cell may include a cell selection instrument. In some variations, the cell selection instrument may include a magnetically activated cell selection instrument.

[0025] In some variations, the work cell may include a sorting instrument. In some variations, the sorting instrument may include a fluorescence-activated cell sorting (FACS) instrument. In some variations, the work cell may include an electroporation instrument. In some variations, the work cell may include a counterflow centrifugal elutriation (CCE) instrument. In some variations, the work cell may include a reagent reservoir.

[0026] In some variations, the cartridge may include a bioreactor module and a selection module. In some variations, the cartridge may include a bioreactor module and a CCE module. In some variations, the cartridge may include a bioreactor module, a selection module, and a CCE module. In some variations, the cartridge may include a bioreactor module, a selection module, and an electroporation module. In some variations, the cartridge may include a bioreactor module, a selection module, a CCE module, and an electroporation module.

[0027] In some variations, the work cell may include an enclosure. In some variations, the enclosure may include an ISO 7 clean room. In some variations, the enclosure may include an ISO 6 clean room. In some variations, the enclosure may include an ISO 5 clean room. In some variations, the enclosure may include a feedthrough.

[0028] In some variations, the method may perform automated manufacturing of the cell product. In some variations, the cell product may include a chimeric antigen receptor (CAR) T cell product. In some variations, the cell product may include a natural killer (NK) cell product. In some variations, the cell product may include a hematopoietic stem cell (HSC) cell product. In some variations, the cell product may include a tumor infiltrating lymphocyte (TIL) cell product. In some variations, the cell product may include a regulatory T (Treg) cell product.

[0029] In some variations, the present disclosure provides a method for processing a solution containing a cell product, performed in an automated system, the method including one or more cell processing steps selected from an enrichment step, a concentration step, a buffer exchange step, a formulation step, a washing step, a selection step, a settling step, an expansion step, a tissue digestion step, an activation step, a transduction step, a transfection step, and a harvesting step, performed sequentially in any order.

[0030]

[0030] In some variations, the concentrating step may include concentrating the selected cell population by conveying the solution to a CCE module of the cartridge via a liquid transfer bus, operating a robot to move the cartridge to a CCE instrument to interface the CCE module with the CCE instrument, and operating the CCE instrument to concentrate the selected cell population in the solution in the CCE module.

[0031]

[0031] In some variations, the washing step may include washing the selected cell population in the solution by conveying the solution to a CCE module of the cartridge via a liquid transfer bus, operating a robot to move the cartridge to a CCE instrument to interface the CCE module with the CCE instrument, and operating the CCE instrument to cause the CCE module to remove culture medium from the solution, introduce culture medium into the solution, and / or replace culture medium in the solution.

[0032]

[0032] In some variations, the selection step may include selecting the selected cell population by conveying the solution via a liquid transfer bus to a selection module on the cartridge, operating a robot to move the cartridge to a selection instrument to engage the selection module with the selection instrument, and operating the selection instrument to cause the selection module to select the selected cell population in the solution.

[0033]

[0033] In some variations, the sorting step may include sorting the cell population by transporting the solution via a liquid transfer bus to a sorting module of the cartridge, operating a robot to move the cartridge to a sorting instrument to interface the sorting module with the sorting instrument, and operating the sorting instrument to cause the sorting module to sort the cell population in the solution.

[0034]

[0034] In some variations, the settling step may include transporting the solution to the bioreactor module of the cartridge via a liquid transfer bus, operating a robot to move the cartridge to the bioreactor instrument to interface the bioreactor module with the bioreactor instrument, and operating the bioreactor instrument to maintain the cells in the bioreactor module.

[0035]

[0035] In some variations, the proliferation step may include propagating the cells by transporting the solution to a bioreactor module of the cartridge via a liquid transfer bus, operating a robot to move the cartridge to the bioreactor instrument, interfacing the bioreactor module with the bioreactor instrument, and operating the bioreactor instrument to propagate the cells in the solution in the bioreactor module by cell replication.

[0036]

[0036] In some variations, the tissue digestion step may include transporting an enzymatic reagent via a liquid transfer bus to a module containing a solution containing the tissue, and the enzymatic reagent digesting the tissue thereby releasing the selected cell population into the solution.

[0037]

[0037] In some variations, the activation step may include activating a selected cell population in solution by transporting an activation reagent via a liquid transfer bus to a module containing the solution containing the cell product.

[0038]

[0038] In some variations, the electroporation step may include conveying the solution to the electroporation module of the cartridge via a liquid transfer bus, operating a robot to move the cartridge to the electroporation instrument to interface the electroporation module with the electroporation instrument, and operating the electroporation instrument to cause the electroporation module to perform electroporation of the selected cell population in the presence of the vector.

[0039] In some variations, the transduction step may include carrying out transduction of the selected cell population in solution by delivering an effective amount of vector via a fluid transfer bus to a module containing the solution containing the cell product. In some variations, the fill / finish step may include delivering a formulation solution via a fluid transfer bus to a module containing the cell product to produce a final cell product, and delivering the final cell product to one or more product collection bags.

[0040] In some variations, the method may include manually or automatically sterilizing the cartridge at the feed-through port. In some variations, the method may include manually or automatically loading one or more of the fluid and the cell product into the cartridge via the sterile fluid transfer port. In some variations, the method may include manually or automatically removing the cell product from the cartridge. In some variations, the cell product may include immune cells. In some variations, the steps are, in order: enrichment step, selection step, activation step, transduction step, expansion step, and harvesting step.

[0041] In some variations, the immune cells may comprise genetically engineered chimeric antigen receptor T cells. In some variations, the immune cells may comprise genetically engineered T cell receptor (TCR) cells. In some variations, the immune cells may comprise natural killer (NK) cells. In some variations, the cell product may comprise hematopoietic stem cells (HSCs). In some variations, the method may include, in sequence, an enrichment step, a selection step, a settling step, a transduction step, and a harvesting step. In some variations, the cell product may comprise tumor infiltrating lymphocytes (TILs). In some variations, the method may include, in sequence, a tissue digestion step, a washing step, an activation step, a proliferation step, and a harvesting step.

[0042] Also described herein is a counterflow centrifugal elutriation (CCE) module that includes a conical element having an inner and outer surface fixedly attached to a distal end of a linear member having an inner and outer surface, the proximal end of the linear member being rotatably attached to a fulcrum to allow extension, retraction, and rotation of the linear member.

[0043]

[0043] Also described herein is a work cell comprising an enclosure, a plurality of instruments, each independently configured to perform one or more cell processing operations on a cartridge, and a robot capable of moving the cartridge between each of the plurality of instruments.

[0044] In some variations, the enclosure may include an air filtration inlet configured to maintain ISO 7 or higher air quality within an interior zone of the work cell. In some variations, the work cell may be automated. In some variations, the instruments may interface with the cartridges to perform cell processing operations on the cartridges. In some variations, the work cell may include a processor. The processor may be configured to control the robot and the plurality of instruments.

[0045] In some variations, the work cell may be configured to receive two or more cartridges. In some variations, the work cell may include a cartridge. In some variations, the cartridge may include multiple modules. In some variations, the cartridge may include a bioreactor module. In some variations, the cartridge may include a cell selection module. In some variations, the cell selection module may include a magnetically activated cell selection module. In some variations, the cartridge may include a sorting module.

[0046] In some variations, the sorting module may include a fluorescence-activated cell sorting (FACS) module. In some variations, the cartridge may include an electroporation module. In some variations, the cartridge may include a counterflow centrifugal elutriation (CCE) module. In some variations, the cartridge may include one or more sterile fluid transfer ports. In some variations, the cartridge may include a fluid transfer bus fluidly coupled to each module. In some variations, the cartridge may include a pump fluidly coupled to the fluid transfer bus.

[0047] In some variations, the work cell may include a pump actuator configured to interface with a pump. In some variations, the work cell may include a bioreactor instrument. In some variations, the bioreactor instrument may include a plurality of slots for cartridges. In some variations, the work cell may include a cell selection instrument. In some variations, the cell selection instrument may include a magnetically activated cell selection instrument. In some variations, the work cell may include a sorting instrument. In some variations, the sorting instrument may include a fluorescence activated cell sorting (FACS) instrument. In some variations, the work cell may include an electroporation instrument.

[0048] In some variations, the work cell may include a counterflow centrifugal elutriation (CCE) instrument. In some variations, the work cell may include reagent reservoirs. In some variations, the cartridge may include a bioreactor module and a selection module. In some variations, the cartridge may include a bioreactor module and a CCE module. In some variations, the cartridge may include a bioreactor module, a selection module, and a CCE module. In some variations, the cartridge may include a bioreactor module, a selection module, and an electroporation module. In some variations, the cartridge may include a bioreactor module, a selection module, a CCE module, and an electroporation module. In some variations, the cartridge may include a second bioreactor module having an internal volume that is two or more times, five or more times, or ten or more times larger than the internal volume of the first bioreactor. In some variations, the enclosure may include a feedthrough. In some variations, the work cell may perform automated manufacturing of a cellular product. In some variations, the system may include multiple bioreactor instruments. Each bioreactor instrument may be configured to receive a single cartridge.

[0049]

[0049] Also described herein is a rotor comprising a first side including a first fluid conduit, a second side including a second fluid conduit opposite the first side, and a cone coupled between the first fluid conduit and the second fluid conduit.

[0050] In some variations, the cone may include a bicone. In some variations, the bicone may comprise a first cone including a first base and a second cone including a second base. The first base may oppose the second base. In some variations, the rotor may include a magnetic portion. In some variations, the rotor may define an axis of rotation. In some variations, at least a portion of the first fluid conduit and at least a portion of the second fluid conduit may extend parallel to the axis of rotation. In some variations, at least a portion of the first fluid conduit and at least a portion of the second fluid conduit may be coaxial.

[0051] In some variations, the cone may comprise a volume of about 10 mL to about 40 mL. In some variations, the cone may comprise a cone angle of about 30 degrees to about 60 degrees. In some variations, at least a portion of the rotor may be optically transparent. In some variations, the rotor may comprise an asymmetric shape. In some variations, the first portion may comprise a cone and the second portion may comprise a paddle shape.

[0052] In some variations, a cartridge for cell processing can include a fluid transfer bath and a plurality of modules. Each module can be fluidly coupled to the fluid transfer bath. The cartridge can include a counterflow centrifugal elutriation (CCE) module including a rotor described herein.

[0053] Also described herein is a rotor comprising a first fluid conduit and a first cone coupled to the first fluid conduit. The first cone may include a first volume. A second fluid conduit may be coupled to the first cone. A second cone may be coupled to the second fluid conduit. The second cone may include a second volume larger than the first volume. A third fluid conduit may be coupled to the second cone.

[0054] In some variations, the first cone may include a first bicone and the second cone may include a second bicone. In some variations, the first bicone may include a third cone including a first base and a fourth cone including a second base. The first base may be opposite the second base. The second bicone may include a fifth cone including a third base and a sixth cone including a fourth base. The third base may be opposite the fourth base.

[0055] In some variations, the rotor may include a magnetic portion. In some variations, at least a portion of the rotor may be optically transparent. In some variations, the first fluid conduit may include an inlet and the third fluid conduit may include an outlet.

[0056]

[0056] Also described herein is a system for cell processing comprising a cartridge having a housing including a rotor configured to separate cells from a fluid, and an instrument including a magnet configured to magnetically rotate the rotor in conjunction with the cartridge.

[0057] In some variations, the cartridge may be configured to move between multiple instruments. In some variations, there may be an air gap between the housing and the magnet. In some variations, the housing may house the rotor. In some variations, the housing may include a consumable component and the magnet may include a durable component.

[0058] In some variations, the magnet can be releasably coupled to the housing. In some variations, the magnet can be configured to move relative to the housing. In some variations, the separated cells can comprise a first size and a first density, and the unseparated cells in the fluid can comprise a second size and a second density different from the first size and the first density. Also described herein is a cartridge for cell processing comprising a liquid transfer bath and a plurality of modules. Each module can be fluidly coupled to the liquid transfer bath. The cartridge can include a counterflow centrifugal elutriation (CCE) module including a rotor described herein.

[0059]

[0059] Also described herein is a method of countercurrent centrifugal elutriation (CCE) that includes moving a rotor defining an axis of rotation toward a magnet, flowing a fluid through the rotor, and magnetically rotating the rotor about the axis of rotation using the magnet while the fluid is flowing through the rotor.

[0060] In some variations, an optical sensor may be used to generate image data of one or more of the fluid and particles within the rotor, and one or more of the rotational speed of the rotor and the flow rate of the fluid may be selected based at least in part on the image data.

[0061] In some variations, the illumination source can be used to illuminate one or more of the fluid and the cells. In some variations, the method can include moving the rotor away from the magnet. In some variations, the method can include moving the rotor toward the illumination source and the optical sensor and moving the rotor away from the illumination source and the optical sensor.

[0062] In some variations, moving the rotor may include using a robot to advance and retract a magnet relative to the rotor. In some variations, rotating the rotor may include a rotational speed of up to 6,000 RPM. In some variations, flowing the fluid may include a flow rate of up to about 150 ml / min during rotation of the rotor.

[0063] Also described herein is a method of magnetically activated cell selection that includes flowing a fluid containing input cells into a flow cell. The set of cells can be labeled with a magnetically activated cell selection (MACS) reagent. The set of cells can be magnetically attracted toward a magnet array for a dwell time. After the dwell time, the set of cells can be flowed out of the flow cell.

[0064] In some variations, the method may include incubating the MACS reagent with the input cells to label the set of cells with the MACS reagent. In some variations, the method may include incubating the MACS reagent at a temperature of about 1°C to about 10°C. In some variations, the method may include flowing the set of cells out of the flow cell by flowing a gas through the flow cell. In some variations, the method may include flowing a fluid without the set of cells out of the flow cell after a residence time. In some variations, the residence time may be at least about 1 minute. In some variations, the magnet array may be disposed outside the flow cell. In some variations, the method may include moving the magnet array relative to the flow cell. In some variations, moving the magnet array may include moving the magnet array away from the flow cell to facilitate flow of the set of cells out of the flow cell. In some variations, the longitudinal axis of the flow cell may be perpendicular to the ground. In some variations, the flow cell may be free of beads.

[0065] Also described herein is a magnetically activated cell selection (MACS) module comprising a flow cell including an elongated cavity having a cavity height. The magnet array can include a plurality of magnets. Each of the magnets can be separated by a separation distance. The ratio of the cavity height to the separation distance can be from about 20:1 to about 1:20.

[0066] In some variations, the flow cell may include a set of linear channels. The linear channels include a first channel parallel to a second channel and a third channel in fluid communication with each of the first and second channels. In some variations, the first channel may include a first cavity height, and the second channel may include a second cavity height. The ratio of the first cavity height to the second cavity height may be from about 1:1 to about 3:7. In some variations, the third channel may have a ratio of the length of the third channel to the diameter of the third channel of from about 2:1 to about 6:1.

[0067] In some variations, a first fluid conduit can be coupled to the inlet of the flow cell and to the outlet of the flow cell. The first fluid conduit can be configured to receive the cell set from the flow cell. A second fluid conduit can be coupled to the inlet of the flow cell and to the outlet of the flow cell. The second fluid conduit can be configured to receive fluid from the flow cell that does not include the cell set.

[0068] In some variations, a cartridge for cell processing can include a fluid transfer bus and multiple modules. Each module can be fluidly coupled to the fluid transfer bus. The cartridge can include a magnetically activated cell selection (MACS) module as described herein.

[0069] Also described herein is a system for cell processing comprising a cartridge including a rotor configured for countercurrent centrifugal elutriation of cells in a fluid. A first magnet can be configured to magnetically rotate the rotor and separate the cells from the fluid in the rotor. The cartridge can further include a flow cell in fluid communication with the rotor and configured to receive the cells from the rotor. A second magnet can be configured to magnetically separate the cells in the flow cell.

[0070] In some variations, the illumination source may be configured to illuminate the cells. The optical sensor may be configured to generate image data corresponding to the cells. In some variations, the system may include one or more of an oxygen deficiency sensor, a leak sensor, an inertial sensor, a pressure sensor, and a bubble sensor. In some variations, the system may include one or more valves and pumps.

[0071]

[0071] In some variations, the separated cells may comprise a first size and a first density, and the unseparated cells of the fluid may comprise a second size and a second density different from the first size and the first density.

[0072] Also described herein is an electroporation module comprising: a fluid conduit configured to receive a first fluid containing cells and a second fluid; an electrode set coupled to the fluid conduit; a pump coupled to the fluid conduit; and a controller including a processor and a memory. The controller can be configured to generate a first signal to introduce the first fluid into the fluid conduit using the pump, generate a second signal to introduce the second fluid into the fluid conduit such that the second fluid separates the first fluid from a third fluid, and generate an electroporation signal to electroporate the cells in the fluid conduit using the electrode set.

[0073] 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 a third fluid into the fluid conduit. The third fluid is separated from the first fluid by the second fluid.

[0074] In some variations, a cartridge for cell processing can include a fluid transfer bus and a plurality of modules. Each module can be fluidly coupled to the fluid transfer bus. The cartridge can include an electroporation module as described herein.

[0075]

[0075] Also described herein is a method of electroporating cells, comprising receiving a first fluid containing cells in a fluid conduit, receiving a second fluid in the fluid conduit so as to separate the first fluid from a third fluid, and applying an electroporation signal to the first fluid to electroporate the cells.

[0076] In some variations, the method may include receiving a third fluid in the fluid conduit separated from the first fluid by the second fluid. In some variations, the first fluid may be substantially static when applying the electroporation signal.

[0077]

[0077] Also described herein is a method for electroporating cells, comprising receiving a first fluid containing cells in a fluid tube, applying a resistance measurement signal to the first fluid using an electrode set, measuring the resistance between the first fluid and the electrode set, and applying an electroporation signal to the first fluid based on the measured resistance.

[0078] In some variations, the method may include receiving a second fluid including a gas into the fluid conduit prior to applying the electroporation signal to the fluid, the first fluid being separated from the third fluid by the second fluid.

[0079] Also described herein is a bioreactor comprising an enclosure including a bottom, a top, and at least one sidewall. A gas permeable membrane can be coupled to one or more of the bottom and sidewalls of the enclosure.

[0080] In some variations, the enclosure may include one or more nesting surfaces curved about the longitudinal axis of the enclosure. In some variations, the one or more nesting surfaces may include a set of concentric toroids. In some variations, the enclosure may include a toroidal shape. In some variations, the enclosure may include a first chamber having a first volume and a second chamber having a second volume. The first chamber is separated from the second chamber, and the first volume is smaller than the second volume. In some variations, the enclosure may include a column extending along the longitudinal axis of the enclosure. In some variations, a cavity may exist between the enclosure and the gas-permeable membrane. In some variations, the gas-permeable membrane may extend along the bottom and sidewalls of the enclosure. In some variations, the outer surface of the gas-permeable membrane may include one or more protrusions.

[0081] In some variations, the bottom of the gas-permeable membrane may include an angle of about 3 degrees to about 10 degrees relative to the bottom of the enclosure. In some variations, the gas-permeable membrane may include a curved surface. In some variations, the gas-permeable membrane may include a set of patterned curved surfaces. In some variations, the set of patterned curved surfaces may have a radius of curvature of about 50 mm to about 500 mm.

[0082] In some variations, a cartridge for cell processing may include a liquid transfer bus and a plurality of modules. Each module may be fluidly coupled to the liquid transfer bus. The cartridge may include a bioreactor module as described herein. In some variations, a system for cell processing may include a cartridge as described herein and further include a bioreactor instrument configured to interface with the cartridge. The bioreactor instrument may include an agitator configured to couple to the bioreactor. The agitator may be configured to agitate the cell culture medium containing the cells. In some variations, a fluidic connector may be configured to couple the bioreactor to the liquid transfer bus. The fluidic connector may include a foldable sidewall. In some variations, the system may include a temperature regulator coupled to the bioreactor. In some variations, the system may include a gas regulator coupled to the bioreactor.

[0083] Also described herein is a fluid connector comprising a first connector including a first proximal end configured to couple to a first fluid device and a first distal end including a first port. The second connector can include a second proximal end configured to couple to a second fluid device and a second distal end including a second port configured to couple to the first port. The first distal end can include a first lumen and the second distal end can include a second lumen. One of the first valve and the second valve can be configured to translate within the first lumen and the second lumen.

[0084] In some variations, the first valve and the second valve may be configured to transition from a closed configuration to an open configuration only when the first valve is mated with the second valve. In some variations, the first port and the second port may be configured to transition between an open configuration and a closed configuration. In some variations, the first connector may include a first port actuator, and / or the second connector may include a second port actuator. In some variations, the second port may be mated to the first port and define a chamber.

[0085] In some variations, one or more of the first connector and the second connector may include a sterilant port configured to couple to a sterilant source. The sterilant port may be configured to be in fluid communication with the first distal end and the second distal end when the second port is coupled to the first port.

[0086] In some variations, the chamber can be configured to receive one or more of the fluid and the sterilant from a sterilant port. In some variations, the sterilant port can be configured to receive a sterilant such that the sterilant sterilizes the first connector and the second connector.

[0087] In some variations, the first connector may include a first valve and the second connector may include a second valve configured to couple to the first valve. In some variations, the first seal may include a first port coupled to a second port and the second seal may include a first valve coupled to the second valve. In some variations, the sterilant may include one or more of vaporized hydrogen peroxide and ethylene oxide.

[0088] In some variations, the fluid connector may include one or more robotic engagement features. In some variations, the first connector may include a first alignment feature and the second connector may include a second alignment feature configured to mate with the first alignment feature in a predetermined axial-rotational configuration. In some variations, one or more of the first fluidic device and the second fluidic device may include an instrument.

[0089] In some variations, the system may further include a robot configured to operate the fluid connector 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 first port signal to couple the first port to the second port using the robot arm. In some variations, the controller may be configured to generate a first valve signal to translate the first valve relative to the second valve using the robot arm, and further to generate a second valve signal to transition the first valve and the second valve to an open configuration. In some variations, the controller may be configured to generate a second port signal to decouple the first port from the second port. Sterility of the fluid connector may be maintained before coupling the first port to the second port and after decoupling the first port from the second port.

[0090] In some variations, a fluid pump can be coupled to the sterilant source. The controller can be configured to generate a first fluid pump signal to circulate fluid into the chamber through the sterilant port. In some variations, the controller can be configured to generate a second fluid pump signal to circulate sterilant into the chamber through the sterilant port to at least sterilize the chamber.

[0091] In some variations, the controller may be configured to generate a third fluid pump signal to remove sterilant from the chamber. In some variations, the controller may be configured to generate a heat sterilization signal to thermally sterilize the fluid connector. In some variations, the controller may be configured to generate a radiation sterilization signal to sterilize the fluid connector using radiation. In some variations, the robot may be configured to couple a fluid connector between at least two of the plurality of instruments and cartridges.

[0092] In some variations, the fluid connector may further include a controller including a memory and a processor and coupled to the robot. The controller may be configured to generate a port signal to couple the first port to the second port using the robot arm, generate a first valve signal to translate the first valve relative to the second valve using the robot arm, and generate a second valve signal to transition the first valve and the second valve to an open configuration.

[0093] Also described herein is a non-transitory computer-readable medium for converting user-defined cell processing operations into cell processing steps to be executed by an automated cell processing system. The non-transitory computer-readable medium may include stored instructions that, when executed on a processor, may perform the steps of receiving an ordered input list of cell processing operations and executing a conversion model on the ordered input list to generate an ordered output list of cell processing steps that can be executed by the system.

[0094]

[0094] In some variations, the ordered output list can be executed by the system to control a robot to move one or more cartridges, each containing a cellular product, between instruments and to control the instruments to perform a cell processing step on each cellular product.

[0095] In some variations, the method may include receiving one or more sets of cell processing parameters. Each set of cell processing parameters is associated with one of the cell processing operations, and each set of cell processing parameters defines characteristics of a cell processing step performed by an instrument in the cell processing step. In some variations, the transformation model may include constraints on the ordered output list determined by the configuration of the automated cell processing system. In some variations, the constraints may include information regarding the configuration of the automated cell processing system. In some variations, the constraints may include one or more of the type and / or number of instruments, the type and / or number of modules on the cartridge, the type and number of reservoirs on the cartridge, the type and / or number of sterile fluid transfer ports on the cartridge, and the number and location of fluid paths between the modules, reservoirs, and sterile fluid transfer ports on the cartridge.

[0096] In some variations, the steps may further include receiving a set of two or more ordered input lists of cell processing operations to perform on two or more cartridges in the automated cell processing system, and executing a transformation model on the set of ordered input lists to generate an ordered output list of cell processing steps. The ordered output list may be executed by the system to control a robot to move two or more cartridges, each containing a cell product, between instruments, and to control the instrument to perform a cell processing step on each cell product in each cartridge.

[0097]

[0097] In some variations, the automated cell processing system may include a non-transitory computer-readable medium as described in any preceding claim.

[0098]

[0098] In some variations, a computer-implemented method for converting user-defined cell processing operations into cell processing steps to be executed by a processor of an automated cell processing system may include receiving an ordered input list of cell processing operations and running a conversion model on the ordered input list to generate an ordered output list of cell processing steps that can be executed by the system.

[0099]

[0099] In some variations, the method may include controlling a robot to move one or more cartridges, each containing a cell product, between instruments and controlling the instruments to perform a cell processing step on each cell product.

[0100] In some variations, the method may include receiving one or more sets of cell processing parameters. Each set of cell processing parameters is associated with one of the cell processing operations, and each set of cell processing parameters defines characteristics of a cell processing step performed by an instrument in the cell processing step. In some variations, the transformation model may include constraints on the ordered output list determined by a configuration of the automated cell processing system. In some variations, the constraints may include information about the configuration of the automated cell processing system.

[0101]

[0101] In some variations, the constraints may include one or more of the type and / or number of instruments, the type and / or number of modules on the cartridge, the type and number of reservoirs on the cartridge, the type and / or number of sterile fluid transfer ports on the cartridge, and the number and location of fluid paths between the modules, reservoirs, and sterile fluid transfer ports on the cartridge.

[0102]

[0102] In some variations, the method may include receiving a set of two or more ordered input lists of cell processing operations to perform on two or more cartridges in an automated cell processing system, running a transformation model on the set of ordered input lists to generate an ordered output list of cell processing steps, controlling a robot to move two or more cartridges, each containing a cell product, between instruments, and controlling the instrument to perform a cell processing step for each cell product in each cartridge.

[0103]

[0103] Additional modifications, features, and advantages of the invention will become apparent from the following detailed description and through practice of the invention. [Brief explanation of the drawings]

[0104] [Figure 1A]

[0104] Block diagram of an exemplary variation of a cell processing system. [Figure 1B]

[0105] FIG. 10 is a block diagram of an exemplary variation of the cartridge. [Figure 2A]

[0106] FIG. 1 is a block diagram of an exemplary variation of a cell processing system. [Figure 2B]

[0106] A perspective view of an exemplary variation of a work cell of a cell processing system. [Figure 2C]

[0106] A perspective view of an exemplary variation of the work cell and cartridge of the cell processing system. [Figure 2D]

[0106] Block diagram of an exemplary variation of a cell processing system. [Figure 2E]

[0106] A block diagram of another exemplary variation of a cell processing system. [Figure 3]

[0107] FIG. 10 is a block diagram of another exemplary variation of a cell processing system. [Figure 4A]

[0108] FIG. 10 is a perspective view of another exemplary variation of a cell processing system. [Figure 4B]

[0108] Another perspective view of another exemplary variation of the cell processing system. [Figure 5]

[0109] FIG. 10 is a perspective view of another exemplary variation of a cell processing system. [Figure 6]

[0110] 1A-1C are schematic diagrams of exemplary variations of the cartridge. [Figure 7]

[0111] FIG. 10 is a schematic diagram of another exemplary variation of the cartridge. [Figure 8A]

[0112] FIG. 10 is a side view of an exemplary variation of the cartridge. [Figure 8B]

[0112] A top view of an exemplary variation of the cartridge. [Figure 8C]

[0112] A side view of an exemplary variation of the cartridge. [Figure 8D]

[0112] A perspective view of an exemplary variation of the cartridge. [Figure 9]

[0113] 10A-10C are side cross-sectional views of exemplary variations of cartridges. [Figure 10A]

[0114] 1 illustrates exemplary variations of rotary valves and actuators. [Figure 10B]

[0114] An exemplary variation of a rotary valve docked to an actuator is shown. [Figure 11A]

[0115] 10A-10C are perspective views of exemplary variations of cartridges including CCE modules in expanded configurations. [Figure 11B]

[0115] FIG. 10 is a cross-sectional side view of an exemplary variation of a CCE module in a storage configuration. [Figure 11C]

[0115] A side cross-sectional view of an exemplary variation of a CCE module in an expanded configuration. [Figure 12A]

[0116] 1 is a perspective view of an exemplary variation of a magnetic activated cell sorting (MACS) instrument including a magnet in an ON configuration. FIG. [Figure 12B]

[0116] Figure 1 is a perspective view of an exemplary variation of a MACS instrument including a magnet in an OFF configuration. [Figure 13A]

[0117] 1A-1C are perspective views of exemplary variations of the cartridge and bioreactor device. [Figure 13B]

[0117] A perspective view of an exemplary variation of a cartridge coupled to a bioreactor device. [Figure 14]

[0118] 1A and 1B are perspective views of an exemplary variation of a bioreactor device including a set of cartridges and cavities configured to receive the cartridges. [Figure 15]

[0119] FIG. 1 is a block diagram of an exemplary variation of a fluid connector system. [Figure 16A]

[0120] 1A-1C are schematic diagrams of exemplary variations of fluid connectors. [Figure 16B] FIG. 16B is a detailed schematic diagram of the fluid connector shown in FIG. 16A. [Figure 16C]

[0120] FIG. 16B is a schematic diagram of the fluid connector shown in FIG. 16A in a coupled configuration. [Figure 16D]

[0120] FIG. 16B is a schematic diagram of the fluid connector shown in FIG. 16A in an open port configuration. [Figure 16E]

[0120] FIG. 16B is a schematic diagram of the fluid connector shown in FIG. 16A receiving a gas. [Figure 16F]

[0120] FIG. 16B is a schematic diagram of the fluid connector shown in FIG. 16A receiving a sterilant. [Figure 16G]

[0120] FIG. 16B is a schematic diagram of the fluid connector shown in FIG. 16A in an open valve configuration. [Figure 16H]

[0120] FIG. 16B is a schematic diagram of the fluidic connector shown in FIG. 16A transferring fluid between fluidic devices coupled to the fluidic connector. [Figure 16I]

[0120] FIG. 16B is a schematic diagram of the fluid connector shown in FIG. 16A in a closed valve configuration. [Figure 16J]

[0120] FIG. 16B is a schematic diagram of the fluid connector shown in FIG. 16A in a closed port configuration. [Figure 16K]

[0120] FIG. 16B is a schematic diagram of the fluid connector shown in FIG. 16A in a disconnected configuration. [Figure 16L]

[0120] FIG. 16B is a schematic diagram of the fluid connector shown in FIG. 16A separated from the sterilant source. [Figure 17A]

[0121] FIG. 1 is a front perspective view of a fluid connector having a closed port configuration. [Figure 17B]

[0121] FIG. 17B is a rear perspective view of the closed port configuration fluid connector shown in FIG. 17A. [Figure 17C]

[0121] FIG. 17C is a rear view of the closed port configuration fluid connector shown in FIG. 17B. [Figure 17D]

[0121] A front perspective view of a fluid connector with an open port configuration. [Figure 17E]

[0121] FIG. 17D is a rear perspective view of the open port configuration fluid connector shown in FIG. [Figure 17F]

[0121] FIG. 17E is a rear view of the open port configuration fluid connector shown in FIG. [Figure 18A]

[0122] FIG. 10 is a side view of a fluid connector in a breakaway configuration. [Figure 18B]

[0122] A cross-sectional side view of a fluid connector in a separated configuration. [Figure 18C]

[0122] A side view of the fluid connector in a coupled configuration. [Figure 18D]

[0122] A side cross-sectional view of a fluid connector in a coupled configuration. [Figure 18E]

[0122] A side view of a fluid connector with an open port configuration. [Figure 18F]

[0122] A side cross-sectional view of a fluid connector with an open port configuration. [Figure 18G]

[0122] Side view of a fluid connector with an open valve configuration. [Figure 18H]

[0122] A side cross-sectional view of a fluid connector with an open valve configuration. [Figure 19]

[0123] 1 is a schematic diagram of an exemplary variation of a fluid connector system. [Figure 20A]

[0124] 1 is a schematic diagram of an exemplary variation of a fluid connector system. [Figure 20B]

[0124] Schematic diagram of an exemplary variation of a fluid connector connection process. [Figure 20C]

[0124] Schematic diagram of an exemplary variation of a fluid connector connection process. [Figure 21]

[0125] FIG. 1 is a block diagram of an exemplary variation of a fluid connector system. [Figure 22]

[0126] FIG. 1 is a block diagram of an exemplary variation of a fluid connector system. [Figure 23]

[0127] FIG. 1 is a block diagram of an exemplary variation of a fluid connector system. [Figure 24A]

[0128] FIG. 1 is a block diagram of an exemplary variation of a fluid connector system. [Figure 24B]

[0128] Schematic diagram of an exemplary variation of a fluid connector connection process. [Figure 24C]

[0128] Schematic diagram of an exemplary variant of the valve. [Figure 25A]

[0129] FIG. 1 is a block diagram of an exemplary variation of a fluid connector system. [Figure 25B]

[0129] Schematic diagram of an exemplary variation of a fluid connector connection process. [Figure 25C]

[0129] Schematic diagram of an exemplary variant of the valve. [Figure 26A]

[0130] 1A-1C are side views of exemplary variations of pump actuators and pumps. [Figure 26B]

[0130] A side view of an exemplary variation of a pump actuator coupled to a pump. [Figure 27]

[0131] 10 is a flowchart of an exemplary variation of a method for transferring fluid using a fluid connector. [Figure 28]

[0132] 1 is a flow chart of an exemplary variation of a cell processing method. [Figure 29]

[0133] 1 is a flow chart of an exemplary variation of a cell processing method. [Figure 30A]

[0134] 1 is a flow chart of an exemplary variation of the cell treatment method for autologous CAR-T cells or engineered TCR cells. [Figure 30B]

[0134] Flowchart of an exemplary variation of the cell treatment method for allogeneic CAR-T cells or genetically engineered TCR cells. [Figure 31]

[0135] 1 is a flow chart of an exemplary variation of a cell treatment method for HSC cells. [Figure 32]

[0136] 1 is a flow chart of an exemplary variation of a cell treatment method for TIL cells. [Figure 33]

[0137] 1 is a flowchart of an exemplary variation of the cell treatment method for NK-CAR cells. [Figure 34A]

[0138] 1 is a flow chart of an exemplary variation of the cell treatment method for Treg cells. [Figure 34B]

[0138] Flow chart of an exemplary variation of the cell treatment method for Treg cells. [Figure 34C]

[0138] Flow chart of an exemplary variation of the cell treatment method for Treg cells. [Figure 35]

[0139] 1 is a flow chart of an exemplary variation of a cell processing method. [Figure 36]

[0140] 10 is a flowchart of an exemplary variation of a method for implementing a transformation model. [Figure 37]

[0141] 1 is an exemplary variation of a graphical user interface associated with an initial process design interface. [Figure 38]

[0142] 10 is an exemplary variation of a graphical user interface associated with creating a process. [Figure 39]

[0143] 10 is an exemplary variation of a graphical user interface associated with an empty process. [Figure 40]

[0144] 10 is an exemplary variation of a graphical user interface associated with adding reagent and consumable containers. [Figure 41]

[0145] 10 is an exemplary variation of a graphical user interface related to process parameters. [Figure 42]

[0146] 10 is an exemplary variation of a graphical user interface associated with a patient weight process parameter. [Figure 43]

[0147] 10 is an exemplary variation of a graphical user interface associated with a pre-processing analysis. [Figure 44]

[0148] 10 is an exemplary variation of a graphical user interface associated with a white blood cell count preprocessing analysis. [Figure 45]

[0149] 10 is an exemplary variation of a graphical user interface associated with process parameter calculations. [Figure 46]

[0150] 10 is an exemplary variation of a graphical user interface associated with a completed process setup. [Figure 47]

[0151] 10 is an exemplary variation of a graphical user interface associated with process operation activation settings. [Figure 48]

[0152] 10 is an exemplary variation of a graphical user interface associated with a completed process operation activation setting. [Figure 49]

[0153] 10 is an exemplary variation of a graphical user interface associated with an initial process operation. [Figure 50]

[0154] 10 is an exemplary variation of a graphical user interface associated with dragging a process action. [Figure 51]

[0155] 10 is another exemplary variation of a graphical user interface associated with dragging a process action. [Figure 52]

[0156] 10 is an exemplary variation of a graphical user interface associated with a filled-in process action. [Figure 53]

[0157] 10 is an exemplary variation of a graphical user interface associated with product monitoring. [Figure 54]

[0158] 10 is another exemplary variation of a graphical user interface associated with product monitoring. [Figure 55]

[0159] FIG. 1 is a block diagram of an exemplary variation of a manufacturing workflow. [Figure 56]

[0160] FIG. 1 is a block diagram of an exemplary variation of a cell separation system. [Figure 57]

[0161] FIG. 1 is a cross-sectional side view of an exemplary variation of a countercurrent centrifugal elutriation (CCE) module. [Figure 58]

[0162] 1A and 1B are side cross-sectional views of exemplary variations of magnetically activated cell selection (MACS) modules. [Figure 59A]

[0163] 1 is a perspective view of an exemplary variation of a CCE system. [Figure 59B]

[0163] A perspective view of an exemplary variation of a CCE system. [Figure 59C]

[0163] A perspective view of an exemplary variation of a CCE system. [Figure 59D]

[0163] A side cross-sectional view of an exemplary variation of a CCE system. [Figure 59E]

[0163] A side cross-sectional view of an exemplary variation of the rotor of the CCE module. [Figure 59F]

[0163] A side cross-sectional view of an exemplary variation of the rotor of the CCE module. [Figure 59G]

[0163] A side cross-sectional view of an exemplary variation of the rotor of the CCE module. [Figure 60A]

[0164] 10A-10C are plan views of exemplary variations of the rotor of the CCE module. [Figure 60B]

[0164] A perspective view of an exemplary variant of the rotor of the CCE module. [Figure 60C]

[0164] A perspective view of an exemplary variant of the rotor of the CCE module. [Figure 60D]

[0164] A side view of an exemplary variant of the rotor of the CCE module. [Figure 60E]

[0164] A perspective view of an exemplary variant of a rotor within a housing. [Figure 60F]

[0164] A plan view schematic diagram of an exemplary variant of the rotor of the CCE module. [Figure 60G]

[0164] A plan view schematic diagram of an exemplary variant of the rotor of the CCE module. [Figure 60H]

[0164] A side view of an exemplary variant of the rotor of the CCE module. [Figure 60I]

[0164] A perspective view of another exemplary variant of the rotor of the CCE module. [Figure 60J]

[0164] FIG. 10 is a perspective view of yet another exemplary variation of the rotor of the CCE module. [Figure 60K]

[0164] FIG. 10 is a schematic plan view of another exemplary variation in rotor dimensions of a CCE module. [Figure 60L]

[0164] An image of an exemplary set of deformations of the rotor of a CCE module. [Figure 61A]

[0165] 1 is a schematic diagram of an exemplary variation of a cell separation process. [Figure 61B]

[0165] Schematic diagram of an exemplary variation of the cell separation process. [Figure 61C]

[0165] Schematic diagram of an exemplary variation of the cell separation process. [Figure 62A]

[0166] FIG. 1 is a perspective view of an exemplary variation of a MACS system in a first configuration. [Figure 62B]

[0166] A perspective view of an exemplary variation of a MACS system in a second configuration. [Figure 62C]

[0166] A side cross-sectional view of an exemplary variation of the MACS system. [Figure 62D]

[0166] A perspective view of an exemplary variation of a MACS system in a second configuration. [Figure 62E]

[0166] A plan view of an exemplary variation of the flow cell and magnet array of the MACS system. [Figure 62F]

[0166] A plan view of an exemplary variation of the flow cell of the MACS system. [Figure 62G]

[0166] Schematic diagram of exemplary variations of flow cells and magnet arrays. [Figure 63A]

[0167] 1A and 1B are perspective views of exemplary variations of magnet arrays. [Figure 63B]

[0167] A perspective view of an exemplary variation of a magnet array. [Figure 63C]

[0167] A perspective view of an exemplary variation of a magnet array. [Figure 63D]

[0167] A perspective view of an exemplary variation of a magnet array. [Figure 63E]

[0167] A perspective view of an exemplary variation of a magnet array. [Figure 64A]

[0168] 1A and 1B are perspective views of exemplary variations of flow cells. [Figure 64B]

[0168] A side cross-sectional view of an exemplary variation of a flow cell. [Figure 64C]

[0168] Schematic diagram of an exemplary variation of the MACS system. [Figure 65A]

[0169] 1 is a schematic diagram of an exemplary variation of a flow cell. [Figure 65B]

[0169] Schematic diagram of an exemplary variation of a flow cell. [Figure 65C]

[0169] Schematic diagram of an exemplary variation of a flow cell. [Figure 66A]

[0170] 1 is a schematic diagram of an exemplary variation of a cell separation process. [Figure 66B]

[0170] Schematic diagram of an exemplary variation of the cell separation process. [Figure 66C]

[0170] Schematic diagram of an exemplary variation of the cell separation process. [Figure 67A]

[0171] 1 is a schematic diagram of an exemplary variation of a cell processing system. [Figure 67B]

[0171] Schematic diagram of an exemplary variation of a cell processing system. [Figure 67C]

[0171] Schematic diagram of an exemplary variation of a cell processing system. [Figure 67D]

[0171] Schematic diagram of an exemplary variation of a cell processing system. [Figure 68A]

[0172] FIG. 1 is a cross-sectional perspective view of an exemplary variation of a bioreactor. [Figure 68B]

[0172] A cross-sectional side view of an exemplary variation of a bioreactor. [Figure 68C]

[0172] A perspective view of an exemplary variation of a bioreactor enclosure. [Figure 68D]

[0172] A plan view of an exemplary variation of the bioreactor enclosure. [Figure 68E]

[0173] 1A and 1B are perspective views of exemplary variations of membranes for bioreactors. [Figure 68F]

[0173] A side view of an exemplary variation of the membrane of the bioreactor. [Figure 68G]

[0173] A perspective view of an exemplary variation of the membrane of the bioreactor. [Figure 68H]

[0173] A bottom view of an exemplary variation of the membrane of the bioreactor. [Figure 69A]

[0174] FIG. 1 is a cross-sectional side view of an exemplary variation of a bioreactor enclosure. [Figure 69B]

[0174] A cross-sectional perspective view of an exemplary variation of a bioreactor enclosure. [Figure 70]

[0175] FIG. 1 is an exploded perspective view of an exemplary variation of a bioreactor. [Figure 71A]

[0176] FIG. 1 is a plan view of an exemplary variation of a bioreactor. [Figure 71B]

[0176] A cross-sectional side view of an exemplary variation of a bioreactor. [Figure 72]

[0177] FIG. 1 is a schematic diagram of an exemplary variation of an electroporation system. [Figure 73]

[0178] FIG. 1 is an exploded perspective view of an exemplary variation of an electroporation module. [Figure 74A]

[0179] FIG. 1 is a schematic diagram of an exemplary variation of the electroporation process. [Figure 74B]

[0179] Schematic diagram of an exemplary variation of the electroporation process. [Figure 75]

[0180] FIG. 1 is a schematic diagram of an exemplary variation of an electroporation process. [Figure 76A]

[0181] 1 is a plot of an exemplary variation of an electroporation process. [Figure 76B]

[0181] A plot of an exemplary variation of the electroporation process. [Figure 76C]

[0181] A plot of an exemplary variation of the electroporation process. [Figure 76D]

[0181] A plot of an exemplary variation of the electroporation process. [Figure 77A]

[0182] 1 is a flowchart of an exemplary variation of a method for separating cells. [Figure 77B]

[0182] A flowchart of an exemplary variation of a method for concentrating cells. [Figure 77C]

[0182] Flowchart of an exemplary variation of the buffer exchange method. [Figure 78]

[0183] 10 is a flowchart of another exemplary variation of a method for separating cells. [Figure 79A]

[0184] 7 is a flowchart of an exemplary variation of a closed-loop method 7900 for separating cells. [Figure 79B]

[0184] A flowchart of an exemplary variation of a closed-loop method 7910 for elutriation of cells. [Figure 79C]

[0184] A flowchart of an exemplary variation of a closed-loop method 7920 for harvesting cells. [Figure 80A]

[0185] 1 is a flowchart of an exemplary variation of a method for separating cells. [Figure 80B]

[0185] A flowchart of an exemplary variation of the method for selecting cells. [Figure 81]

[0186] 10 is a flowchart of another exemplary variation of a method for separating cells. [Figure 82A]

[0187] 1 is a flow chart of an exemplary variation of a method for preparing a bioreactor. [Figure 82B]

[0187] A flowchart of an exemplary variation of a method for loading a bioreactor. [Figure 82C]

[0187] A flowchart of an exemplary variation of a method for preparing a bioreactor. [Figure 82D]

[0187] A flowchart of an exemplary variation of a calibration method for a bioreactor. [Figure 82E]

[0187] A flowchart of an exemplary variation of a method for mixing reagents. [Figure 82F]

[0187] A flowchart of an exemplary variation of a method for mixing reagents. [Figure 82G]

[0187] A flowchart of an exemplary variation of a method for culturing cells. [Figure 82H]

[0187] A flowchart of an exemplary variation of a method for refrigerating cells. [Figure 82I]

[0187] A flowchart of an exemplary variation of a method for obtaining a sample. [Figure 82J]

[0187] A flowchart of an exemplary variation of a method for culturing cells. [Figure 82K]

[0187] Flowchart of an exemplary variation of the medium exchange method. [Figure 82L]

[0187] A flowchart of an exemplary variation of a method for controlling gas. [Figure 82M]

[0187] A flow chart of an exemplary variation of a method for controlling pH. [Figure 83]

[0188] 1 is a flow chart of an exemplary variation of a method for electroporating cells. [Figure 84]

[0189] 10 is a flowchart of another exemplary variation of a method for electroporating cells. [Figure 85]

[0190] 1A-1C are schematic diagrams of exemplary variations of fluid connectors. [Figure 86]

[0191] 10A-10C are schematic diagrams of exemplary variations of fluid connector ports. [Figure 87]

[0192] 10A-10C are schematic diagrams of exemplary variations of the fluid connector connection process. [Figure 88]

[0193] 10A-10C are schematic diagrams of exemplary variations of the fluid connector connection process. [Figure 89]

[0194] 1A-1C are schematic diagrams of exemplary variations of fluid connectors. [Figure 90A]

[0195] 10A-10C are side views of exemplary variations of fluid connectors. [Figure 90B]

[0195] FIG. 90B is a perspective view of the fluid connector shown in FIG. 90A. [Figure 90C]

[0195] FIG. 90B is a side cross-sectional view of the fluid connector shown in FIG. 90A. [Figure 91A]

[0196] 10A-10C are side views of exemplary variations of fluid connectors. [Figure 91B]

[0196] FIG. 91B is a perspective view of the fluid connector shown in FIG. 91A. [Figure 91C]

[0196] FIG. 91B is a side cross-sectional view of the fluid connector shown in FIG. 91A. [Figure 91D]

[0197] 10A-10C are side views of exemplary variations of fluid connectors. [Figure 91E]

[0197] FIG. 91D is a perspective view of the fluid connector shown in FIG. [Figure 91F]

[0197] FIG. 91D is a side cross-sectional view of the fluid connector shown in FIG. [Figure 92A]

[0198] 10A-10C are side views of exemplary variations of fluid connectors. [Figure 92B]

[0198] FIG. 92B is a perspective side view of the fluid connector shown in FIG. 92A. [Figure 92C]

[0198] FIG. 92B is a perspective view of the fluid connector shown in FIG. 92A. [Figure 92D]

[0198] FIG. 92B is a side cross-sectional view of the fluid connector shown in FIG. 92A. [Figure 93A]

[0199] 1A and 1B are perspective views of exemplary variations of fluid connectors. [Figure 93B]

[0199] FIG. 93B is a perspective view of the fluid connector shown in FIG. 93A. [Figure 94A]

[0200] 1A and 1B are perspective views of exemplary variations of fluid connectors. [Figure 94B]

[0200] FIG. 94B is a perspective view of the fluid connector shown in FIG. 94A. [Figure 95A]

[0201] 1A and 1B are perspective views of exemplary variations of fluid connectors. [Figure 95B]

[0201] FIG. 95B is a perspective view of the fluid connector shown in FIG. 95A. [Figure 95C]

[0201] Detailed side view of a port in an open port configuration. [Figure 95D]

[0201] Detailed side view of a port in a closed port configuration. [Figure 96A]

[0202] 1A and 1B are plan views of exemplary variations of fluidic devices. [Figure 96B]

[0202] A side view of an exemplary variation of a fluidic device coupled to a robot. [Figure 96C]

[0202] A perspective view of an exemplary variation of a fluidic device held by a robot. [Figure 97A]

[0203] 1 is a perspective view of an exemplary variation of a MACS module. [Figure 97B]

[0203] A cross-sectional perspective view of an exemplary variation of a MACS module. [Figure 97C]

[0203] A side cross-sectional view of an exemplary variation of a MACS module. [Figure 98]

[0204] 1 is a flow chart of an exemplary variation of a cell processing method. [Figure 99]

[0205] 1 is a flow chart of an exemplary variation of a cell processing method. [Figure 100]

[0206] 1 is a flow chart of an exemplary variation of a cell processing method. [Figure 101]

[0207] 1 is a flow chart of an exemplary variation of a cell processing method. [Figure 102]

[0208] 1 is a schematic diagram of an exemplary variation of a cell processing system. [Figure 103A]

[0209] 1 is a perspective view of an exemplary variation of a sterile fluid transfer device. [Figure 103B]

[0209] A perspective view of an exemplary variation of a sterilization fluid transfer device. DETAILED DESCRIPTION OF THE INVENTION

[0105]

[0210] Described herein are systems and methods for processing and manufacturing cellular products for biomedical applications. Cell processing methods and systems can include transferring a cartridge containing a cellular product between multiple instruments within a work cell. One or more instruments are configured to interface with the cartridge to perform cell processing steps on the cellular product, thereby enabling the system (e.g., work cell) to perform the cell processing steps on the cellular product. In some variations, multiple cell processing steps can be performed within a single cartridge. For example, a robotic arm can be configured to transfer the cartridge between multiple instruments for different cell processing steps. The cartridge can include multiple cell processing devices (e.g., modules). These devices can be, for example, a bioreactor, a counterflow centrifugal elutriation (CCE) module, a magnetic cell sorter (e.g., a magnetically activated cell selection module), an electroporation device (e.g., an electroporation module), a sorting module (e.g., a fluorescence-activated cell sorting (FACS) module), an acoustic flow cell module, a centrifugation module, a microfluidic enrichment module, or a combination thereof. In some variations, the system can process two or more cartridges in parallel. For example, a bioreactor may include multiple slots configured to interface with multiple cartridges simultaneously, and typically, one process step (e.g., cell culture in the bioreactor) may be rate-limiting in the operation of the cell processing system. The cell processing systems described herein may reduce operator intervention and increase throughput by using robotics to automate the transfer of cartridges (and cell products) between instruments. However, in some variations, cartridges may be transferred manually between instruments. Additionally, the use of multiple bioreactors may enable the system to process multiple cartridges for multiple patients simultaneously, thereby increasing the throughput of the system.Additionally, the automated cell processing system can facilitate the transfer of sterile fluid between the cartridge and other components of the instrument or system, such as a fluid connector (e.g., a sterile fluid transfer port), a reagent reservoir, a second cartridge, a sampling container (e.g., a sterile fluid transfer device), combinations thereof, etc.

[0106] Work Cell

[0211] In some variations, a system (e.g., a work cell) for cell processing can include multiple instruments, each independently configured to perform one or more cell processing operations on a cartridge. A robot can be configured to move cartridges between each of the multiple instruments. The instruments can include one or more of a bioreactor instrument, a cell selection instrument (e.g., a magnetically activated cell selection instrument), a sorting instrument (e.g., a fluorescence-activated cell sorting (FACS) instrument), an electroporation instrument, a counterflow centrifugal elutriation (CCE) instrument, a reagent reservoir, etc. The system can perform automated manufacturing of a cellular product.

[0107]

[0212] The cartridge can be portable and configured to facilitate automated sterile cell processing using a work cell and robotics. For example, the cartridge can be configured to move relative to one or more instruments in a work cell to perform different cell processing steps. In some variations, the cartridge can be configured to move instruments relative to the cartridge. In some variations, the cartridge can include multiple modules, including one or more of a bioreactor module, a cell selection module (e.g., a magnetically activated cell selection module), a sorting module (e.g., a fluorescence-activated cell sorting (FACS) module), an electroporation module, and a counterflow centrifugal elutriation (CCE) module. The cartridge can further include one or more of a sterile fluid transfer port, a fluid transfer bus fluidly coupled to each module, and a pump fluidly coupled to the fluid transfer bus.

[0108]

[0213] In some variations, a method of processing a solution containing a cell product may include cell processing steps of digesting tissue with an enzymatic reagent to release a selected cell population into the solution, concentrating the cells with a CCE instrument, washing the cells with a CCE instrument, selecting cells in the solution with a selection instrument, sorting the cells in the solution with a sorting instrument, differentiating or expanding the cells in a bioreactor, activating the cells with an activation reagent, electroporating the cells, transducing the cells with a vector, and completing the cell product.

[0109] Cell Selection System

[0214] The cell processing systems 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 characteristics such as size and / or density using a counterflow centrifugal elutriation device. It is also possible to separate cells based on the presence of predetermined antigens using, for example, a magnetically activated cell selection device. In some variations, a cell selection system including modules for these separation methods can facilitate one or more cell processing steps, including, but not limited to, cell concentration, cell dilution, cell washing, buffer replacement, and magnetic separation. The cell selection systems described herein can increase throughput and cell yield output in a compact, portable design. For example, before magnetically separating the cells, a cell suspension may be mixed with a magnetic reagent in excess or at a predetermined concentration (e.g., cells / ml). Similarly, after magnetically separating the cells, the cells may be washed with a solution (e.g., an appropriate buffer solution).

[0110]

[0215] In some variations, the cell separation system may include a rotor configured for countercurrent centrifugal elutriation of cells in a fluid, a first magnet configured to magnetically rotate the rotor to separate cells from the fluid in the rotor, a flow cell in fluid communication with the rotor and configured to receive cells from the rotor, and a second magnet configured to magnetically separate cells in the flow cell.

[0111]

[0216] In some variations, the CCE module is integrated into a cartridge, enabling the cell processing system to separate cells based on cell size and / or density. In some variations, the cell separation system may include a housing including a rotor configured to separate cells from a fluid (e.g., separate cells of different sizes and / or densities from cells present in the fluid) and a magnet configured to magnetically rotate the rotor. The housing may be configured to move relative to the magnet, or vice versa (e.g., move the magnet relative to the housing). The CCE modules described herein can perform cell separation within a compact, portable housing, and the magnet may be located outside the housing (e.g., the magnet is located within the CCE instrument).

[0112]

[0217] In some variations, a compact rotor that may be useful for cartridge integration may include input and output fluid conduits extending from the rotor to opposite sides of a rotor housing. For example, the rotor may include a first side including a first fluid conduit and a second side opposite the first side including a second fluid conduit. An elutriation chamber (e.g., a cone) may be coupled between the first and second fluid conduits.

[0113]

[0218] In some variations, a method of separating cells from a fluid may include moving a rotor defining an axis of rotation toward a magnet, flowing a fluid through the rotor, rotating (e.g., magnetically) the rotor about the axis of rotation using the magnet while flowing the fluid through the rotor, and moving the rotor away from the magnet.

[0114]

[0219] In some variations, a method of separating cells from a fluid can include flowing a fluid containing cells into a flow cell. The set of cells can be labeled with magnetic particles. The set of cells can be magnetically attracted toward a magnet array during a residence time and can flow out of the flow cell after the residence time.

[0115]

[0220] In some variations, the flow cell can include an elongated cavity having a cavity height, and the magnet array includes a plurality of magnets, each separated by a separation distance. A predetermined ratio of cavity height to separation distance can optimize magnetic separation of cells within the flow cell.

[0116] Electroporation

[0221] In some variations, the electroporation modules described herein can be configured to facilitate one or more of cell transduction and transfection. As detailed herein, a fluid volume containing cells (e.g., a first batch) can be physically separated from subsequent fluid volumes containing cells (e.g., a second batch, a third batch) by a gas (e.g., an air gap). Separate application of electroporation signals (e.g., voltage pulses, waveforms) to each of the individual fluid batches can improve electroporation efficiency and thus increase throughput. In some variations, active field compensation can similarly improve electroporation efficiency and throughput.

[0117]

[0222] In some variations, the cell processing device may include a fluid conduit configured to receive a first fluid containing cells and a second fluid (e.g., gas, oil), an electrode set coupled to the fluid conduit, a pump coupled to the fluid conduit, and a controller including a processor and memory. The controller may be configured to generate a first signal to introduce the first fluid into the fluid conduit using the pump, generate a second signal to introduce the second fluid into the fluid conduit such that the second fluid separates the first fluid from a third fluid, and generate an electroporation signal to electroporate cells in the fluid conduit using the electrode set.

[0118]

[0223] In some variations, a method of electroporating cells may include receiving a first fluid containing cells into a fluid conduit, receiving a second fluid containing a gas into 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.

[0119]

[0224] In some variations, a method of electroporating cells may include receiving a first fluid containing cells in a fluid tube, applying a resistance measurement signal to the first fluid using an electrode set, measuring resistance between the first fluid and the electrode set, and applying an electroporation signal to the first fluid based on the measured resistance.

[0120] Bioreactor

[0225] In some variations, the bioreactor may include an enclosure including a bottom and sidewalls, and a gas-permeable membrane coupled to one or more of the bottom and sidewalls of the enclosure. The gas-permeable membrane may support cell culture. In some variations, the cell processing system may include the bioreactor and an agitator coupled to the bioreactor. The agitator may be configured to agitate the bioreactor based on orbital motion.

[0121] Fluid Connector

[0226] Currently, there are no automated, multi-use sterile fluid connector solutions in cell therapy manufacturing that allow a sterile fluid connector set to undergo multiple cycles of connection and disconnection to a system. For example, conventional sterile fluid connectors are typically single-use devices and are therefore expensive and labor-intensive. Generally, the fluid connectors described herein include multiple sealed enclosures between the sterile portion (e.g., the lumen or cavity of the fluid connector) and the external (e.g., non-sterile) ambient environment, facilitating sterility control of the fluid connector and its associated devices. 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, the fluid connectors can be sterilized using sterilants without damaging the cell product or other biological material.

[0122]

[0227] In some variations, the sterile manufacturing systems described herein utilize one or more sterile fluid connectors and can be configured for robotic manipulation, such as by a robotic arm. The sterile fluid connectors described herein enable automated, sterile, and metered fluid transfer for automating cell therapy manufacturing. Automating cell therapy manufacturing can also reduce per-patient manufacturing costs, lower the risk of process failure, and enable commercial-scale patient demand for cell therapy. In some variations, the sterile fluid connectors can increase one or more of sterility, efficiency, and speed by eliminating human operators from the manufacturing process. The automated and integrated sterilization processes described herein can be applied to the fluid connectors to maintain system sterility. For example, the fluid connectors can maintain sterility over multiple connection / disconnection cycles between separate sterile, closed-volume fluidic devices (e.g., enclosures, containers, vessels, cartridges, instruments, bioreactors, closed vessels, sealed chambers). Thus, the systems, devices, and methods described herein can reduce the complexity of the sterilization process, reduce energy use, and increase sterilization efficiency.

[0123]

[0228] In some variations, the fluidic connector may include a first connector configured to mate with a second connector (e.g., a male connector and a female connector). The proximal ends of each of these connectors may be configured to connect (e.g., be in fluid communication with, form a fluid pathway) with a respective fluidic device to transfer one or more of a fluid (e.g., liquid and / or gas) and a biological material (e.g., a cellular product) between the fluidic devices. The distal ends of these connectors may include ports configured to mate with each other. The fluidic connector may also include sterilant ports within the distal ends of the first and second connectors configured to facilitate sterilization of the chambers. The fluidic connectors can be sterilized as desired before or after connection to ensure sterility. In this manner, the fluidic connectors may be reused for multiple connection and disconnection cycles.

[0124]

[0229] In some variations, a system (e.g., a work cell) utilizing the fluidic connectors described herein may include a robot configured to operate the fluidic connectors, and a controller configured to control the robot to manipulate (e.g., move, connect, open, close, disconnect) the first and second connectors together (without human intervention) while maintaining sterility of the fluidic connectors and a plurality of fluidic devices, thereby further reducing the risk of contamination. The fluidic devices may be one or more of an instrument, a cartridge, etc.

[0125] Cell Processing Control

[0230] Described herein are systems and methods for manufacturing cellular products for biomedical applications using automated systems. Conventional semi-automated solutions for cell processing do not allow users to define the biological process. Instead, users select from a limited set of predefined machine processes and process control parameters. Currently, no scalable manufacturing solutions exist for cell therapy manufacturing. For example, cell therapy manufacturing is traditionally performed in batches (i.e., one product is manufactured in a single room / set, with the necessary processing tools located internally). This is 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's product in a single multi-function processing tool. However, existing solutions (e.g., Miltenyi Prodigy) do not allow users to define the biological process. Furthermore, the manual work required by conventional solutions increases the risk of product contamination and human error.

[0126]

[0231] In some variations, the systems described herein can be used to convert a set of cell therapy biological manufacturing processes into a set of machine instructions suitable for automated execution. For example, a method for converting user-defined cell processing operations into cell processing steps to be executed by a processor of an automated cell processing system can include receiving an ordered input list of cell processing operations and executing a transformation model on the ordered input list to generate an ordered output list of cell processing steps that can be executed by the system. As used herein, a transformation model can refer to an algorithm, process, or transformation configured to convert a set of cell processing steps into a set of machine or hardware instructions for the system. In some variations, a robot can be controlled to move one or more cartridges, each containing a cell product, between instruments, and the instruments can be controlled to perform a cell processing step for each cell product. Thus, the present systems and methods enable biologists to define a manufacturing process in biological terms and have the system convert this biological model (e.g., process definition) into a set of machine-executable instructions.

[0127]

[0232] The comprehensive automation of the closed systems described herein can reduce process failure rates and costs. For example, comprehensive automation can reduce manufacturing time (e.g., residence time) and increase throughput compared to traditional manual methods. For example, multiple processes (e.g., 10 or more) can be performed simultaneously. The methods described herein can also reduce the likelihood of contamination and user error. Thus, the systems, devices, and methods described herein can increase one or more of the following: automation, reproducibility, reliability, process flexibility, instrument throughput, and process scalability of cell processing, while reducing one or more of labor costs and process time.

[0128] I. System

[0233] Described herein are systems and devices configured to perform cell processing steps to produce a cell product (e.g., a cell therapy product). In some variations, the cell processing system may include multiple instruments, each independently configured to perform one or more cell processing operations on a cartridge (e.g., a fluidic device), and a robot capable of moving the cartridge between each of the multiple instruments. The use of a robot and a controller can facilitate one or more of the automation, efficiency, and sterility of the cell processing system.

[0129]

[0234] In some variations, a system for cell processing can include multiple instruments, each independently configured to perform one or more cell processing operations on a cartridge. A robot can move the cartridge between each of the multiple instruments. In some variations, the system can be a work cell that includes an enclosure.

[0130]

[0235] 1A is a block diagram of a cell processing system 100 including a work cell 110 and a controller 120. In some variations, the work cell 110 may include one or more of an instrument 112, a cartridge 114 (e.g., consumables, fluidic devices), a robot 116 (e.g., a robotic arm), a reagent reservoir 118, a fluid connector 132, a sterilant source 134, a fluid source 136, a pump 138, a sensor 140, and a sterilant 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.

[0131]

[0236] In some variations, the work cell can include a fully or at least partially closed enclosure within which one or more cell processing steps are performed in a fully or at least partially automated process. In some variations, the work cell can be an open system without an enclosure, which can be configured for use in a clean room, biosafety cabinet, or other sterile location. In some variations, a robot 116 can be used to move the cartridge 114 to reduce manual handling in the cell processing steps. In some variations, the work cell can be configured to perform sterile fluid transfer into and out of the cartridge in a fully or partially automated process. For example, one or more fluids can be stored in the sterile fluid transfer device 142. In some variations, the sterile fluid transfer device can be a portable consumable that is movable within the system 100. The sterile fluid transfer devices and fluid connectors described herein enable automated, sterile, and quantitative fluid transfer for automating cell therapy manufacturing. In some variations, the work cell enclosure may be configured to meet International Organization for Standardization (ISO) standards ISO 7 or higher (e.g., ISO 6 or ISO 5). An advantage of meeting ISO 7 or higher standards is that the system can be used in facilities that do not meet ISO 7 standards (i.e., are not clean rooms or other well-filtered air spaces). Optionally, the facility may be an ISO 8 or ISO 9 facility. In some variations, the volume of the work cell is approximately 800 m 3 Less than 700m 3 Less than 600m 3 Less than 500m 3 Less than 300m 3 Less than 250m 3 Less than 200m 3 Less than 150m 3 Less than 100m 3 Less than 50m 3 Less than 25m 3 Less than 10m3 Less than and about 5m 3 It may include less than, as well as all ranges and sub-values ​​therebetween.

[0132]

[0237] In some variations, the robot 116 may be configured to manipulate the consumable cartridges 114 and fluid connectors 132 between different instruments to perform a predetermined sequence of cell processing steps. In some variations, the same consumable cartridge 114 may be received by different instruments 112 and / or multiple cartridges 114 may be processed in parallel.

[0133]

[0238] In some variations, cartridges 114 can contain cell products from different donors or for different recipients. A cell product from a single donor can be split among multiple cartridges 114 if necessary to generate enough product for therapeutic use or if the donor is providing product to multiple recipients (e.g., for allogeneic transplants). A cell product for a single recipient can be split among multiple cartridges 114 if necessary to generate enough product for therapeutic use in that recipient. If necessary to generate several cell products through unique genetic modifications, a cell product for a single recipient can be split among multiple cartridges 114 and then optionally recombined in a specific ratio for use in therapy in that recipient. For example, fluidic connectors 132 can be coupled between two or more cartridges 114 to transfer cell products and / or fluids between the cartridges 114. Additionally, fluid connectors 132 may be coupled between a set of fluid-carrying components (e.g., cartridges 114, reagent reservoirs 118, fluid sources 136, sterile fluid transfer devices 142, fluid tubing, containers, vessels, etc.) of system 100. For example, a first fluid connector may be coupled between a first cartridge and the sterile fluid transfer device, and a second fluid connector may be coupled between the sterile fluid transfer device and a second cartridge.

[0134]

[0239] 1B , cartridge 114 may include one or more of bioreactor 150, cell separation system 152, electroporation module 160, fluid transfer bus 162, sensor 164, and fluidic connector 166, as described in detail herein. Cell separation system 152 may include one or more of rotor 154, flow cell 156, and magnet 158. In some variations, magnet 158 ​​may include one or more magnets and / or magnet arrays. For example, cell separation system 152 may include a first magnet configured to magnetically rotate rotor 154 and a second magnet (e.g., a magnet array) configured to magnetically separate cells within flow cell 156.

[0135] Work Cell

[0240] In some variations, the work cell 110 may include an at least partially closed enclosure (e.g., housing) within which one or more automated cell processing steps are performed. For example, the work cell 110 may be configured to transfer sterilizing fluids into and out of the cartridge 114 in a fully or partially automated process. In some variations, the work cell 110 may not include an enclosure but may be configured for use in a clean room, biosafety cabinet, or other appropriately clean or sterile location. In some variations, the work cell 110 may include a feedthrough access biosafety cabinet, quality control instrumentation, pumps, consumables (e.g., fluidic devices), fluid connectors, consumable feedthroughs, and a sterilization system (e.g., sterilant source and / or generator, fluid source, heater / dessicator, aerator).

[0136]

[0241] 2A is a block diagram of a cell processing system including a work cell 203. The work cell 203 may include an enclosure 202 with four walls, a bottom, and a roof. The work cell may be divided into an interior zone 204 with feedthrough 206 access and quality control (QC) instrumentation 212. An air filtration inlet (not shown) can provide high-efficiency particulate air (HEPA) filtration to provide ISO 7 or higher air quality in the interior zone 204. This air filtration can maintain sterile cell processing in an ISO 8 or ISO 9 manufacturing environment. The work cell 203 may also have an air filter at the air outlet to maintain the room's ISO rating. In some variations, the work cell 203 may further include within the interior zone 104 a bioreactor instrument 214, a cell selection instrument 216 (e.g., MACS), an electroporation instrument (EP) 220, a counterflow centrifugal elutriation (CCE) instrument 222, a sterile fluid transfer instrument 224 (e.g., fluid connector), a reagent reservoir 226, and a sterilization system 260. The reagent reservoir 226 may be accessible by a user via a sample pickup port 228. A robot 230 (e.g., support arm, robotic arm) may be configured to transfer one or more cartridges 250 (e.g., consumables) from one instrument to another and / or to or from a reagent reservoir. In some variations, the work cell 203 may include one or more movable barriers 213 (e.g., access, doors) configured to facilitate access to one or more of the instruments within the work cell 203.

[0137]

[0242] In some variations of methods according to the present disclosure, a human operator can insert one or more empty cartridges 250 into the feedthrough 206 via cartridge port 207. The cartridges 250 can be pre-sterilized, or the feedthrough 206 can sterilize the cartridges 250 using ultraviolet radiation (UV) or a chemical sterilant provided as a steam, spray, or cleaning solution. The feedthrough 206 chamber can optionally be configured to automatically spray, wash, irradiate, or otherwise treat the cartridges (e.g., with ethanol and / or isopropyl alcohol solution, vaporized hydrogen peroxide (VHP)) to maintain sterility (e.g., ISO 7 or higher) of the interior zone 204. The cartridges 250 are transferred to the biosafety cabinet 206, where an input cell product can be provided and inserted into the cartridges 250 via a sterilant transfer port. The user can then return the cartridges 250 (via the robot 230) to the feedthrough 206 and initiate automated processing using a computer processor in a computer server rack (e.g., the controller 120). The robot 230 may be configured to move the cartridge 250 in a predetermined sequence through multiple instruments and stations through components of the work cell 200. Upon completion of cell processing, the cartridge 250 containing the processed cell product may be returned to the feedthrough 206 for retrieval by a user. In some variations, the exterior of the enclosure 202 may include an input / output device 208 (e.g., a display, a touch screen).

[0138]

[0243] Figure 2B is a perspective view of a work cell 205 of a cell processing system. Figure 2C is a perspective view of a cell processing system illustrating a cartridge 250 (e.g., any of the cartridges described herein) installed in work cell 205 (e.g., any of the work cells described herein). Multiple cartridges may be inserted into work cell 205 simultaneously to perform one or more cell processing operations on these cartridges in parallel.

[0139]

[0244] In some variations, the height of work cell 205 can include greater than about 1 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 about 100 m, and greater than about 100 m, and all values ​​and ranges therebetween. In some variations, one or more of the length and width of work cell 205 can include greater than about 1 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 about 100 m, and greater than about 100 m, and all values ​​and ranges therebetween.

[0140]

[0245] FIG. 2D is a schematic diagram of a variation of work cell 200. Work cell 200 may include an enclosure 202 with four walls, a bottom, and a roof. The work cell may be divided into an interior zone 204 with feedthrough 206 access, a biosafety cabinet (BSC) 208, a computing server rack 210 (e.g., controller 120), and quality control (QC) instrumentation 212. An air filtration inlet (not shown) can provide high-efficiency particulate air (HEPA) filtration to provide ISO 7 or higher air quality in interior zone 204. This air filtration can maintain sterile cell processing in an ISO 8 or ISO 9 manufacturing environment. The work cell may also have an air filter at the air outlet to maintain the room's ISO rating. In some variations, the work cell 200 may further include within the interior zone 204 an instrument 211 (e.g., located in a universal instrument bay), a bioreactor instrument 214, a cell selection instrument 216 (e.g., MACS, cell selection system), a cell sorting instrument 218 (e.g., FACS), an electroporation instrument (EP) 220, a counterflow centrifugal elutriation (CCE) instrument 222, a sterilization fluid transfer instrument 224 (e.g., fluid connectors), reagent reservoirs 226, and a sterilization system 260 including one or more of a sterilant source, a fluid source, and a pump. The reagent reservoirs 226 may be accessible by a user via a sample pickup port 228. A robot 230 (e.g., support arm, robotic arm) may be configured to move one or more cartridges 250 (e.g., consumables) from one instrument to another or to a reagent reservoir.

[0141]

[0246] In some variations, a human operator can insert one or more cartridges 250 into the feedthrough 206. The cartridges 250 can be pre-sterilized, or the feedthrough 206 can sterilize the cartridges 250 using ultraviolet radiation (UV) or a chemical sterilant provided as a spray or wash. The feedthrough 206 chamber can optionally be configured to automatically spray, wash, irradiate, or otherwise treat the cartridges (e.g., with ethanol and / or isopropyl alcohol solution) to maintain the sterility of the interior zone 204 (e.g., ISO 7 or higher) or the sterility of the biosafety cabinet 208 (e.g., ISO 5 or higher). The cartridges 250 are transferred to the biosafety cabinet 206, where an input cell product can be provided and inserted into the cartridges 250 using a sterile fluid transfer device 224 (e.g., a fluid connector). The user can then return the cartridges 250 to the feedthrough 206 and initiate automated processing using a computer processor in the computer server rack 210 (e.g., controller 120). The robot 230 may be configured to move the cartridge 250 to multiple instruments and stations in a predetermined sequence. The components of the workcell 200 are controlled by a computer processor in the computer server rack 210. Additionally or alternatively, the sequence in which the cartridge 250 moves within the workcell 200 may not be predetermined. For example, the movement of the cartridge 250 may be determined based on one or more of the results of previous steps, sensor values, predetermined thresholds (e.g., based on a quality control system), etc. Upon completion of cell processing, the cartridge 250 containing the processed cell product may be returned to the feedthrough 206 for retrieval by a user. Additionally or alternatively, the cell product 250 containing the processed cell product may be transferred (via a fluidic connector) to a second cartridge (e.g., a single-use cartridge) and stored in the reagent reservoir 226 for retrieval by a user.

[0142]

[0247] In some variations, cells from a patient and starting reagents can be inserted into a cartridge (e.g., a single-use cartridge) by a human operator in a biosafety cabinet that is separate from or integrated into the work cell. In some variations, the cartridges described herein containing the cell product and reagents are closed so they can be moved through non-sterile fields without contamination. Automated decontamination routines may also be performed on the cartridge. For example, the cartridge may be placed in a feedthrough that can facilitate decontamination of the cartridge before entering the ISO7 environment of the work cell.

[0143]

[0248] FIG. 2E is a top schematic view of another variation of work cell 201. Work cells 200, 201, and 203 may include an enclosure 202 with four walls, a bottom, and a roof. The work cell may be divided into an interior zone 204 with feedthrough 206 access, a biosafety cabinet (BSC) 208, a computing server rack 210 (e.g., controller 120), and quality control (QC) instrumentation 212. An air filtration inlet (not shown) can provide high-efficiency particulate air (HEPA) filtration to provide ISO 7 or higher air quality in interior zone 204. This air filtration can maintain sterile cell processing in an ISO 8 or ISO 9 manufacturing environment. The work cell may also have an air filter at the air outlet to maintain the room's ISO rating. In some variations, the work cell 200 may further include within the interior zone 104 an instrument 211 (e.g., located in a universal instrument bay), a bioreactor instrument 214, a cell selection instrument 216 (e.g., MACS), a cell sorting instrument 218 (e.g., FACS), an electroporation instrument (EP) 220, a counterflow centrifugal elutriation (CCE) instrument 222, a sterile fluid transfer instrument 224, and a reagent reservoir 226. The reagent reservoir 226 may be accessible by a user via a sample pickup port 228 (e.g., a door that can facilitate bulk loading of the sterile fluid transfer instrument 224). A robot 230 (e.g., a support arm, a robotic arm) may be configured to move one or more cartridges 250 (e.g., consumables) from one instrument to another or to a reagent reservoir.

[0144]

[0249] In some variations of methods according to the present disclosure, a human operator can insert one or more empty cartridges 250 into the feedthrough 206. Additionally or alternatively, pre-filled cartridges may be inserted into the feedthrough 206. The cartridges 250 can be pre-sterilized, or the feedthrough 206 can sterilize the cartridges 250 using ultraviolet radiation (UV) or a chemical sterilant provided as a spray or wash. The feedthrough 206 chamber can optionally be configured to automatically spray, wash, irradiate, or otherwise treat the cartridges (e.g., with ethanol and / or isopropyl alcohol solution) to maintain the sterility of the interior zone 204 (e.g., ISO 7 or higher) or the sterility of the biosafety cabinet 208 (e.g., ISO 5 or higher). The cartridges 250 are transferred to the biosafety cabinet 106, where an input cell product can be provided and inserted into the cartridges 250 via a sterile fluid transfer port. The user can then return cartridge 250 to feedthrough 206 and initiate automated processing using a computer processor in computer server rack 210 (e.g., controller 120). Robot 230 can be configured to move cartridge 250 to multiple instruments and stations in a predetermined sequence. The components of workcell 200 are controlled by the computer processor in computer server rack 210. At the end of cell processing, cartridge 250 containing the processed cell product can be returned to feedthrough 206 for collection by the user.

[0145]

[0250] In some variations, one or more components of a sterilization system (e.g., sterilant source, pump) can be coupled to the work cell. For example, FIG. 3 is a block diagram of a cell processing system 300 including a work cell 310, a sterilization system 320, a fluidic connector 330, and a fluidic device 340. In some variations, the fluidic device 340 can include a main (e.g., consumable) feedthrough and a fluidic device (e.g., reagent) feedthrough. The sterilization system 320 can include a sterilant source 322, a pump 324, and a heater (e.g., desiccant / drier) 326. For example, the heater 326 can be configured to vent under a predetermined set of conditions. The sterilization system 320 can be coupled to and in fluid communication with one or more of the work cell 310, the fluidic connector 330, and the fluidic device 340. In some variations, a robot (not shown) can be configured to manipulate and operate the cell processing system 300. For example, the fluidic connector 330 can be coupled to one or more of the fluidic device 340 and an instrument (not shown). Sterilization system 320 can be used to sterilize and / or vent one or more of work cell 310, fluid connector 330, and fluidic device 340 by circulating one or more sterilants and fluids (e.g., hot air, vaporized hydrogen peroxide (VHP)). In some variations, sterilization system 320 can include one or more of vaporized hydrogen peroxide (VHP), electron beam (e-beam) sterilization, dry heat decontamination, and steam-in-place sterilization. In some variations, sterilization system 320 can provide a sterility assurance level (SAL) of at least 10-3 SAL.

[0146]

[0251] 4A and 4B show perspective views of a cell processing system 400 including cartridges 400, 402, feedthroughs 410, 412, and fluid connectors 420, 422 (e.g., sterile fluid transfer devices). For example, cartridge 400 is shown in feedthrough 410 in FIG. 16A after a robot (not shown) has moved cartridge 400 to fluid connector 420.

[0147] robot

[0252] In general, a robot may include any mechanical device capable of moving cartridges from one location to another. For example, a robot may include a mechanical manipulator (e.g., an arm) that is in a fixed position or attached to a linear rail or a two- or three-dimensional rail system. In some variations, the robot includes a robot shuffling system. In other variations, the robot includes a wheeled device. In some variations, the system includes two or more robots of the same or different types (e.g., two robotic arms each independently configured to move cartridges between instruments). The robot may also include end effectors for precise manipulation of various cartridges, scanning barcodes, or reading radio-frequency identification tags (RFID).

[0148]

[0253] FIG. 5 is a perspective view of a cell processing system 500. In this system, a robotic arm moves consumable cartridges between slots of various instruments, each configured to perform a different cell processing step. In some variations, the same consumable cartridge can be accepted by various instruments. System 500 may include a modular design to accommodate various instrument configurations. In some variations, multiple cartridges can be processed in parallel. Each cartridge can contain cells from a different donor or contain a cell product intended for a different recipient. For example, if a donor provides products to multiple recipients (e.g., for allogeneic transplants), a cell product from a single donor can be split into multiple cartridges to generate a predetermined amount of cell product for use in therapy. In some variations, a cell product intended for a single recipient can be split into multiple cartridges to generate a predetermined amount of product for use in therapy in that recipient. In some variations, a cell product intended for a single recipient can be split into multiple cartridges to generate predetermined amounts of several cell products through unique genetic modifications and recombine in specific ratios for use in therapy in that recipient.

[0149] cartridge

[0254] Generally, the cell processing systems described herein can include one or more cartridges containing one or more modules configured to interface with one or more instruments. A robot (e.g., a robotic arm) can be configured to move the cartridge and / or instrument to perform one or more cell processing steps. For example, a cartridge can include a bioreactor module and / or a fluid connector (e.g., a sterile fluid transfer port) coupled by the robot to a bioreactor instrument in a work cell. Once a given processing step is complete, the cartridge can be moved by the robot to another instrument in the work cell, to which another cartridge can be coupled. Thus, portable cartridges and shareable instruments can increase the efficiency, throughput, and flexibility of cell manufacturing processes.

[0150]

[0255] In some variations, the cartridge may optionally provide a self-contained device capable of performing one or more cell processing steps. Modules may be integrated into a fixed configuration within the cartridge. Additionally or alternatively, modules may be configurable or movable within the cartridge, allowing various cartridges to be assembled from shared modules. Similarly, a cartridge may be a single closed unit with fixed components for each module. Alternatively, a cartridge may include configurable modules coupled by configurable fluidic, mechanical, optical, and electrical connections. In some variations, one or more sub-cartridges, each containing a set of modules, may be configured to be assembled to perform various cell processing workflows. Each module may be provided in a separate housing or integrated with other modules within a cartridge or sub-cartridge. While this disclosure generally depicts modules as separate components for simplicity, they may be arranged in any suitable configuration. For example, components of different modules may be interspersed, with each module being defined by a set of connected components that collectively perform a predetermined function. However, components of each module may or may not be physically grouped within the cartridge. In some variations, multiple cartridges can be used to process a single cell product, which can be done by transferring the cell product from one cartridge to another of the same or different type, and / or splitting the cell product among many cartridges, and / or pooling multiple cell products into fewer cartridges.

[0151]

[0256] Typically, each instrument in the system interfaces with one or more modules on the cartridge. For example, the electroporation module on the cartridge (if present) is transferred by the system to the electroporation instrument and interfaces with the electroporation instrument to perform the electroporation step on the cell product, and may also interface with common components such as fluid bus line components (e.g., pumps, valves, sensors, etc.). An advantage of such a split-module / instrument design is that expensive components (e.g., motors, sensors, heaters, lasers, etc.) can be maintained within the instrument in the system during processing of multiple cartridges. In this variation, disposable cartridges can be used, eliminating the need to sterilize cartridges between uses. Furthermore, the ability to simultaneously utilize multiple instruments in parallel with multiple cell manufacturing processes can increase utilization of shared instruments (e.g., electroporation instruments, CCE instruments, MACS instruments, sterile fluid transfer instruments, FACS instruments, etc.). In contrast, conventional semi-automated instruments (e.g., the Miltenyi Prodigy) have instrument components that are idle and not capable of simultaneous parallel use.

[0152]

[0257] FIG. 6 is a schematic diagram of a cartridge 600, which may be a consumable product made from cost-effective materials that are recyclable or have limited use. The cartridge 600 may include a small bioreactor module 614a, a large bioreactor module 614b, a cell selection module 616, a cell sorting module 618, an electroporation module 620, and a fluid transfer bus 624 fluidly coupled to a counterflow centrifugal elutriation (CCE) module 622. In some variations, the cell selection module 616 may be a magnetically activated cell sorting (MACS) module. The cell sorting module 618 may include a fluorescence-activated cell sorting (FACS) module. The cartridge 600 may include a housing 602 that makes the cartridge self-contained and, optionally, protects the contents from contamination. Sterile liquid transfer ports (SLTPs) 606a-606k may be fluidly coupled to reservoirs 607a-607k. Each reservoir may be a flexible bag or a rigid container. In some variations, the flexible bag can be configured to hold a large volume and allow fluid transfer, but can collapse when fluid is forced out and expand when fluid is forced in, so that liquid or gas does not displace the transferred fluid and maintain pressure within the reservoir.

[0153]

[0258] In some variations, the liquid transfer bus 624 includes valves V1-V28 and may further include tubing fluidly linking these valves to each other and to each module. The valves shown coupled to four fluid lines are 4 / 2 (four-port, two-position) valves, and the valves shown coupled to three fluid lines are 3 / 2 (three-port, two-position) valves. The internal flow paths of the valves are indicated in the legend. The cartridge may further include a first pump 632a and a second pump 632b. Each of these pumps exposes tubing outside the housing 602 to allow each pump to interface with a pump actuator (e.g., a rotor) within one of the instruments of the system (e.g., a work cell). The liquid transfer bus 624 may be fluidly coupled to a reservoir 607d and a product bag, which are fluidly coupled to the STLP 606d and product input tubing lines 627a-627b. An operator can load a cell product into reservoir 607d by connecting product input tubing 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 allow a system according to the present disclosure (e.g., work cell 110) to automatically add fluid to reservoir 607d. For example, one or more fluid-carrying containers, such as reservoirs 607a-607k or bags, can receive fluid using the SLTP. Additionally or alternatively, the SLTP can be configured to periodically sample one or more of the fluid-carrying containers. The cartridge can further include collection bags 626a-626c fluidly coupled to fluid transfer bus 624 via valves V17-V19. Cartridge 600 can be configured to allow an operator to remove collection bags 626a-626c after cell processing by the system is complete.

[0154]

[0259] FIG. 7 is a schematic diagram of another variation of cartridge 700. For example, cartridge 700 may include a reduced feature set compared to cartridge 600. Cartridge 700 may include a fluid transfer bus 724 that may be fluidly coupled to a bioreactor module 714, a counterflow centrifugal elutriation (CCE) module 722, and a module 716 selected from a cell selection module, a cell sorting module, an electroporation module, or any other cell processing module. Cartridge 700 may include a housing 702 and sterile liquid transfer ports (SLTPs) 706a-706f (e.g., fluid connectors). Each of SLTPs 706a-706f is fluidly coupled to a reservoir 707a-707f, which may be a flexible bag or a rigid container, respectively. SLTP 706g is fluidly coupled to bioreactor module 714, allowing a system or operator direct access to the bioreactor. Reservoir 707c may be fluidly coupled to SLTP 707c and product input tubing line 727. In some variations, liquid transfer bus 724 may include fourteen valves V1-V3, V9, V11-V12, V17-V23, and V28, and may further include tubing fluidly coupling these valves to each other and / or to each module. The cartridge may further include collection bags 726a-726c fluidly coupled to liquid transfer bus 724 via valves V17-V19. The cartridge may further include pump 732. Pump 732 has exposed tubing outside of housing 702 to allow each pump to interface with a pump actuator within the system (e.g., work cell).

[0155]

[0260] 8A and 8B , respectively, show side and top views of another cartridge variation. In some variations, cartridge 800 may include bioreactor 814, pump 816, and counterflow centrifugal elutriation (CCE) module 822. Cartridge 800 may include blank spaces 818, 819, and 820 configured to accommodate one or more additional modules, such as a cell selection module, a cell sorting module, an electroporation module, or a miniature bioreactor module. In some variations, the blank spaces may define empty volumes of the cartridge reserved for accommodating modules at other times. In some variations, cartridge 800 may include two or more additional bioreactors and / or reservoirs within blank spaces 818, 819, and 820. Along near the surface of cartridge 800, there may be fluidic connectors 806a-806j (e.g., SLTPs) fluidically coupled to reservoirs 807a-807f. Reservoirs 807b and 807e may contain fluid (e.g., buffer or media). Along the top surface are product input tubing lines 827a-d, which may be fluidly connected to reservoirs 807a, 807b, 807e, and 807f, respectively. Fluid transfer bus 824 may fluidly connect the STLPs, reservoirs, and product input tubing to the modules via tubing.

[0156]

[0261] In some variations, the exterior dimensions of the housing 802 may include about 225 mm x about 280 mm x about 385 mm, about 225 mm x about 295 mm x about 385 mm, and about 450 mm x about 300 mm x about 250 mm, and all values ​​and sub-ranges therebetween. In some variations, the volume of the cartridge 800 may include about 10%, about 20%, about 30% or less, and all ranges and sub-ranges therebetween. In some variations, the volume of the cartridge 800 may include about 10%, about 20%, about 30%, about 50%, about 100%, about 200% or more, and all ranges and sub-ranges therebetween.

[0157]

[0262] In some variations, cartridge 800, as shown in the side view of Figure 8C and the perspective view of Figure 8D, can include a MACS module 818. For example, bioreactor module 814 can include ports 815a-815f, including pH and dissolved oxygen (DO) sensors (ports 815a and 815b), gas input line 815c, output line 815d, each with a sterile filter at the rear of the connector, and coolant input line 815e and output line 815f from the bioreactor apparatus interface when interfaced with bioreactor module 814 (for heat exchange). For example, gas input line 815c can be configured for gas transfer to the fluid (e.g., via headspace gas control or a gas-permeable membrane).

[0158]

[0263] 9 shows a side cross-sectional view of cartridge 900. In some variations, cartridge 900 may include an enclosure (e.g., housing), a bioreactor 914, one or more pumps 916, a valve 930, a cell selection module 917, and a counterflow centrifugal elutriation (CCE) module 922. In some variations, cell selection module 616 may be a magnetically activated cell selection (MACS) module 917. The cartridge may further include a collection bag 926. Cartridge 900 may optionally include a blank configured to accommodate one or more additional modules, such as a cell selection module, a cell sorting module, an electroporation module 918, etc. In some variations, cartridge 900 may include one or more bioreactors and / or reservoirs within the blank.

[0159]

[0264] In some variations, the cartridge may include one or more valves. In some variations, a valve 1000 on the cartridge may be configured to receive an actuator 1010 provided by the instrument (shown in FIG. 10A). Upon insertion of the cartridge into the instrument, the valve 1000 may be configured to dock with the actuator 1010 (shown in FIG. 10B) so that rotation of the actuator 1010 switches the valve 1000 from one position to another. In some variations, the valve may be configured to pinch a section of flexible tubing. The pinch valve may include a closing mechanism, and an external actuator may be configured to operate in conjunction with the pinch valve (e.g., using a linear solenoid) to open and close the valve. While the valve itself may be configured to be disposable, the actuator may be integrated into an instrument configured to repeatedly process the cartridge.

[0160] Reagent storage container

[0265] In some variations, the system includes a reagent reservoir (or multiple reagent reservoirs) for storing reagents, including, but not limited to, cell culture media, buffers, cytokines, proteins, enzymes, polynucleotides, transfection reagents, non-viral vectors, viral vectors, antibiotics, nutrients, cryoprotectants, solvents, cellular material, and pharmaceutically acceptable excipients. Additionally or alternatively, waste materials may be stored in the reagent reservoir. In some variations, in-process samples extracted from one or more cartridges may be stored in the reagent reservoir. The reagent reservoir may include one or more temperature-controlled compartments (e.g., a freezer, cooler, water bath, warming chamber, or the like, at temperatures of, for example, about -80°C, about -20°C, about 4°C, about 25°C, about 30°C, about 37°C, and about 42°C). The temperatures in these compartments can be varied during the cell manufacturing process to heat or cool the reagents. In variations of the disclosed methods, cartridges can be moved to the reagent reservoirs by a robot (or manually by an operator). The reagent reservoirs interface with one or more sterilizing fluid transfer ports on the cartridge to dispense reagents or substances into the cartridge. Optionally, fluids are added to or removed from the cartridge before, during, or after adding or removing reagents. In some variations, the system includes a sterilizing fluid transfer device, which is also configured to transfer fluids into or out of the cartridge automatically, manually, or semi-automatically. An operator can manually supply reagents to the sterilizing fluid transfer station, or a robot can supply reagents (e.g., from a feedthrough or other location). In some cases, the robot moves one or more reagents from the reagent reservoirs to the sterilizing fluid transfer station. The reagent reservoirs may have automatic doors to provide robotic access to sterilizing fluid transfer devices and / or other reagent reservoirs, each optionally under independent closed-loop temperature control. These devices and reservoirs may be configured for pick-and-place movement by the robot. In some variations, the reagent reservoirs may include one or more sample pickup areas.For example, the robot may be configured to move one or more reagents to or from one or more of the sample pickup areas.

[0161]

[0266] A variety of materials can be used to construct the cartridge and cartridge housing, including metal, plastic, rubber, and / or glass, or combinations thereof. The cartridge, its components, and its housing can be formed by molding, machining, extrusion, 3D printing, or any combination thereof. The cartridge may include commercially available components (e.g., tubing, valves, accessories), which can be attached to or integrated into custom components or devices. The cartridge housing can provide an additional layer of enclosure to further protect the sterility of the cell product. An operator can load or unload the cartridge in an ISO 5 or higher environment using aseptic techniques that ensure the sterility of the cartridge contents when the cartridge is opened. In some variations, an operator can load or unload the cartridge using manual sterile connections (e.g., sterile tubing welding). A robotic system can also perform aseptic loading and unloading of liquids from the cartridge using sterile fluid transfer instruments and sterile fluid transfer ports on the cartridge.

[0162] Countercurrent Centrifugal Elutriation

[0267] Countercurrent centrifugal elutriation (CCE) is a technique used to separate cells based on characteristics such as size and / or density. Countercurrent centrifugal elutriation combines centrifugation and countercurrent elutriation. Centrifugation corresponds to the process of sedimentation under the influence of a centrifugal force field, while countercurrent elutriation corresponds to the separation process by washing. Separation occurs in a conical (e.g., biconical, funnel-shaped) elutriation chamber. Particles (e.g., cells) in a fluid conveyed to the elutriation chamber are subjected to two opposing forces: centrifugal force, which propels the fluid away from the axis of rotation, and fluid velocity (e.g., countercurrent flow), which propels the fluid toward the axis of rotation. By varying the flow rate and centrifugal force, particle (e.g., cell) separation can be achieved. For example, particles can be separated based on characteristics such as size and density, as described in further detail herein.

[0163]

[0268] Countercurrent centrifugal elutriation (CCE) can perform multiple operations useful for cell therapy manufacturing workflows, including, but not limited to, cell washing, cell concentration, media / buffer exchange, transduction, and separating white blood cells from other blood components (e.g., platelets and red blood cells). In some variations, the fluid source for the cell separation process (e.g., an apheresis bag) can include a suspension of white blood cells, red blood cells, platelets, and plasma. To separate immune cells of interest, white blood cells can be isolated and subsequently magnetically labeled for magnetic separation. The white blood cell separation step can be performed in a CCE module to separate cells based on size and density, and magnetic separation can be performed in a MACS module. In some variations, integrating the CCE module into the cartridge enables the cell processing system to separate cells based on one or more of cell cycle progression (e.g., G1 / M phase cells over G0, S, G2 phase cells) and cell type (e.g., white blood cells versus red blood cells and / or platelets).

[0164]

[0269] Generally, a rotor configured to rotate can include an elutriation chamber (e.g., conical, biconical). A fluid containing a suspension of cells can be injected into the rotor under continuous flow. When cells are introduced into the cone (e.g., biconical), they migrate according to their sedimentation rate to a gradient location where the effects of two forces acting on the cells are balanced. Smaller cells (e.g., platelets) with lower sedimentation rates can be rapidly washed toward the axis of rotation with increasing flow velocity. These smaller cells can be output (e.g., swept) from the cone. Relatively larger (or denser) cells (e.g., red blood cells) flow through the cone at a relatively slow speed and reach equilibrium at the elutriation boundary where centrifugal and traction forces are balanced, and the flow rate is relatively slow due to the widening of the cone. The largest or densest cells (e.g., white blood cells) remain near the entrance to the chamber where centrifugal force and fluid velocity are high. By gradually increasing the flow rate, successively larger or denser cell fractions (e.g., platelets → red blood cells → white blood cells) can be output from the rotor. By continuously increasing the flow rate, eventually all cells are elutriated from the cone.

[0165]

[0270] 56 is a block diagram of a cell separation system 5600 including a work cell 5610 and at least one cartridge 5620. In some variations, the work cell 5610 may include one or more of a counterflow centrifugal elutriation (CCE) instrument 5632 (e.g., a first magnet), a magnetically activated cell selection (MACS) instrument 5642 (e.g., a magnet array, a second magnet), a fluidic connector 5652, a pump 5654, an imaging system including an optical sensor 5660 and an illumination source 5662, a sensor 5664, and a processor 5670. In some variations, the cartridge 5620 may include one or more of a CCE module 5630 (e.g., a rotor), a MACS module 5640 (e.g., a flow cell), and a fluidic connector 5650 (e.g., a sterile fluid transfer port, a fluidic transfer bus). For example, a cartridge for cell processing may include a fluidic transfer bus and multiple modules, each module fluidly linked to the fluidic transfer bus. These modules may include any of the CCE or MACS modules described herein. In some variations, a robot (not shown) may be configured to move the cartridge 5620 between various positions within the work cell 5610 to perform various cell processing steps.

[0166]

[0271] In some variations, the imaging system (e.g., optical sensor 5660, illumination source 5622) may be configured to generate image data corresponding to one or more of the CCE module 5630 and the MACS module 5640. For example, as described in detail herein, image data of fluid flow through the rotor of the CCE module 5630 may be analyzed and used to control the fluid flow rate and / or the rotational speed of the rotor. In some variations, the optical sensor 5660 may be a CMOS / CCD sensor having, for example, a resolution of about 100 μm, a working distance of about 40 mm to about 100 mm, and a focal length of less than about 8 mm. The optical sensor 5660 may be configured to operate in synchronization with the illumination source 5662. In some variations, the optical sensor 5660 may include one or more of a colorimeter, a turbidity sensor, and an optical density sensor. In some variations, the illumination source 5662 may operate as a strobe light configured to output light pulses synchronized with the rotational speed of the rotor of the CCE module 5630.

[0167]

[0272] In some variations, the sensor 5664 may include one or more of an optical density sensor configured to measure the strength of the fluid, a leak detector configured to detect moisture and / or leaks, an inertial sensor configured to measure vibrations, a pressure sensor (e.g., a photoelectric sensor) configured to measure pressure in the fluid line, a bubble sensor configured to detect the presence of bubbles in the fluid line, a colorimetric sensor, a vibration sensor, etc.

[0168]

[0273] In some variations, the fluid connector 5652 may include one or more valves configured to control fluid flow between the work cell and the cartridge 5620. The processor 5670 may correspond to a controller (e.g., a processor and memory) described in detail herein. The processor 5670 may be configured to control one or more of the CCE instrument 5632, the MACS instrument 5642, the pump 5654, the fluid connector 5652 (e.g., a valve), the optical sensor 5660, the illumination source 5662, and the sensor 5664.

[0169]

[0274] In some variations, a system 5600 for cell processing can include a cartridge 5600 including a rotor of a CCE module 5630 configured for counterflow centrifugal elutriation of cells in a fluid. A first magnet of the CCE instrument 5632 can be configured to magnetically rotate the rotor to separate cells from the fluid in the rotor. The cartridge can further include a flow cell of a MACS module 5640 coupled to the rotor and configured to receive cells from the rotor. A second magnet of the MACS instrument 5642 can be configured to magnetically separate cells in the flow cell.

[0170]

[0275] In some variations, the illumination source 5662 may be configured to illuminate the cells. The optical sensor 5660 may be configured to generate image data corresponding to the cells. In some variations, the system 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, the system 5600 may include one or more valves and pumps.

[0171]

[0276] 57 is a cross-sectional side view of a counter-flow centrifugal elutriation (CCE) module 5700. The CCE module 5700 includes a housing 5710 (e.g., enclosure), a rotor 5720 configured to rotate relative to the housing 5710, and one or more fluid ports 5730 (e.g., fluid inlet, fluid outlet). In some variations, the CCE module 5700 is portable and can be configured to move within the work cell 5610 and cartridge 5620. For example, a robot can move the CCE module 5700 between various tools in the work cell 5610.

[0172]

[0277] 58 is a side cross-sectional view of a magnetically activated cell selection (MACS) module. The MACS module includes a housing 5810 (e.g., an enclosure), 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 fluid port 5820 and the second fluid port 5830. As described in detail herein, the flow cell 5810 may include a cavity (e.g., a chamber) containing one or more channels (e.g., linear channels, laminar fluid flow channels). In some variations, the cavity of the flow cell 5810 may be substantially empty. For example, the flow cell 5810 is free of mesh, beads, serpentine channels, etc. In some variations, the flow cell 5810 may have a longitudinal axis aligned perpendicular to the ground. That is, the flow cell 5810 is oriented vertically and the first fluid port 5820 is positioned at a higher elevation than the second fluid port 5830 so that gravity can drive fluid flow through the flow cell 5810. In some variations, the MACS module 5800 is portable and can be configured to move within the work cell 5610 and cartridge 5620. For example, a robot can move the MACS module 5630 between various instruments in the work cell 5610.

[0173]

[0278] 59A and 59B are perspective views of a system 5900 (e.g., a CCE system) for cell processing. System 5900 includes a CCE module 5930 (e.g., a cartridge) including a housing 5931 and a rotor 5910, a CCE instrument 5932, an optical sensor 5960, and an illumination source 5962. In some variations, CCE instrument 5932 may include a magnet configured to magnetically rotate rotor 5910 within CCE module 5930. To facilitate illumination by illumination source 5962 and image data generation by optical sensor 5960, one or more portions of housing 5931 and rotor 5910 may be optically transparent.

[0174]

[0279] In some variations, a system 5900 for cell processing can include a cartridge 5930 including a housing 5931 containing a rotor 5910 configured to separate cells from a fluid. An instrument 5932 including a magnet can be configured to interface with the cartridge 5930 to magnetically rotate the rotor 5910. The cartridge 5930 can be configured to transfer a cellular product between multiple instruments. In some variations, the housing 5931 can house the rotor 5910. In some variations, the housing 5931 can include one or more openings 5937 configured to facilitate visualization (e.g., imaging) of the rotor 5910. FIGS. 59A and 59B show the magnet 5932 adjacent to but not attached to the housing 5931. FIG. 59C is a perspective view of the rotor 5910 and housing 5931, with the magnet 5932, optical sensor 5960, and illumination source 5962 not shown.

[0175]

[0280] In some variations, cartridge 5930 (e.g., housing 5931, 5910) may include a consumable component, such as a disposable component, a limited use component, or a single-use component. In some variations, magnet 5932 may include a durable component that may be reused multiple times. In some variations, magnet 5932 may be removably coupled to housing 5931. For example, housing 5931 may be movable relative to magnet 5932 to facilitate magnetic coupling between magnet 5932 and multiple cartridges 5930. Additionally or alternatively, magnet 5932 may be configured to move relative to housing 5931.

[0176]

[0281] 59D is a cross-sectional side view of a CCE module 5930. In some variations, a housing 5931 of a rotor 5910 can 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, the second side 5935 being opposite the first side 5933. A rotor 5910 (including a cone or bi-cone as described in detail herein) can be coupled between the first fluid port 5912 and the second fluid port 5914. In some variations, the CCE module 5930 can include an air gap 5902 between the housing 5931 and the magnet 5932. That is, the cartridge 5930 and the magnet 5932 can be coupled without contact. As a result, the cartridge does not need to be mechanically coupled to the magnet 5932 to perform counter-flow centrifugal elutriation. Thus, rotor 5910 may be less sensitive to alignment with magnet 5932 and may experience less vibration between rotor 5910 and magnet 5932. Furthermore, the space between rotor 5910 and magnet 5932 allows second fluid port 5914 to extend closer to second side 5935 of housing 5931, thereby allowing fluid to flow on either side of rotor 5910.

[0177]

[0282] In some variations, counterflow centrifugal elutriation can be performed by system 5900 by moving magnet 5932 toward (or away from) rotor 5910. The rotor can define an axis of rotation (e.g., coaxial with first fluid port 5912 and second fluid port 5914). Fluid can flow through the rotor via first fluid port 5912 and second fluid port 5914. While fluid flows through rotor 5910, magnet 5932 can magnetically rotate the rotor about the axis of rotation. The rotor can move away from the magnet. For example, moving rotor 5910 can include advancing and retracting rotor 5910 relative to magnet 5932 using a robot (not shown).

[0178]

[0283] In some variations, fluid can enter rotor 5910 through first fluid port 5912 along a first side 5933 of rotor 5910. After countercurrent centrifugal elutriation through rotor 5910, fluid can exit rotor 5910 through second fluid port 5914 along a second side 5935 of rotor 5910.

[0179]

[0284] In some variations, the counterflow centrifugal elutriation can be visualized by an optical sensor 5960 and an illumination source 5962 to manage and modify cell separation in real time based on predetermined criteria in a closed loop to maximize elutriation efficiency. In some variations, the optical sensor 5960 can be configured to image any portion of the rotor through which fluid flows (e.g., the first fluid conduit, the second fluid conduit, the third fluid conduit, the first bicone, the second bicone). 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 optical sensor can image the output of the cone to identify non-target cells being elutriated.

[0180]

[0285] In some variations, one or more of the rotor rotation speed and the fluid flow rate can be selected based at least in part on the image data. For example, the rotor can include a rotation speed of up to 6,000 RPM. For example, the fluid can include a flow rate of up to about 150 ml / min while the rotor is rotating. In some variations, the rotor can be moved toward the illumination source 5962 and the optical sensor 5960. Additionally or alternatively, the rotor 5910 can be moved away from the illumination source 5962 and the optical sensor 5960.

[0181]

[0286] FIG. 59E is a cross-sectional side view of a rotor 5910 including a first fluid port 5912 (e.g., a fluid conduit, inlet) and a second fluid port 5914 (e.g., a fluid conduit, outlet). In some variations, the first fluid port 5912 and the second fluid port 5914 may extend parallel to each other and / or to the axis of rotation of the rotor 5910. In some variations, the first fluid port 5912 and the second fluid port 5914 are located on opposite sides of the rotor 5910, which can simplify fluid routing and cartridge design and further reduce manufacturing costs. For example, each of these ports includes only one lumen, thereby simplifying fluid seals. Conventionally, a drive motor is fixedly mechanically coupled to the second side of the rotor, creating complex fluid paths (including inlets and outlets) on the first side of the rotor. FIGS. 59F and 59G are cross-sectional side views of a rotor 5910 disposed within a housing 5931.

[0182]

[0287] FIG. 60A is a plan view of a rotor 6000 that may be used with any of the CCE systems, CCE modules, cartridges, housings, combinations thereof, etc. described herein. The rotor 6000 may include a first fluid conduit 6010, a cone 6020 (e.g., a bicone), a second fluid conduit 6030, a magnetic portion 6040 (e.g., a magnet), and a housing 6050. Fluid may flow sequentially through the first fluid conduit 6010, the cone 6020, and the second fluid conduit 6030. In some variations, the magnetic portion 6040 may 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., through the page of FIG. 60A ). In some variations, at least a portion of the first fluid conduit 6010 and at least a portion of the second fluid conduit 6030 may be coaxial.

[0183]

[0288] In some variations, the cone 6020 may include a bi-cone having a first cone including a first base surface and a second cone including a second base surface, with the first base surface facing the second base surface. In some variations, the bi-cone may include 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 include a generally stepped shape. For example, one or more cones may include stacked circular steps. In some variations, the cones of the rotor may include a single cone.

[0184]

[0289] In some variations, at least a portion of the rotor 6000 may be optically transparent to facilitate visualization and / or imaging of the rotor 6000 and / or the fluid (e.g., cells) within the rotor 6000. For example, portions of the cone 6020 and the first and second fluid conduits 6010, 6030 may be transparent.

[0185]

[0290] In some variations, the cone may include a volume of about 10 ml to about 40 ml. In some variations, the cone may include a cone angle of about 40 degrees to about 60 degrees.

[0186]

[0291] In some variations, the cone includes a first cone (e.g., a distal cone) and a second cone (e.g., a proximal cone), where the first cone can be larger than the second cone. In some variations, the length of the first cone can be about 60 mm to about 90 mm. In some variations, the length of the proximal cone can be about 15 mm to about 40 mm. In some variations, the diameter of the cone (e.g., the maximum diameter of the cone) can be about 15 mm to about 40 mm.

[0187]

[0292] In some variations, rotor 6000 may include an asymmetric shape. In some variations, a first portion (e.g., a first end) of rotor 6000 may include a cone 6020 and a second portion (e.g., a second end) may include a paddle shape.

[0188]

[0293] In some variations, the cone can have a length of at least about 4 cm (e.g., about 9 cm to about 12 cm), a cone diameter of about 5 cm or less (e.g., about 3 cm to about 5 cm), a fluid flow rate of up to about 100 ml / min (e.g., about 60 ml / min to about 100 ml / min), and a rotational speed of less than about 3000 RPM. The first and second cones can be generally straight (as opposed to convex or concave) in shape.

[0189]

[0294] 60B and 60C are perspective views and FIG. 60D is a side view of rotor 6002 including first fluid conduit 6012, cone 6022, second fluid conduit 6032, and housing 6052. FIG. 60E is a perspective view of rotor 6002 disposed within housing 6090.

[0190]

[0295] 60F is a plan view of a rotor 6004 having two cones (e.g., two bi-cones). The rotor 6004 is configured to perform elutriation of cells (e.g., red blood cells, leukoreduction transfusion products) in the second cone for the purpose of recycling the buffer solution for reuse. The rotor 6004 can 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 fluid conduit 6023, and a third fluid conduit 6032 coupled to the second cone 6024. The first cone 6022 can include a first volume, and the second cone 6024 can include a second volume that is larger than the first volume. In some variations, the ratio of the second volume to the first volume can be between about 2:1 and about 5:1. The fluid can 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, the rotor 6004 can include a magnetic portion 6042.

[0191]

[0296] In some variations, the first cone 6022 may include a first bicone and the second cone 6024 may include a second bicone. In some variations, the first bicone may include a third cone including a first base and a fourth cone including a second base opposite the first base. In some variations, the second bicone may include a fifth cone including a third base and a sixth cone including a fourth base opposite the third base.

[0192]

[0297] In some variations, portions of the rotor 6004 may be optically transparent, such as the first cone 6022, the second cone 6024, and at least portions 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.

[0193]

[0298] In some variations, cells enter the first cone 6022, and elutriation of red blood cells (RBCs) 6030 can occur in the second cone 6024. Because the second cone 6024 extends away from the axis of rotation (the center of the housing 6052), the RBCs 6030 can be concentrated at the inlet 6025 of the second cone 6024 by centrifugation. The large volume of the second cone 6024 reduces the velocity of the fluid (e.g., buffer solution), which can reduce the forces acting on the RBCs 6030 in the second cone 6024. By recirculating the fluid (e.g., buffer solution), elutriation of a high concentration of RBCs can be performed using less fluid (e.g., buffer solution). In some variations, white blood cells 6040 can be collected from the first cone 6022. An optical sensor can be configured to image the first cone 6022 to generate imaging data used to identify the boundary between the WBCs 6040 and the RBCs 6030. In some variations, the recirculating fluid can be passed through a filter to remove small particles (eg, platelets) using less fluid (eg, buffer).

[0194]

[0299] Figure 60G is a top view and Figure 60H is a side view of a rotor 6005 having two cones (e.g., two bi-cones) and configured to perform elutriation of cells (e.g., red blood cells) in the second cone. A rotor having two cones can facilitate recycling 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.

[0195]

[0300] Figure 601 is a perspective view of rotor 6006, including cone 6024 and housing 6054. Figure 60J is a perspective view of rotor 6007, including cone 6026 and housing 6056. Figure 60K is a schematic plan view of rotor 6008 and corresponding dimensions. Figure 60L is an image of a set of rotors with various dimensions.

[0196]

[0301] 11A-11C illustrate another variation of a counterflow centrifugal elutriation (CCE) module 1100. FIG. 11A is a perspective view of a cartridge 1110 including the CCE module 1100 in an expanded configuration configured to receive a CCE instrument. FIGS. 11B and 11C are side cross-sectional views of the CCE module 1100 in a retracted and expanded configuration, respectively. In some variations, the CCE module may include a conical element having an inner and outer surface fixedly attached to the distal end of a linear member having an inner and outer surface. The proximal end of the linear member may be rotatably attached to a fulcrum to allow the linear member to expand, retract, and / or rotate. For example, FIG. 11C illustrates the linear member extending out of the cartridge housing and then rotating to generate centrifugal force. The cell product can be conveyed between the inner and outer surfaces of the linear member (optionally within a tube) to the conical element against the centrifugal force generated by the rotation of the linear member and delivered to an opening at the distal end of the inner surface of the conical element. By counterflow of the solution and sedimentation of the cells under the centrifugal force, the cells in the cell product can be separated based on the ratio of their hydrodynamic cross section to their mass. Then, by increasing the flow rate and / or decreasing the rotation of the linear member, the cells can be selectively returned to the proximal end of the linear member through the voids in the inner surface of the linear member. These selected cells can be directed into a tube that returns them to the cartridge. After the concentration and / or washing steps are performed, the linear member can be retracted into the housing, resulting in the storage configuration shown in FIG. 11B.

[0197] Magnetic cell selection

[0302] In general, the systems and methods described herein can select cells based on magnetically labeled cells corresponding to cells bearing a predetermined antigen. For example, a cell suspension of interest can be immunologically labeled with magnetic particles (e.g., magnetic beads) configured to selectively bind to the surface of the cells of interest. When the cell suspension flows through a flow cell, the labeled cells can generate a large magnetic moment. The flow cell can be placed in proximity to a magnet array (e.g., permanent magnets, electromagnets). The magnet array generates a gradient magnetic field within the flow cell to attract the labeled cells for separation, capture, recovery, and purification. The magnet array can be configured to generate non-uniform magnetic fields at the edges and interfaces of the individual magnets, covering the entire volume of the flow cell, so that the magnetophoretic force is equal to the attractive force exerted by the fluid flowing through the flow cell.

[0198]

[0303] 61A-61C are schematic diagrams of a magnetic cell separation (e.g., magnetically activated cell selection) system and process. The magnetic cell separation system may include a flow cell 6110 including an inlet 6130 and an outlet 6132, a magnet array 6120, a first fluid source 6140 (e.g., an input sample source), a second fluid source 6142 (e.g., a buffer source), a third fluid source 6150 (e.g., a target cell reservoir), a fourth fluid source 6152 (e.g., a waste reservoir), and a valve set 6134. As shown in step 6100, a set of cells 6160, 6170, including labeled cells 6160 (e.g., magnetically labeled cells) and unlabeled cells 6170, may flow into the flow cell 6110. For example, the set of cells 6160 may be labeled with a magnetically activated cell selection (MACS) reagent. The MACS reagent may be incubated with the set of cells to label (e.g., attach, bind) the cells to the MACS reagent. As described in detail herein, the magnet array 6120 can be positioned external to the flow cell 6110 such that it is movable relative to the flow cell 6110. For example, the magnet array 6120 can be moved away from the flow cell 6110 to facilitate the flow of the cell set 6160 out of the flow cell 6110. Conventional flow cells include a tortuous path that includes mesh and / or beads that capture cells. However, it is difficult to recover labeled cells from conventional flow cell configurations. In contrast, the flow cell 6110 described herein does not include a tortuous path of beads, mesh, etc., and therefore allows for efficient sequential selection using either positive or negative selection. In some variations, the flow cell can include a generally layered channel as described in detail herein.

[0199]

[0304] In step 6102, the magnet array 6120 may magnetically attract the set of cells 6160 toward the magnet array 6120 for a predetermined dwell time and / or based on a measured amount of magnetically separated cells. In some variations, the dwell time may be at least 1 minute (e.g., at least 2 minutes, at least 3 minutes, at least 5 minutes). Unlabeled cells 6170 are not magnetically attracted to the magnet array 6120 and may exit the outlet 6132 of the flow cell 6110 into the fourth fluid source 6152. In some variations, the fluid within the flow cell (e.g., cells 6160, 6170) may be held statically within the flow cell 6110 for a dwell time before the fluid (e.g., cells 6170) flows out the outlet 6132. In some variations, the length of the flow cell 6110 may be oriented substantially perpendicular to the ground to facilitate fluid flow through the flow cell 6110 using gravity. In step 6104 , the magnetic coupling between the magnet array 6120 and the cells 6160 is released after a dwell time, allowing the cells 6160 to flow into the third reservoir 6150 .

[0200]

[0305] In some variations, cells may remain attached to the surface of the flow cell after the magnet array 6120 is removed due to static friction. Therefore, gas may be flowed through the flow cell 6110 to assist cell collection into the third reservoir 6150. Gas flow through the flow cell can improve cell recovery more than forcing liquid through the flow cell. Because the interface created by the gas (e.g., bubbles, air gap) is maintained by gravity, relatively wide flow cells can be implemented, further improving cell recovery over horizontally oriented flow cells. The MACS modules described herein can be configured to perform positive and / or negative selection by altering the order of steps.

[0201]

[0306] Additionally or alternatively, an optical sensor may be configured to image the flow cell to generate imaging data that is used to identify the amount of cells magnetically attracted to the magnet array. When a predetermined amount of cells is measured by the optical sensor, the fluid containing the labeled cells may be allowed to flow out of the flow cell.

[0202]

[0307] FIG. 62A is a perspective view of a MACS module 6200 in a first configuration. The MACS module 6200 (and any of the MACS modules described herein) can be a component of any of the cartridges described herein. For example, a cartridge for cell processing can include a fluid transfer bath and multiple modules, each fluidly linked to the fluid transfer bath. The 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 further include a magnet array 6220 including multiple magnets. Each magnet can be separated by a separation distance, as shown in FIGS. 62G, 63D, and 63E, although the magnets of the magnet array 6220 shown in FIGS. 62A-62E are in contact with adjacent magnets.

[0203]

[0308] Figure 62G is a schematic diagram of a flow cell 6210 and a magnet array 6220. In some variations, the flow cell 6210 may include a cavity height 6202 and a cavity width 6204. Fluid may be configured to flow through the flow cell 6210 in a first direction 6206. The magnet array 6220 may include a plurality of magnets, each having a width 6222. In some variations, adjacent magnets may be separated by a predetermined separation distance 6224. Each magnet pair may have the same or a different separation distance 6224. As shown in Figure 62G, the orientation (e.g., poles) of the magnets in the magnet array 6220 may include a predetermined pattern.

[0204]

[0309] In some variations, the ratio of cavity height 6202 to separation distance 6224 can include about 20:1 to about 1:20, about 10:1 to about 1:10, about 5:1 to about 1:5, and about 3:1 to about 1:3, including all values ​​and subranges therebetween. In some variations, actuator 6240 (e.g., linear, rotary) can be configured to move magnet array 6220 relative to flow cell 6210. In some variations, the orientation (e.g., poles) of the magnets in magnet array 6220 can include a predetermined pattern (e.g., a Halbach array).

[0205]

[0310] In some variations, the magnet array 6220 can move relative to the flow cell 6210, or vice versa. Figure 62A shows the MACS module 6200 in an open configuration, and Figure 62B shows the MACS module 6200 in a closed configuration. Figure 62B is a perspective view of the MACS system 6200 in a second configuration that can magnetically attract labeled cells toward the magnet array 6220. In the second configuration, magnetic field lines generated by the magnet array traverse the flow channel and apply a magnetophoretic force to magnetically labeled cells injected into the channel. Figure 62C is a side cross-sectional view of the MACS system 6200 including the magnet array 6220. Figure 62D is a perspective view of the MACS system 6200 in the second configuration. Figure 62E is a plan view of the flow cell 6210 and magnet array 6220 of the MACS system. Figure 62F is a plan view of the flow cell 6210 of the MACS system.

[0206]

[0311] 63A-63E are perspective views of magnet array sets 6300, 6310, 6320, 6330, and 6340. One or more of the size, strength, shape, spacing, and orientation of the magnets in the magnet array can be configured to generate a magnetic field that attracts magnetically labeled cells. Additionally or alternatively, the magnet array can include a high-permeability material configured to increase or decrease the magnetic field strength and magnetic field gradient within the flow cell. This material can be disposed between the magnets and the flow cell. Additionally or alternatively, this material can be disposed within the flow cell and / or on one or more sides of the flow cell.

[0207]

[0312] Figures 64A and 64B are perspective and side cross-sectional views, respectively, of a MACS module 6400 including a flow cell 6410 and a magnet array 6420. The flow cell 6410 includes a set of linear channels 6412, 6414, 6416, including a first channel 6412 parallel to a second channel 6416 and a third channel 6416 in fluid communication with each of the first channel 6412 and the second channel 6416. As shown in Figure 64B, the third channel 6416 can be disposed between the first channel 6412 and the second channel 6414 to define a volume in which fluids from the first channel 6412 and the second channel 6416 interact (e.g., mix). In some variations, the flow cell 6410 may include a first inlet 6430 coupled to the first channel 6412 and configured to receive a first fluid 6460 (e.g., cells). A second inlet 6431 may be coupled to the second channel 6414 and configured to receive a second fluid 6470 (e.g., a buffer solution). The flow cell 6410 may include a first outlet 6432 coupled to the first channel 6412 and a second outlet 6433 coupled to the second channel 6414.

[0208]

[0313] The magnet array 6420 is disposed external 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 so that the fluid flows in a generally vertical direction.

[0209]

[0314] In some variations, the first channel 6412 can have different dimensions than the second channel 6414. For example, the first cavity height of the first channel 6412 can be greater than the second cavity height of the second channel 6414. For example, the ratio of the first cavity height to the second cavity height can include about 1:1 to about 3:7, about 1:1 to about 2:3, and about 2:3 to about 3:7, and all values ​​and subranges therebetween. Because the cavity height of the first channel 6412 is greater than the second channel 6414, the fluid flowing through the first channel 6412 can have a slower flow rate than the second channel 6414. In some variations, the third channel 6416 can have a ratio of the length of the third channel 6416 to the diameter of the third channel 6416 that can include about 2:1 to about 6:1, about 2:1 to about 3:1, about 3:1 to about 4:1, about 4:1 to about 5:1, about 5:1 to about 6:1, and about 3:1 to about 5:1, including all values ​​and subranges therebetween.

[0210]

[0315] 64B , a first fluid 6462 may flow generally in a first direction within the flow cell 6410. The magnetic attractive force generated by the magnet array 6420 may pull magnetically labeled cells 6416 within the first fluid 6462 from the first channel 6412 toward the second channel 6414 (e.g., toward the magnet array 6420), such that the cells 6416 may separate from other portions of the first fluid 6462 within the third channel 6416. Similarly, a second fluid 6470 (e.g., a buffer solution) may flow through the second channel 6414. As the cells 6416 flow toward the magnet array 6420, they displace the second fluid 6470 flowing through the third channel 6416, such that a portion of the second fluid 6470 may flow into the first channel 6412. In this manner, the magnetically labeled cells 6416 are magnetically separated from the first fluid 6462 and the second fluid 6470 can facilitate removal of the first fluid 642 that does not contain the cells 6416 .

[0211]

[0316] In some variations, a set of fluid loops can be coupled to the flow cell to enable multiple cell separation cycles. Figure 64C is a schematic diagram of a MACS module including 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 a set of cells from the outlet 6432 of the flow cell 6410 and collect and / or recirculate them via the inlet 6430 of the flow cell 6410. A second fluid conduit 6490 can be coupled to the inlet 6431 of the flow cell 6410 and the outlet 6433 of the flow cell 6410 to recirculate fluids such as buffer and uncollected magnetically labeled cells. The second fluid conduit 6490 can be configured to receive fluids from the flow cell 6410 that do not contain the set of cells. Based on the number of cycles performed, labeled cells of higher purity can be recovered. For example, one cell separation cycle may result in about 80% cell purity, a second cell separation cycle may result in about 96% cell purity, a third cell separation cycle may result in about 99.2% cell purity, and a fourth cell separation cycle may result in about 99.84% cell purity.

[0212]

[0317] In some variations, centrifugal force can be applied to the magnetic cell separation process to further attract the labeled cells toward the magnetic array independent of fluid flow rate to maintain throughput. Figures 65A-65C are schematic diagrams of a MACS module 6500 that uses centrifugal force to facilitate the cell separation process. Figure 65A shows a flat flow cell 6510 configured to be rolled to form a generally cylindrical shape 6512. The flow cell 6510 may include a curved channel 6520.

[0213]

[0318] FIG. 65B shows a cylindrical flow cell 6510 concentrically surrounded by (e.g., nested within) a cylindrical magnet array 6530. In FIG. 65B, only a cross section of the magnet array 6530 is shown for clarity. The flow cell 6510 can be spaced a predetermined separation distance from the magnet array 6530. The flow cell 6510 can thus be configured to rotate 6550 about its longitudinal axis to generate an outward centrifugal force on the fluid 6540 in the flow channel 6520, toward the magnet array 6530. During the cell separation process, the fluid can be subjected to a set of forces shown in FIG. 65C. These forces include bulk fluid forces 6560 in the axial (e.g., bulk flow) direction, centrifugal forces 6570 (e.g., proportional to the net particle-system buoyant force) directed radially outward from the center of rotation, and magnetic forces 6580 (e.g., proportional to the net particle-system magnetic attractive force) directed radially outward from the center of rotation. In some variations, labeled cells may have a higher density than unlabeled cells. Thus, centrifugal forces may preferentially push labeled cells toward the magnet 6530, further increasing the specificity and efficiency of cell separation.

[0214]

[0319] 66A through 66C are schematic diagrams of a cell separation system and process. The magnetic cell separation system may include a flow cell 6610 including a channel 6620 (shown diagrammatically flattened for clarity) and a magnet array 6630. As shown in step 6600, a set of cells 6640, 6642, including labeled cells 6640 (e.g., magnetically labeled cells) and unlabeled cells 6642, may flow into the channel 6620 of the flow cell 6610. For example, the set of cells 6640 may be labeled with a magnetically activated cell selection (MACS) reagent. The magnet array 6630 may be positioned external to the flow cell 6610 such that it is movable relative to the flow cell 6610. For example, the magnet array 6630 may be movable away from the flow cell 6610 to facilitate the exit of the set of cells 6640 from the flow cell 6610.

[0215]

[0320] In step 6602, the flow cell 6650 can be rotated to generate centrifugal force that pushes the cells 6640, 6642 toward the magnet array 6630. In some variations, the length of the flow cell 6610 can be oriented substantially perpendicular to the ground so that gravity facilitates fluid flow through the flow cell 6610. In step 6604, the magnet array 6630 can magnetically attract the set of cells 6640 toward the magnet array 6630 for a predetermined dwell time as described herein. Unlabeled cells 6642 are not magnetically attracted to the magnet array 6630 and can exit the flow cell 6610, for example, into a waste container. In some variations, the fluid within the flow cell (e.g., cells 6160, 6170) can be statically held within the flow cell 6110 for a dwell time before the fluid (e.g., cells 6170) flows out of the outlet 6132. In some variations, the magnetic coupling between the magnet array 6630 and the cells 6640 is released after a dwell time, allowing the cells 6640 to be recovered.

[0216]

[0321] Figures 12A and 12B show a magnet and MACS module 1210 of a magnet-containing MACS instrument 1200. The magnet is shown in the ON configuration in Figure 12A and in the OFF configuration in Figure 12B.

[0217] Bioreactor

[0322] The bioreactors described herein can include vessels configured to culture mammalian cells. Generally, cells and gene therapy products can be grown in the bioreactor to generate a clinical dose that can then be administered to a patient. Numerous biological and environmental factors can be controlled to optimize the proliferation rate and success of cell growth. The bioreactor modules described herein allow for one or more of monitoring, adjusting, and / or controlling cell growth (e.g., to facilitate consistent and efficient cell growth).

[0218]

[0323] 67A is a schematic diagram of a cell processing system 6700 (e.g., a bioreactor module) including one or more of a bioreactor 6710, one or more sensors 6720, an agitator 6730, a temperature regulator 6740, and a gas regulator 6750. In some variations, the sensors 6720 can be configured to monitor (e.g., measure, detect, determine) one or more characteristics of the bioreactor module 6700 and the cells within the bioreactor 6710. For example, the sensors 6720 can include one or more of a pH sensor, a dissolved oxygen (DO) sensor, a temperature sensor, a glucose sensor, a lactose sensor, a cell density sensor, a humidity sensor, combinations thereof, etc. One or more of the sensors can be a non-invasive optical sensor.

[0219]

[0324] 67B-67D are schematic diagrams of a cell processing system including a work cell 6760, a bioreactor system 6700 (e.g., a bioreactor apparatus), a cartridge 6770, an agitator 6730, and a fluidic connector 6780. In some variations, the cartridge 6770 for cell processing may include a fluid transfer bus and multiple modules (e.g., a bioreactor module, a CCE module, a MACS module, an EP module). Each module may be fluidically linked to the fluid transfer bus. The bioreactor module may include at least one bioreactor.

[0220]

[0325] The bioreactor instrument 6700 can be configured to interface with the cartridge 6770. In some variations, the bioreactor instrument 6700 can include an agitator 6730 configured to couple to the bioreactor. The agitator can be configured to agitate the cell culture medium containing the cells. In some variations, a fluidic connector 6780 can be configured to couple the bioreactor system 6700 and the work cell 6760.

[0221]

[0326] FIG. 67B illustrates a cartridge 6770 including a bioreactor disposed within a work cell 6760. The bioreactor 6700 can be separated from the work cell 6760. As shown in FIG. 67C, once the fluidic connector 6780 couples the work cell 6760 to the bioreactor 6700 (e.g., to create a sterile flow path), the cartridge 6770 can be moved into the bioreactor 6700. For example, as shown in FIG. 67D, the cartridge 6770 can be coupled to (e.g., placed on) an agitator 6730 and then agitated. In some variations, the fluidic connector 6780 can include a set of collapsible sidewalls (e.g., accordion-like) configured to receive and distribute the agitation of the agitator 6730 while not transmitting such movement to the work cell 6760. That is, the fluidic connector 6780 can function as a bellows to maintain a connection between the work cell 6760 and the bioreactor 6700 without agitating the work cell 6760. In some variations, a fluid connector 6780 can couple a bioreactor (eg, of cartridge 6770) to a fluid transfer bus.

[0222]

[0327] In some variations, the agitator may be configured to generate motion (e.g., orbital, rotational, linear) relative to the bioreactor to mix the culture as needed to promote interaction between reagents and cells. For example, orbital motion may be used to create a homogenous culture volume so that a small sample taken from the culture may be representative of the entire culture. In some variations, the agitator 6730 may include one or more impellers. The agitator 6730 may be configured to provide mixing at varying intensities during a regular cycle of culture.

[0223]

[0328] In some variations, such as the toroidal bioreactors described herein, which include geometries that can promote continuous, gentle fluid flow around the entire circumference, orbital motion can promote increased interactions within the cell culture, thereby supporting homogeneous mixing while minimizing shear stress transmitted to the cells.

[0224]

[0329] In some variations, the temperature regulator 6740 can be configured to control the temperature of the bioreactor and corresponding process. The temperature regulator 6740 can be coupled to the bioreactor. For example, the temperature regulator 6740 can control the temperature of the cell culture between about 2°C and about 40°C to ensure that the culture is heated to physiological conditions when desired and cooled to slow metabolic processes (e.g., to keep the cells dormant). For example, the thermal regulator 6740 can include a circulating coolant coupled to a heat exchanger coupled to a thermal interface (e.g., a heating plate / cooling plate).

[0225]

[0330] In some variations, a gas regulator 6750 can be coupled to the bioreactor and configured to control the gas composition of the bioreactor and corresponding process using one or more of clean dry air (CDA), carbon dioxide, and nitrogen. The gas regulator 6750 can be coupled to the bioreactor. For example, the sensor 6720 and the gas regulator 6750 can provide closed-loop gas control of the bioreactor module 6700. In some variations, the CDA can include oxygen, such as pure oxygen. In some variations, the gas regulator can include a manifold coupled to one or more gas sources. The manifold can include a solenoid coupled to a valve (e.g., a restrictive orifice) configured to control gas flow through the bioreactor 6710. The solenoid can be configured to pulse-control the amount and composition of gas received through the manifold. Additionally or alternatively, one or more of a proportional valve and a mass flow controller (MFC) can be configured to measure and control gas flow to the manifold. In some variations, the gas regulator 6750 may include one or more sensors to measure the gas mixture and / or flow rate. Additionally or alternatively, these sensors may be configured for closed-loop control of the gas flow through the gas regulator.

[0226]

[0331] In some variations, the gas regulator 6750 can be used to control the pH of the bioreactor 6710 using the pH measured by the pH sensor. For example, the gas regulator 6750 can control the free hydrogen ions and pH of the culture by controlling the CO2 concentration of the gas contacting the cell culture in response to the measured pH. In some variations, the pH of the bioreactor 6710 can be about 5.5 to about 8.5. To adjust the pH, one or more of the CO2 composition of the gas in the bioreactor 6710, a buffer, and a reagent (e.g., an acid or a base) can be used. In some variations, the dissolved oxygen concentration of the bioreactor 6710 can be about 0% to about 21%. The nitrogen composition of the gas in the bioreactor 6710 can be used to adjust the dissolved oxygen concentration. For example, the dissolved carbon dioxide concentration can be adjusted by controlling both the agitator in the bioreactor and the flow rate and composition of the gas contacting the cell culture.

[0227]

[0332] In some variations, a gas regulator 6750 can be used to control the oxygen concentration (e.g., below atmospheric levels) in the bioreactor 6710 using dissolved oxygen measured by a dissolved oxygen sensor. For example, the gas regulator 6750 can control the nitrogen concentration of the gas contacting the cell culture to create hypoxic conditions.

[0228]

[0333] 68A and 68B are cross-sectional perspective views of a bioreactor 6800 including an enclosure 6810 including a bottom 6812, a sidewall 6814, and a top 6816. A gas permeable membrane 6820 can be coupled to one or more of the bottom 6812 and the sidewall 6814 of the enclosure 6810. In some variations, the enclosure 6810 can include a first chamber 6830 having a first volume and a second chamber 6832 having a second volume. The first chamber 6830 is separated from the second chamber 6832, and the first volume is smaller than the second volume. In some variations, the first chamber 6830 can be concentrically nested within the second chamber 6832. For example, the nesting of the chambers can increase the overall working volume range (e.g., 100:1). The first chamber 6830 may include a recessed shape with a beveled bottom to promote fluid pooling in the center of the first chamber 6830 during aspiration. In some variations, the bottom 6812 may be disposed on a temperature regulator (not shown), such as a thermoelectric element. In some variations, the enclosure 6810 may be constructed of a thermally conductive material, such as metal (e.g., aluminum).

[0229]

[0334] In some variations, the bioreactor 6800 can be coupled to a gas regulator (not shown) to facilitate gas transfer (e.g., into or out of the culture) through the gas permeable membrane 6820. The gas permeable membrane 6820 can be configured to retain the cell culture. Gas can diffuse through the surface of the culture in contact with the gas permeable membrane, allowing for increased oxygenation of the cell culture and removal of gaseous metabolic by-products from the cell culture, thereby increasing the potential for metabolic activity. For example, the gas permeable membrane 6820 allows dissolved oxygen to diffuse into the culture in close proximity to the cell bed where oxygen can be consumed. In some variations, the bioreactor can be coupled to both a first gas regulator to facilitate gas transfer through the gas permeable membrane and a second gas regulator to facilitate control of the headspace gas composition.

[0230]

[0335] In addition to gas transfer, the gas reactors described herein can be configured to efficiently control the temperature of the cell culture using conductive thermal interfaces (e.g., gas permeable membrane 6820, enclosure 6810) along both the bottom and side walls of the bioreactor.

[0231]

[0336] In some variations, the first chamber 6830 may include a working volume of about 10 ml to about 100 ml. In some variations, the first chamber 6830 may include a total volume of about 10 ml to about 130 ml. In some variations, the second chamber 6832 may include a working volume of about 100 ml to about 1000 ml. In some variations, the second chamber 6832 may include a total volume of about 100 ml to about 1400 ml. In some variations, the first chamber 6830 may include a diameter of about 10 mm to about 100 mm and a height of about 10 mm to about 100 mm. In some variations, the second chamber 6832 may include a diameter of about 100 mm to about 250 mm and a height of about 10 mm to about 100 mm.

[0232]

[0337] As shown in FIG. 68B, the bottom 6822 of the gas permeable membrane 6820 can be angled from about 3 degrees to about 10 degrees relative to the bottom 6812 of the enclosure 6810. Similarly, FIGS. 69A and 69B show a sloped base. For example, due to the slope of the bottom 6822, the chambers 6830, 6832 deepen toward the center of the bioreactor 6800. This promotes cell growth near the center of the bioreactor 6800 and can assist in one or more of cell sampling, cell transfer, cell recovery, etc. In some variations, orbital motion of the bioreactor 6800 can promote cell aggregation near the center of the bioreactor 6800, thereby increasing cell-to-cell interactions.

[0233]

[0338] In some variations, the gas-permeable membrane 680 can include curved surfaces. In some variations, the gas-permeable membrane can include a set of patterned curved surfaces. For example, the set of patterned curved surfaces can have a radius of curvature of about 50 mm to about 500 mm.

[0234]

[0339] In some variations, the bioreactor can be configured to facilitate monitoring (e.g., temperature, pH, dissolved oxygen) and fluid flow (e.g., gas composition, fluid transfer) between chambers. As shown in FIG. 68C, the enclosure 6810 can include one or more nesting surfaces surrounding the longitudinal axis (e.g., center) of the enclosure 6810. For example, the nesting surfaces can include a set of concentric rings. The enclosure 6810 can have a ring shape. FIG. 68C is a perspective view of the enclosure 6810 including a set of openings 6818 (e.g., holes, apertures, slits, slots), and FIG. 68D is a bottom view. In some variations, the openings 6818 allow gas transfer and / or heat transfer between components and chambers of the bioreactor 6800. Additionally or alternatively, one or more sensors can be coupled to the openings 6818. For example, the openings 6818 can be coupled to a non-contact sensor (e.g., pH, DO), such as an optical sensor (not shown) configured to determine fluorescent spots disposed on a surface of the bioreactor. In some variations, one or more of the sensor and fluid connector may be introduced through opening 6818.

[0235]

[0340] In some variations, the gas permeable membrane extends along the bottom 6812 and sidewalls 6814 of the enclosure 6810, as shown in FIG. 68B. In some variations, the gas permeable membrane extends only along the bottom 6812 of the enclosure 6810. FIG. 68E is a perspective view and FIG. 68F is a side view of a gas permeable membrane 6820, the outer surface of which includes one or more protrusions 6824 (e.g., protrusions, spacers, ribs, etc.). The protrusions 6824 are also shown in the perspective view of FIG. 68G and the bottom view of FIG. 68H. The protrusions 6824 contact the enclosure 6810 and define a cavity between the enclosure 6810 and the gas permeable membrane 6820. That is, protrusions 6824 can be configured to mechanically separate enclosure 6810 from a portion of gas permeable membrane 6820 to facilitate heat transfer from enclosure 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), ethyl vinyl acetate (EVA), and can have a thickness of about 0.1 mm to about 0.4 mm, about 0.2 mm to about 0.3 mm, about 0.25 mm, and all ranges and subvalues ​​therebetween.

[0236]

[0341] Figure 69A is a side cross-sectional view of a bioreactor enclosure 6910. The enclosure 6910 includes a first chamber 6912, a second chamber 6914, and a column 6916 extending along the longitudinal axis of the enclosure 6910. Figure 69B is a cross-sectional perspective view of the enclosure 6910 showing the nested curves of the enclosure 6910. The column 6916 can be configured in combination with agitation, such as orbital motion, to facilitate cell culture.

[0237]

[0342] Figure 70 is an exploded perspective view of a bioreactor 7000 including an enclosure 7010, a gas permeable membrane 7020, and an upper portion 7030. The upper portion 7030 may be constructed of a material such as polyethylene.

[0238]

[0343] Figure 71A is a top view of a bioreactor 7100 including a first chamber 7110 and a second chamber 7120. Figure 71B is a side cross-sectional view of the bioreactor 7100.

[0239]

[0344] Figures 13A and 13B are perspective views of a cartridge 1300 and a bioreactor instrument interface 1310. In Figure 13B, the bioreactor instrument interface 1310 is coupled to the cartridge 1300.

[0240]

[0345] 14 is a perspective view of a bioreactor device 1410 including cartridges 1400, 1402, 1404 and a set of cavities 1420, 1422, 1424 configured to receive each cartridge. In some variations, each cartridge can be docked to allow for simultaneous growth, culture, and settling steps.

[0241] Electroporation Module

[0346] In some variations, the electroporation module can be configured to facilitate intracellular transfer of macromolecules (e.g., transfection by electroporation). The electroporation module can include a continuous-flow or batch-mode chamber and one or more electrode sets for applying direct or alternating current to the chamber. A discharge from one or more capacitors or a current source can generate a current within the chamber sufficient to facilitate the transfer of cellular products, such as polynucleotides, proteins, nucleoprotein complexes, or other macromolecules, into the cells. As with the other modules described herein, one or more components used in a process step (here, electroporation) can be provided on the cartridge or in an instrument associated with the cartridge. For example, one or more capacitors and / or batteries can be provided in the module on the cartridge or in the instrument. In some variations, the electroporation module can be configured to apply an electric field to a cell suspension under continuous flow in a microfluidic device, as described, for example, in Garcia et al. Sci. Rep. 6:21238 (2016).

[0242]

[0347] Additionally or alternatively, intracellular delivery of macromolecules may be achieved by other methods, such as mechanoporation. It should be understood throughout this disclosure that variations including an electroporation module may alternatively or additionally include a mechanoporation module or other modules configured to perform any suitable method of delivering macromolecules into cells. Mechanoporation can be achieved, for example, by applying transient fluid pressure to a solution containing cells or by applying physical pressure to cells (e.g., with a microneedle). For example, International Patent Applications WO 2017 / 041051 and WO 2017 / 123663 present exemplary methods of mechanoporation by passing a cell suspension through a constriction, and these applications are incorporated herein by reference. Mechanoporation can also be achieved by applying a vortex to a cell suspension in a microfluidic device.

[0243]

[0348] 72 is a schematic diagram of an electroporation module 7200 (e.g., an electroporation system). The electroporation module 7200 includes an electroporation chamber 7210 (which may include fluid conduits), a pump 7220, an inlet 7230, an outlet 7232, a pinch valve set 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 sensor set 7250 (e.g., a bubble sensor), a controller (e.g., a processor and memory) configured to control the module 7200, and a signal generator 7270 configured to send an electroporation signal (e.g., a voltage pulse) to the electroporation chamber 7210.

[0244]

[0349] In some variations, fluid conduit 7210 can be configured to receive a first fluid containing cells and a second fluid. An electrode set 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 first fluid is separated from a third fluid by the second fluid, and generate an electroporation signal to electroporate cells in fluid conduit 7210 using the electrode set.

[0245]

[0350] 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 a third fluid into the fluid conduit 7210. The third fluid may be separated from the first fluid by the second fluid. In some variations, the cartridge for cell processing may include a liquid transfer bus and multiple modules, such as the electroporation module 7200. Each module may be fluidically linked to the liquid transfer bus.

[0246]

[0351] The sensor set 7250 may be configured to measure a fluid change in the fluid line, such as a change from a first fluid to a second fluid (e.g., liquid to air) in the fluid line. The module 7200 may further include a valve set configured to ensure that fluid does not flow back into the electroporation module 7210 and / or the fluid source 7240. The electroporation chamber 7210 may include a cavity configured to hold a fluid to be electroporated and an electrode set for applying an electroporation signal to the fluid. For example, the signal generator 7270 may generate square valve pulses as described in more detail herein.

[0247]

[0352] In some variations, the electroporation module 7200 (e.g., valve 7234, pump 7220, sensor 7250, and controller 7260) can be configured to control fluid flow through the electroporation chamber 7210 discontinuously (e.g., in a batch process). For example, a first batch of cells can be electroporated and physically separated from a second batch of cells by an intermediate fluid such as air or a fluid such as oil. Separating the cell batches can reduce mixing of transfected and non-transfected cells and also ensure a fixed batch volume. That is, a fluid gap can form a visually verifiable boundary between the cell batches to reduce diffusion and mixing between electroporated and non-electroporated cells. Separating the cell batches can reduce the time cells are exposed to certain cytotoxic reagents (e.g., electroporation buffer), thereby improving performance.

[0248]

[0353] In some variations, electroporation of cell batches can be performed when the system is substantially static (e.g., substantially free of fluid flow). In contrast, conventional continuous-flow electroporation has an upper fluid flow rate limit that correlates with transfection efficiency. The batch processing described herein transfers cell batches into and out of the electroporation chamber 7210 at a predetermined rate, increasing the overall throughput of the system 7200 without reducing electroporation efficiency. Furthermore, the electroporation system 7200 does not use precisely controlled flow rates / pulse rates as required by continuous-flow electroporation systems.

[0249]

[0354] FIG. 73 is an exploded perspective view of an electroporation module 7300, which may include an electrode 7310, a fluid conduit 7320 (e.g., an electroporation chamber), a substrate 7330 (e.g., an alloy bus bar), a housing 7340, and a fastener 7350. In some variations, the fluid conduit 7320 may be configured to hold a fluid volume of about 0.4 ml to about 3.5 ml. The electroporation module 7300 is a parallel plate design. In some variations, the electrodes may comprise stainless steel and be separated by an insulating gasket. In some variations, the electrodes may be polished and / or coated with a non-reactive material (e.g., gold, platinum) to reduce the gradual buildup of biological material (e.g., charged molecules, DNA, proteins) on the electrode surface.

[0250]

[0355] In general, a method for electroporating cells may include receiving a first fluid containing cells into a fluid conduit, receiving a second fluid into 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 separated from the first fluid by the second fluid within the fluid conduit. In some variations, the first fluid may be substantially static when applying the electroporation signal.

[0251]

[0356] 74A-74B are schematic diagrams of variations of electroporation processes 7400, 7402. Method 7400 can include loading 7410 cells into an electroporation chamber 7450. For example, in step 7412, a first fluid can be injected into the electroporation chamber by opening valve v1 and using a pump to generate negative pressure (valves v2 and v3 are closed). In step 7414, a second fluid (e.g., gas, oil) can separate the first fluid from a third fluid to generate a first batch of cells for electroporation. For example, valve v2 can be opened and the pump can generate negative pressure while valves v1 and v3 are closed. In some variations, the loading volume can be about 1 ml to about 3 ml, and the injection time can be about 8 seconds to about 15 seconds (at a rate of about 20 ml / min). In step 7420, electroporation of cells in the first fluid can be performed with the valves closed and the pump turned off. In step 7430, valves v1 and v2 are closed, valve v3 is opened, and with the pump generating positive pressure, the cells in the first fluid can be flushed out of the electroporation chamber 7450 and output.

[0252]

[0357] FIG. 74B illustrates another configuration in which a pump disposed between the input and the electroporation chamber can be configured for unidirectional injection. Method 7402 can include introducing (7411) cells into electroporation chamber 7450. For example, in step 7416, a first fluid can be injected into the electroporation chamber by opening valves v1 and v4 and the pump generating positive pressure (valves v2 and v3 are closed). In step 7418, a second fluid (e.g., gas, oil) can separate the first fluid from a third fluid to generate a first batch of cells for electroporation. For example, valves v1 and v3 can be closed while valves v2 and v4 are open and the pump generating positive pressure. In step 7422, electroporation of cells in the first fluid can be performed with the valves closed and the pump turned off. In step 7432, valves v1 and v4 are closed, valves v2 and v3 are opened, and with the pump generating positive pressure, the cells in the first fluid can be forced out of the electroporation chamber 7450 and output.

[0253]

[0358] In some variations, the impedance / resistance between the electrodes of an electroporation system can increase over time due to electrode passivation / degradation caused by the adhesion of charged biological material (e.g., charged molecules, DNA, proteins) to the electrode surface. Active field compensation can be used to ensure a consistent electric field strength is applied to cells across multiple cell batches. This can alleviate the need for electrode surface modification to reduce passivation.

[0254]

[0359] FIG. 75 is a circuit diagram of a resistor divider network for an electroporation process 7500. For example, the voltage V chip The set of cells can be placed in an electroporation chamber 7510 to which a fluid resistance R can be applied. bcorresponds to the fluid (e.g., cell mixture) resistance. Assuming uniform cell distribution and assuming that electroporation is performed on each fluid batch of the same volume, the fluid resistance R b must be consistent. R i corresponds to the resistance between the fluid and the electrode, which increases over time throughout the electroporation process. In a conventional electroporation process, V ps is constant. However, R i increases over time, the voltage applied to the fluid decreases over time, resulting in a decrease in the electric field strength.

[0255]

[0360] Fluid resistance R b Due to the variations in , and the small number of pulses that can be applied, interpolation to compensate for the reduction in field strength may not accurately compensate for electrode passivation.

[0256]

[0361] In some variations, a method for electroporating cells may include receiving a first fluid containing cells into a fluid conduit, applying a resistance measurement signal to the first fluid using an electrode set, measuring the resistance between the first fluid and the electrode set, and applying an electroporation signal to the first fluid based on the measured resistance. In some variations, a second fluid containing a gas may be received into 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.

[0257]

[0362] Figures 76A through 76D are plots 7600, 7602, 7604, and 7606 of measurement and electroporation waveforms. Figure 76A shows a first resistance measurement pulse 7620 with a low voltage and wide pulse width. Figure 76B shows a second resistance measurement pulse 7622 with a high voltage and narrow pulse width. Figure 76C shows a third resistance measurement pulse 7624 with a continuous low voltage waveform for monitoring impedance changes continuously over time. Figure 76D shows a fourth resistance measurement pulse 7626 with a low AC voltage waveform for monitoring impedance changes continuously over time. Each resistance measurement pulse reduces the voltage and / or pulse width to avoid inducing electroporation in the cells. By monitoring the voltage and current of the applied resistance measurement pulses, changes in resistance can be measured and the electroporation pulses applied to the cell batch can be compensated accordingly.

[0258]

[0363] In some variations, the electroporation signal can include about 1 pulse to about 50 pulses, the voltage can be about 100 V to about 700 V, the pulse width can be about 100 μs to about 1 ms, the pulse interval can be about 5 seconds to about 30 seconds, the resistance pulse voltage can be about 10 V to about 40 V, and the resistance pulse width can be about 10 μs to about 50 μs.

[0259]

[0364] For example, an 8-batch electroporation run can receive one electroporation pulse per batch. Each electroporation pulse can have an electric field strength of about 0.5 kV / cm to about 2.0 kV / cm. The resistance measurement pulse applied before each batch can have an electric field strength of less than about 0.2 kV / cm so that the resistance measurement pulse does not induce electroporation.

[0260] Sterile Liquid Transfer Device

[0365] Generally, the sterile fluid transfer devices described herein can be configured to store fluids for transfer to another component of a cell processing system, such as a cartridge or bioreactor. In some variations, the sterile fluid transfer device can include a portable consumable configured to be moved using a robot. For example, a robot can be configured to move the sterile fluid transfer device from a reagent storage container to an ISO7 space for a sterile fluid transfer fixture within the cell processing system. The sterile fluid transfer device enables automated, sterile, and quantitative fluid transfer for automating cell therapy manufacturing.

[0261]

[0366] 103A and 103B are perspective views of a sterile fluid transfer device 10300 including a fluid cavity 10310 (e.g., container, vessel), a fluid connector 10320 (e.g., fluid connector), and a pump 10330. Fluid stored in the fluid cavity 10310 can be transferred into and out of the sterile fluid transfer device 10300 via the fluid connector 10320 using the pump 10330. In some variations, the sterile fluid transfer device 10300 can include an engagement mechanism 10340 (e.g., a robot mount) to facilitate robotic arm control.

[0262] Fluid Connector

[0367] Generally, the sterile fluid connectors described herein form a sterile fluid pathway between at least two fluidic devices, enabling fluid transfer that can be at least one of sterile fluid transfer, fully automated fluid transfer, and precisely metered fluid transfer (e.g., precise control of fluid volume transferred). In some variations, a robot can be configured to couple the fluid connector between at least two of the plurality of instruments and one or more cartridges. In some variations, the robot can be configured to operate a fluid controller to open and close a set of ports and valves in the fluid connector. Using a robot and a controller to operate the fluid connector can facilitate automation and sterility of a cell processing system.

[0263]

[0368] In some variations, a system may include a robot configured to operate a fluid connector as described herein and a controller including a memory and a processor. The controller may be coupled to the robot. The controller may be configured to generate a port signal to couple a first port to a second port using the robot arm, generate a first valve signal to translate a first valve relative to a second valve using the robot arm, and generate a second valve signal to transition the first valve and the second valve to an open configuration.

[0264]

[0369] In some variations, a fluid pump can be coupled to the sterilant source, and the controller can be configured to generate a first fluid signal to circulate fluid into the chamber through the sterilant port, the controller can be configured to generate a second fluid signal to circulate sterilant into the chamber through the sterilant port to at least sterilize the chamber, and the controller can be configured to generate a third fluid signal to remove sterilant from the chamber.

[0265]

[0370] In some variations, the controller may be configured to generate a port signal to couple the first port to the second port using the 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.

[0266]

[0371] The fluid connector can also accommodate multiple connection cycles in a sterile system and can be controlled without human intervention. For example, the fluid connector can include one or more of an engagement mechanism to facilitate robotic arm control and an alignment mechanism to ensure proper connection between the connector components. FIG. 15 is a block diagram of an exemplary variation of a fluid connector system 1500 including a fluid connector 1510, a first fluidic device 1520, a second fluidic device 1522, a sterilant source 1530, a fluid source 1532, a robot (e.g., a robotic arm) 1540, and a controller 1550. The fluid connector 1510 can be removably coupled (e.g., connected and / or disconnected, attached / detached) to each of the first fluidic device 1520, the second fluidic device 1522, the sterilant source 1532, the fluid source 1532, and the robot 1540. In some variations, the fluidic device can include one or more of a cartridge and a sterile fluid transfer device. For example, the sterile fluid transfer device can be in fluid communication with the cartridge via the fluid connector. As described in detail herein, the separate portions (e.g., male connector, female connector) of the fluid connector 1510 can be removably coupled to one another. The robot 1540 can be configured to physically manipulate (e.g., removably couple) one or more of the fluid connector 1510, the first fluidic device 1520, the second fluidic device 1522, the sterilant source 1530, and the fluid source 1532 in a predetermined manner. For example, the robot 1540 can connect the fluid connector 1510 between the first fluidic device 1520 and the second fluidic device 1522. The robot 1540 can also connect the sterilant source 1530 and / or the fluid source 1532 to a sterilant port of the fluid connector 1510. In some variations, the robot 1540 can control one or more valves and / or ports of the fluid connector 1510 to initiate a sterilization process of one or more portions of the fluid connector 1510, for example, using a sterilant from the 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 the fluid transfer and sterilization.

[0267]

[0372] FIG. 16A is a schematic diagram of an exemplary variation of a fluid connector 1600. The fluid connector 1600 can include a lumen extending along its length. The lumen is disposed between a first fluid device 1630 and a second fluid device 1640 to allow fluid flow through the fluid connector 1600. In some variations, the first fluid device 1630 and the second fluid device 1640 can be aseptically connected and disconnected using the fluid connector 1600. The fluid devices 1630, 1640 can include closed sterile devices and can be the same or different types of fluid devices. For example, the fluid devices 1630, 1640 can include one or more of a sterile fluid transfer device and a consumable. In some variations, the fluid connector 1600 can include a first connector 1610 including a first proximal end 1612 and a first distal end 1614. The first proximal end 1612 may be configured to couple to a first fluidic device 1630. The first distal end 1614 may include a first port 1616, a first housing 1617, and a first valve 1618. The first housing 1617 may be configured to receive the first port 1616 in a closed configuration as described in detail herein.

[0268]

[0373] The fluid connector 1600 may further include a second connector 1620 including a second proximal end 1622 and a second distal end 1624. The second proximal end 1622 may be configured to couple to a second fluid device 1640. The second distal end 1624 may include a second port 1626, a second housing 1627, and a second valve 1628. The second housing 1627 may be configured to receive the second port 1626 in a closed configuration. In FIG. 16A , the first connector 1610 includes a sterilant port 1650 configured to couple to a sterilant source (not shown). Additionally or alternatively, the second connector 1620 may also include the sterilant port 1650. As described in detail herein, when the second port 1626 is coupled to the first port 1616, the sterilant port 1650 can be configured to be in fluid communication with the first distal end 1614 and the second distal end 1624.

[0269]

[0374] In some variations, the fluidic devices 1630, 1640 may include a sterilant chamber and a sterilant port configured to receive a sterilant. The sterilant chamber can house a fluidic device connector (not shown) configured to mate with the proximal end of the first connector 1610 or the second connector 1620. The fluidic devices 1630, 1640 may receive a sterilant similar to the fluidic connector 1600.

[0270]

[0375] 16B is a detailed schematic diagram of a first connector 1610 including a first port housing 1617 and a chamber 1615. The chamber 1615 may be defined by a cavity enclosed by one or more of the distal ends 1614, 1624. For example, the chamber 1615 in FIG. 16B may include a portion of the first connector 1610 between the first valve 1618 and the first port 1616 in the closed configuration (e.g., the first distal end 1614). In some variations, the first chamber 1615 is approximately 1 cm 3 ~about 5cm 3 When the first connector 1610 is coupled to the second connector 1620 and the ports 1616, 1626 are in an open configuration (e.g., as shown in FIG. 16D ), the chamber 1616 may include a portion of the fluid connector 1600 between the first valve 1618 and the second valve 1628 (e.g., the first distal end 1614 and the second distal end 1624). The chamber 1615 may include a closed volume configured to receive a fluid, such as a sterilant, from a sterilant port 1650. In some variations, the sterilant port 1650 may include an inlet 1652 and an outlet 1654. Methods of using fluid connectors are described in further detail in connection with FIGS. 16C-16L and 27.

[0271]

[0376] In some variations, the fluid connector 1600 may include one or more alignment and robotic engagement features configured to facilitate robotic manipulation, as detailed herein. In some variations, the fluid connector 1600 may be connected to one or more sensors, pumps, and valves to facilitate fluid transfer and monitoring.

[0272]

[0377] In some variations, the components of the fluidic connector in contact with the fluid can be USP Class VI compliant for cell processing and / or GMP applications. In some variations, the components of the fluidic connector can be constructed from materials including, but not limited to, cyclic olefin copolymer (COC), polychlorotrifluoroethylene, polyetherimide, polysulfone, polystyrene, polycarbonate, polypropylene, silicone, polyetheretherketone, polymethylmethacrylate, nylon, acrylic, polyvinyl chloride, vinyl, phenolic resins, petroleum-based polymers, glass, polyethylene terephthalate, metals, stainless steel, titanium, aluminum, cobalt chromium, chromium, silicates, glasses, alloys, ceramics, carbohydrate polymers, mineraloids, and combinations or composites thereof.

[0273]

[0378] Figures 17A through 18D show exterior and interior views of variations of the fluid connector. Figure 17A shows a front perspective view of a fluid connector 1700 in a closed port configuration. Figure 17B shows a rear perspective view of the fluid connector 1700, and Figure 17C shows a rear view. In general, the fluid connector may include multiple internal seals to reduce contamination and aid in sterilization, and alignment features to aid in proper alignment of the fluid connector components.

[0274]

[0379] The fluid connector 1700 may include a lumen extending along its length. In some variations, the fluid connector 1700 may comprise a first connector 1710 including a first proximal end 1712 and a first distal end 1714. The first proximal end 1712 may be configured to couple to a first fluid device (not shown for clarity). The first proximal end 1712 may 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. In FIG. 17A , the first housing 1717 is shown holding the first port 1716 in a closed configuration. The first connector 1710 further includes sterilant ports 1750, 1752 configured to couple to a sterilant source (not shown for clarity). In some variations, the sterilant ports 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 within the sterilant port when not connected to a robot or actuator. The first connector 1710 may include a first alignment feature 1760, such as a set of protrusions on the first distal end 1714 of the first connector 1710. The alignment feature can ensure that small positioning errors due to robotic manipulation do not affect the operation of the fluid connector.

[0275]

[0380] The fluid connector 1700 may further comprise a second connector 1720 including a second proximal end 1722 and a second distal end 1724. The second proximal end 1722 may be configured to couple to a second fluid device (not shown for clarity). The second proximal end 1722 may include a Luer connector or any other suitable connector. The second distal end 1724 may include a second port 1726 and a second housing 1727. In FIG. 17A , the second housing 1727 is shown holding the second port 1726 in a closed configuration. The second connector 1720 may further include a second alignment feature 1762, such as a hole set 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-rotational configuration to assist in mating of the first connector 1710 and the second connector 1720 .

[0276]

[0381] The first port 1716 and the second port 1726, which are respectively held within a first housing 1717 at the first distal end 1714 and a second housing 1727 at the second distal end 1724, facilitate robotic control as the ports 1716, 1726 are not separable from the fluid connector 1700, thus reducing the risk of failure of automated robotic operation.

[0277]

[0382] In some variations, the first connector 1710 may include a first robotic engagement mechanism 1770, and the second connector 1720 may include a second robotic engagement mechanism 1772. The robotic engagement mechanisms 1770, 1772 may be configured to be operated by a robot (e.g., robot 1540), such as a robotic arm. In some variations, as shown in FIGS. 17A-17F , the robotic engagement mechanisms 1770, 1772 may be operably coupled to the first port 1716 and the second port 1726, respectively, and configured to actuate the ports 1716, 1726 into closed and open port configurations. Additionally or alternatively, a user may manually actuate the robotic engagement mechanisms 1770, 1772 to actuate each port 1716, 1726.

[0278]

[0383] Figure 17D is a front perspective view of the fluid connector 1700 in an open port configuration. Figure 17E is a rear perspective view of the fluid connector 1700 in an open port configuration, and Figure 17F is a rear view. In the open port configuration, the first valve 1718 of the first connector 1710 and the second valve 1728 of the second connector 1720 are shown in Figure 17D.

[0279]

[0384] 18A is a side view and FIG. 18B is a cross-sectional side view of a fluid connector 1800 in a disconnected configuration. In some variations, the fluid connector 1800 can include a first connector 1810 including 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) on a second connector 1820. The fluid connector 1800 can include a second connector 1820 including a second housing 1827 including a second port 1826. The first connector 1810 and the second connector 1820 can be axially aligned, and the alignment feature can assist in rotational alignment of the first connector 1810 with respect to the second connector 1820. The first valve 1818 may include a first valve stem 1819 and the second valve 1828 may include a second valve stem 1829 .

[0280]

[0385] 18C is a side view and FIG. 18D is a side cross-sectional view of the fluid connector 1800 with the first housing 1817 and second housing 527 together in a coupled configuration, but with the first connector 1810 and second connector 1820 not in fluid communication because the first port 1816 and second port 1826 are both in a closed configuration. A first alignment feature on each connector 1810, 1820 can be configured to ensure axial and / or rotational alignment of the first connector 1810 and second connector 1820.

[0281]

[0386] Figure 18E is a side view of fluid connector 1800 in an open port configuration, and Figure 18F is a cross-sectional side view. First port 1817 and second port 1827 each transition from a closed configuration to an open configuration. This creates a closed interior volume within the distal end of each connector 1810, 1820. Because first valve 1818 and second valve 1828 are each in a closed configuration, fluid flow between first connector 1810 and second connector 1820 is blocked and is restricted in each half by the automatic shut-off valves on either side.

[0282]

[0387] FIG. 18G is a side view and FIG. 18H is a cross-sectional side view of the fluid connector 1800 in an open valve configuration, in which the first valve 1818 is coupled to the second valve 1828. For example, the second valve 1828 can be translated toward the first valve 1818 along the longitudinal axis of the second connector 1820. As shown in FIGS. 18G and 18H, the second connector 1820 can be axially compressed to translate the second valve 1828 toward the first valve 1818. The first valve 1818 coupled to the second valve 1828 forms a radial seal, and the first valve stem 1819 and the second valve stem 1829 contact to allow fluid communication between the first connector 1810 and the second connector 1820.

[0283]

[0388] 19 through 26B are schematic diagrams of variations of a fluid connector system for coupling fluidic devices. In some variations, the fluidic connector may include a first connector configured to couple to any one of a plurality of second connectors. FIG. 19 is a schematic diagram of an exemplary variation of a fluidic connector system 1900 including a first connector 1910, a plurality of second connectors 1920, 1921, 1922, a first fluidic device 1930 (e.g., a sterile fluid transfer device), a second fluidic device 1940 (e.g., a consumable), and a robot 1960 (e.g., a robotic arm, a 3DOF robot). The first connector 1910 may be coupled in fluid communication with the first fluidic device 1930, and the second connectors 1920, 1921, 1922 may be coupled in fluid communication with the second fluidic device 1940. The first connector 1910 and the second connectors 1920, 1921, 1922 may each include a port 1916 configured to mate with 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.

[0284]

[0389] In some variations, to facilitate an efficient shared fluid connection between the fluidic device, the fluid connector, and the sterilization system, the robot 1960 may be configured to couple to one or more of a sterilant source, a fluid source, and a pump. For example, FIG. 96A is a plan view of a fluidic device 9600 (e.g., a sterilization fluid transfer device) including a fluid port 9610 configured to couple to a fluid source (not shown) and a sterilization port 9620 configured to couple to a sterilant source (not shown). FIGS. 96B and 96C are side and perspective views, respectively, of the fluidic device 9600 coupled to 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.

[0285]

[0390] In some variations, the fluid connector may include a third connector disposed between the first and second connectors. Figure 20A is a schematic diagram of an exemplary variation of a fluid connector system 2000 including a first connector 2010, a plurality of second connectors 2020, 2021, 2022, a third connector 2070 (e.g., an instrument, a sterile enclosure), a first fluidic device 2030 (e.g., a sterile fluid transfer device), a second fluidic device 2040 (e.g., a consumable), and a robot 2060 (e.g., a robotic arm, a 3DOF robot, a 1DOF robot). The first connector 2010 may be coupled in fluid communication with the first fluidic device 2030, and the second connectors 2020, 2021, 2022 may be coupled in fluid communication with the second fluidic device 2040. The third connector 2070 may be coupled between the first connector 2010 and one of the second connectors 2020, 2021, 2022. The third connector 2070 may include a lumen configured to receive and circulate a sterilant through one or more of the first connector 2010, the second connectors 2020, 2021, 2022, and the third connector 2070. In some variations, one or more of the first fluidic device 2030 and the first connector 2010 may be simplified by non-removably coupling the sterilization port 2052 to a sterilant source and / or a fluid source.

[0286]

[0391] The robot 2060 may include one or more end effectors 2062, 2064, 2066 configured to manipulate and / or couple to one or more of the first fluidic device 2030, the first connector 2010, and the third connector 2070. For example, the first fluidic device 2030 may include one or more fluid ports 2050 configured to couple to the end effector 2062. Similarly, the third connector 2070 may include one or more sterilization ports 2052 configured to couple to the robot 2060 (e.g., the end effector 2064). In some variations, the robot 2060 may be configured to couple to one or more of a sterilant source, a fluid source, and a pump to facilitate an efficient shared fluid connection between the fluidic devices, the fluid connectors, and the sterilization system.

[0287]

[0392] 20B and 20C are schematic diagrams of the fluid connector connection process. In FIG. 20B, at 2002, a 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 couple the second connector 2020 directly to the first connector 2010.

[0288]

[0393] 20C , at 2002, a third connector 2070 can be coupled to the distal end of the first connector 2010 and the distal end of the second connector 2020. At 2005, the first connector 2010 and the second connector 2020 can each be translated relative to one another through the third connector 2070. At 2007, the second connector 2020 can be further translated toward the first connector 2010 to directly couple the first connector 2010 to the second connector 2010. FIG. 20C further illustrates that the first port 2090 and the second port 2092 can transition between a closed port configuration and an open port configuration.

[0289]

[0394] 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 is a block diagram of an exemplary variation of a fluid connector system 2100 including a first connector 2110, a plurality of second connectors 2120, 2121, 2122, a third connector 2170 (e.g., an instrument, a sterile enclosure), a first fluidic device 2130 (e.g., a sterile fluid transfer device), a second fluidic device 2140 (e.g., a consumable), a first robot 2160, and a second robot 2166. The first connector 2110 may be coupled in fluid communication with the first fluidic device 2130, and the second connectors 2120, 2121, 2122 may be coupled in fluid communication with the second fluidic device 2140. The third connector 2170 may be coupled between the first connector 2110 and one of the second connectors 2120, 2121, 2122. The third connector 2170 may include a lumen configured to receive and circulate a sterilant through one or more portions of the first connector 2110, the second connectors 2120, 2121, 2122, and the third connector 2170. In some variations, one or more of the first fluidic device 2130 and the first connector 2110 may be simplified by non-removably coupling the third connector 2170 to a sterilant source and / or a fluid source.

[0290]

[0395] 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 an efficient shared fluid connection between the fluidic device, the fluid connector, and the sterilization system.

[0291]

[0396] In some variations, the fluid connector may include a sterilant source coupled to a plurality of second connectors. Figure 22 is a block diagram of an exemplary variation of a fluid connector system 2200 including a first connector 2210, a plurality of second connectors 2220, 2221, 2222, a first fluidic device 2230 (e.g., a sterilant transfer device), a second fluidic device 2240 (e.g., a consumable), a robot 2260, a sterilant source 2290 including one or more valves, and a sterilant switch 2292. The first connector 2210 may be coupled in fluid communication with the first fluidic device 2230, and the second connectors 2220, 2221, 2222 may be coupled in fluid communication with the second fluidic device 2240. The robot 2260 may include one or more end effectors 2262, 2264 configured to manipulate and / or couple to one or more of the first fluidic device 2230 and the first connector 2210. For example, the first fluidic device 2230 may include one or more fluid ports 2250 configured to couple to the end effector 2262. In some variations, the sterilant source 2290 may be coupled to a switch 2292. To facilitate an efficient shared fluid connection between the fluidic device, the fluid connector, and the sterilization system, the switch 2292 may be coupled to each of the second connectors 2220, 2221, 2222. In some variations, a sterilant conduit may be routed from the switch 2292 through the second fluidic device 2240 to each of the second connectors 2220, 2221, 2222.

[0292]

[0397] 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 exemplary variation of a fluidic connector system. Figure 23 is a block diagram of an exemplary variation of a fluidic connector system 2300 including a first connector 2310, a plurality of second connectors 2320, 2321, 2322, a first fluidic device 2330 (e.g., a sterilant liquid transfer device), a second fluidic device 2340 (e.g., a consumable), a robot 2360, a sterilant valve set 2390 disposed within the housing of the second fluidic device 2340, and a sterilant switch 2392. The first connector 2310 may be coupled in fluid communication with the first fluidic device 2330, and the second connectors 2320, 2321, 2322 may be coupled in fluid communication with the second fluidic device 2340. The robot 2360 may include one or more end effectors 2362, 2364 configured to manipulate and / or couple to one or more of the first fluidic device 2330 and the first connector 2310. For example, the first fluidic device 2330 may include one or more fluid ports 2350 configured to couple to the end effector 2362. In some variations, the sterilant valve 2390 may be coupled to the switch 2392. To facilitate an efficient shared fluid connection between the fluidic device, the fluid connector, and the sterilization system, the switch 2392 may be coupled to each of the second connectors 2320, 2321, 2322 via the sterilant valve 2390. In some variations, a sterilant conduit may be routed from the switch 2392 through the second fluidic device 2340 to each of the second connectors 2320, 2321, 2322.

[0293]

[0398] In some variations, the fluid connector can include a sterilant source coupled to a plurality of second connectors, each of which can have a sterilant port (e.g., a sterilant valve) and a sterilant conduit through the fluidic device. Figure 24A is a block diagram of an exemplary variation of a fluid connector system 2400 including a first connector 2410, a plurality of second connectors 2420, 2421, 2422, a first fluidic device 2430 (e.g., a sterilant transfer device), a second fluidic device 2440 (e.g., a consumable), 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 may include one or more end effectors 2462, 2464 configured to manipulate and / or couple to one or more of the first fluid device 2430 and the first connector 2410. For example, the first fluid device 2430 may include one or more fluid ports 2450 configured to couple to the end effector 2462.

[0294]

[0399] In some variations, each of the second connectors 2420, 2421, 2422 may include a sterilant port 2494, 2496, 2498 that includes a valve coupled to the distal end of the second connector 2420, 2421, 2422. In some variations, a sterilant conduit may be routed from the switch 2492 through the second fluidic device 2440 to each of the sterilant ports 2494, 2496, 2498. In some variations, a sterilant source (not shown) may be coupled to the switch 2492. To facilitate an efficient shared fluid connection between the fluidic device, the fluid connector, and the sterilization system, the switch 2492 may be coupled to each of the second connectors 2420, 2421, 2422 via the sterilant ports 2494, 2496, 2498.

[0295]

[0400] Figure 24B is a schematic diagram of the fluid connector connection process 2402, 2404, 2406 when a first connector 2410 is mated 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 the sterilant valve 2494. In some variations, when the first connector 2410 is mated to the second connector 2420 at 2404 and 2406, the valve 2494 may transition to an open valve configuration.

[0296]

[0401] In some variations, the plurality of second connectors may include one or more pneumatic sterilant valves and a sterilant pathway through the fluidic device. Figure 25A is a block diagram of an exemplary variation of a fluid connector system 2500 including a first connector 2510, a plurality of second connectors 2520, 2521, 2522, a first fluidic device 2530 (e.g., a sterilant transfer device), a second fluidic device 2540 (e.g., a consumable), a robot 2560, and a sterilant switch 2592 coupled to a sterilant source (not shown). The first connector 2510 may be coupled in fluid communication with the first fluidic device 2530, and the second connectors 2520, 2521, 2522 may be coupled in fluid communication with the second fluidic device 2540.

[0297]

[0402] In some variations, each of the second connectors 2520, 2521, 2522 may include a pneumatic sterilant port 2594, 2596, 2598 that includes a valve coupled to the distal end of the second connector 2520, 2521, 2522. In some variations, a sterilant conduit may be routed from the switch 2592 through the second fluidic device 2540 to each of the sterilant ports 2594, 2596, 2598. In some variations, a sterilant source (not shown) may be coupled to the switch 2592. To facilitate an efficient shared fluid connection between the fluidic device, the fluid connectors, and the sterilization system, the switch 2592 may be coupled to each of the second connectors 2520, 2521, 2522 via the sterilant ports 2594, 2596, 2598.

[0298]

[0403] 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 fluidic device 2530, the first connector 2510, and the sterilant ports 2594, 2596, 2598. For example, the first fluidic 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 for pneumatic actuation of the sterilant ports 2594, 2596, 2598. Pneumatic actuation of the sterilant ports may allow for a reduced number of check valves between the sterilant ports 2594, 2596, 2598 and the switch 2592 to form the sterilant conduits.

[0299]

[0404] Figure 25B is a schematic diagram of the fluid connector connection process 2502 and 2504 when a first connector 2510 is mated 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 25C is a detailed schematic diagram of the sterilant valve 2594. In some variations, when the first connector 2510 is mated to the second connector 2520 in 2504 and the valve 2594 is pneumatically actuated, the valve 2594 may transition to an open valve configuration.

[0300] Liquid Transfer Bath

[0405] Generally, the modules of the cartridge can be fluidly coupled to one another directly or via one or more fluid transfer buses to allow one or more of the following to be transferred between the modules: cellular product (i.e., one or more solutions containing the cellular product), fluids, and reagents. In some variations, the fluid transfer buses can include portions of the cartridge configured to control the flow and distribution of the cellular product between the modules and reservoirs. The fluid transfer buses can include one or more of a fluid manifold, fluid conduits (e.g., tubing), one or more valves (including, but not limited to, one or more of a 2 / 2 valve, a 3 / 2 valve, a 3 / 3 valve, a 4 / 2 valve, and a rotary selector valve).

[0301]

[0406] To transfer cell products, reagents, or fluids within the cartridge, any pump or other structure can generate a pressure differential between fluids in one portion of the cartridge and fluids in another portion of the cartridge. For example, the cartridge can include one or more pumps, be pre-inserted with pressurized fluid contained behind a valve, and be connected to a fluid source or fluid sink. The cartridge can include one or more mechanical pumps (e.g., linear pumps, peristaltic pumps, gear pumps, screw pumps, plunger pumps) or multiple portions of a single pump (i.e., a pump can be coupled with a pump actuator). External pressure can be applied to the cartridge, tubing within the cartridge, or pouches within the cartridge (i.e., applying pressure to either the liquid within the pouch or the pouch's headspace gas). In some variations, the arrangement of cartridge components can facilitate gravity-based fluid transfer within the cartridge (e.g., gravity-fed pumping). While one advantage of the disclosed variations is that they may reduce operator intervention, the disclosed systems and methods can also employ manual operation within a designed workflow or as a supplement to automated operation in the event of imperfect system operation. For example, some process steps may include manual intervention, such as the input or output of a fluid. An operator can intervene in an automated process to correct device operation (e.g., manually compressing a bag to flush remaining fluid through the system). Fluids can include liquids and / or gases, and compressed gas provided externally or within a pressurized chamber can be used to generate liquid flow, such as the transfer of a solution containing a cell product, from one module to another.

[0302]

[0407] In some variations, the fluid transfer bus may be configured to deliver one or more cell products to each of a series of modules in an order set by the cartridge design or determined by operation of the system by one or more processors. Similarly, some cartridge variations may have the advantage that the order of cell processing steps and process parameters of cell therapy processing steps are controlled by a controller rather than set by the cartridge. In some variations, the fluid transfer bus may be controlled (e.g., by configuring the state of one or more valves attached to the fluid bus) to deliver cell products to modules in any of a variety of sequences or to bypass one or more modules. In some variations, a module may be used more than once in 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 may include 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 cultivation step, washing the CCE module with a washing solution, and transferring the cell product to the CCE module for a second enrichment step.

[0303]

[0408] In some variations, one or more fluid transfer buses may be fluidly coupled to multiple bags or reservoirs used to provide solutions or reagents, store cellular products, or collect waste fluids or reagents.

[0304]

[0409] In some variations, the cartridge may include one or more pumps that can be fluidly coupled to the fluid transfer bus and / or one or more modules. The one or more pumps may include a motor operably coupled to control the circuitry and a power source (e.g., a battery or an electrical connector for a power source external to the cartridge). In some variations, the pump may be split between a pump on the cartridge and a pump actuator on one or more instruments of the system. The pump may be an opening in the cartridge, around which tubing is disposed and configured to receive a pump actuator (e.g., a peristaltic rotor). Separating the pump components that contact the cellular product (e.g., tubing) from the pump components that perform the actions of the cellular product (i.e., a pump actuator, such as a peristaltic rotor) can simplify the cartridge's compactness. For example, Figures 26A and 26B show the pump head 2610 and pump 2610 of the cartridge in a separated configuration (Figure 13A) and a combined configuration.

[0305]

[0410] In some variations, one or more pumps 146 (e.g., fluid pumps) may generate a predetermined fluid flow rate for circulating the sterilant and / or fluid. In some variations, the pumps may include one or more positive displacement pumps (e.g., peristaltic pumps, diaphragm pumps, syringe pumps), centrifugal pumps, combinations thereof, etc. One or more fluid sources may be coupled to the pumps.

[0306]

[0411] In some variations, the pump may be configured to receive a pump signal (generated by the controller) configured to circulate the sterilant for a dwell time sufficient to sterilize at least a portion of the fluid connector. For example, the pump may be configured to circulate the sterilant for at least 10 seconds. In some variations, the pump may be configured to receive a pump signal configured to circulate a non-sterilant gas (e.g., an inert gas, air) to remove the sterilant.

[0307]

[0412] In some variations, in a discontinuous flow pump (e.g., a peristaltic pump), peristaltic flow can be generated, for example, when a tube contacts and releases between rollers. In some variations, closed-loop feedback from a flow sensor can be used to compensate for the peristaltic flow to generate a substantially continuous flow. For example, a flow sensor can be coupled to a fluid line to measure the flow rate. A controller can receive the measured flow rate and generate a pump signal for the pump based on a proportional correction function configured to reduce the "ripple" measured by the flow sensor. Additionally or alternatively, the controller may perform cyclic error correction on the pump signal to reduce cyclic errors that may be unique to each pump. For example, the flow sensor can measure and determine the cyclic error of the pump. A pump signal including cyclic error correction can correspond to a waveform including the inverse of the error. The resulting pump flow can compensate for fluctuations in the flow rate.

[0308] controller

[0413] In some variations, 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. Controller 120 may be configured to control (e.g., operate) work cell 110. Controller 120 may include multiple devices. For example, work cell 110 may house one or more components of controller 120 (e.g., processor 122, memory 124, communication device 126), while one or more components of controller 120 (e.g., input device 128, display 130) may be provided remotely from work cell 110.

[0309] Processor

[0414] A processor described herein (e.g., processor 122) can process data and / or other signals to control one or more components of a system (e.g., workcell 110, controller 120). A processor may be configured to receive, process, compile, calculate, store, access, read, write, and / or transmit data and / or other signals. Additionally or alternatively, a processor may be configured to control one or more components of a device and / or one or more components of a controller (e.g., a console, touchscreen, personal computer, laptop, tablet, server).

[0310]

[0415] In some variations, the processor may be configured to access or receive data and / or other signals from one or more of the workcell 110, the server, the controller 120, and a storage medium (e.g., memory, flash device, memory card, database). In some variations, the processor may be any suitable processing device configured to operate and / or execute an instruction set or code, and may include one or more data processors, image processors, graphics processing units (GPUs), physical processing units, digital signal processors (DSPs), analog signal processors, mixed-signal processors, machine learning processors, deep learning processors, finite state machines (FSMs), compression processors (e.g., data compression to reduce data rates and / or memory requirements), encryption processors (e.g., for secure wireless data transfer), and / or central processing units (CPUs). The processor may be, for example, a general-purpose processor, a field programmable gate array (FPGA), an application specific integrated circuit (ASIC), a processor board, etc. The processor may be configured to run and / or execute application processes and / or other modules, processes, and / or functions associated with the system.The underlying device technology can come in a wide variety of component types (e.g., metal-oxide semiconductor field-effect transistor (MOSFET) technologies such as complementary metal-oxide semiconductor (CMOS), bipolar technologies such as emitter-coupled logic (ECL), polymer technologies (e.g., silicon-conjugated polymer and metal-conjugated polymer-metal structures), mixed analog and digital, etc.).

[0311]

[0416] The systems, devices, and / or methods described herein may be implemented by software (executed on hardware), hardware, or a combination thereof. Hardware modules may include, for example, general-purpose processors (or microprocessors or microcontrollers), field-programmable gate arrays (FPGAs), and / or application-specific integrated circuits (ASICs). Software modules (executed on hardware) may be expressed in a variety of software languages ​​(e.g., computer code), including structured text, typed documents, C, C++, C#, Java, Python, Ruby, Visual Basic, and / or other object-oriented, procedural, or other programming languages ​​and development tools. Examples of computer code include, but are not limited to, microcode or microinstructions, machine instructions such as those generated by a compiler, code used to create Web services, and files containing high-level instructions executed by a computer using an interpreter. Additional examples of computer code include, but are not limited to, control signals, encryption code, and compression code.

[0312] memory

[0417] The cell processing systems and devices described herein may include memory (e.g., memory 124) configured to store data and / or information. In some variations, the memory may include one or more of random access memory (RAM), static RAM (SRAM), dynamic RAM (DRAM), memory buffer, erasable programmable read-only memory (EPROM), electrically erasable read-only memory (EEPROM), read-only memory (ROM), flash memory, volatile memory, non-volatile memory, combinations thereof, etc. In some variations, the memory may store instructions for causing a processor to perform modules, processes, and / or functions associated with the device, such as image processing, image display, sensor data, data and / or signal transmission, data and / or signal reception, and / or communications, etc. Some variations described herein may relate to computer storage products with a non-transitory computer-readable medium (which may also be referred to as a non-transitory processor-readable medium) having instructions or computer code for performing various computer-implemented operations. A computer-readable medium (or processor-readable medium) is non-transitory in that it does 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 (which may also be referred to as code or an algorithm) may be designed and constructed for one or more specific purposes. In some variations, a memory may be configured to store received data and / or data generated by a controller and / or workcell. In some variations, a memory may be configured to store data temporarily or permanently.

[0313] Input devices

[0418] In some variations, the display may include and / or be operatively coupled to an input device 128 (e.g., a touchscreen) configured to receive input data from a user. For example, user input to the input device 128 (e.g., a keyboard, buttons, touchscreen) may be received and processed by a processor (e.g., processor 122) and memory (e.g., memory 124) of the system 100. The input device may include at least one switch configured to generate a user input. For example, the input device may include a touch surface upon which a user may input corresponding to the user input (e.g., touching a finger against the touchscreen). An input device including a touch surface may be configured to detect contact and movement on the touch surface using any of a number of touch-sensitivity technologies, including capacitive, resistive, infrared, optical imaging, dispersive signal, acoustic pulse recognition, and surface acoustic wave technologies. In variations of input devices that include at least one switch, the switch may comprise, for example, at least one of a button (e.g., hard key, soft key), a touch surface, a keyboard, an analog stick (e.g., a joystick), a directional pad, a mouse, a trackball, a jog dial, a step switch, a rocker switch, a pointer device (e.g., a stylus), a motion sensor, an image sensor, and a microphone. The motion sensor can receive user movement data from an optical sensor and classify user gestures as user input. The microphone can receive audio data and recognize the user's voice as user input.

[0314]

[0419] In some variations, the cell processing system may optionally include one or more output devices, such as an acoustic device and a tactile device, in addition to a display. The acoustic device may output any system data, alarms, and / or notifications audibly. For example, the acoustic device may output an audible alarm if a malfunction is detected. In some variations, the acoustic device may include at least one of a speaker, a piezoelectric acoustic device, a magnetostrictive speaker, and / or a digital speaker. In some variations, a user may communicate with other users using the acoustic device and a communication channel. For example, a user may form an acoustic communication channel (e.g., a VoIP phone call).

[0315]

[0420] Additionally or alternatively, the system may include a haptic device configured to provide additional sensory output (e.g., force feedback) to the user. For example, the haptic device may generate a haptic response (e.g., vibration) to confirm a user input to an input device (e.g., a touch surface). As another example, the haptic feedback may indicate that the user input is overridden by the processor.

[0316] communication devices

[0421] In some variations, a controller may include a communications device (e.g., communications device 126) configured to communicate with other controllers and one or more databases. The communications device may be configured to connect the controller to another system (e.g., the Internet, a remote server, a database, a workcell) via a wired or wireless connection. In some variations, the system may be in communication with other devices over one or more wired and / or wireless networks. In some variations, the communications device may include a radio frequency receiver, transmitter, and / or optical (e.g., infrared) receiver and transmitter configured to communicate with one or more devices and / or networks. The communications device may communicate wired and / or wirelessly.

[0317]

[0422] A communication device may include RF circuitry configured to receive and transmit RF signals. RF circuitry converts between electrical and electromagnetic signals and can communicate with communication networks and other communication devices via electromagnetic signals. RF circuitry may include well-known circuits for performing these functions. These circuits may include, but are not limited to, an antenna system, an RF transceiver, one or more amplifiers, a tuner, one or more oscillators, a digital signal processor, a codec chipset, a subscriber identity module (SIM) card, memory, etc.

[0318]

[0423] Wireless communication through any of these devices may use any of a number of communication standards, protocols, and technologies.These include, but are 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-HSPADA), 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 802.11n, etc.), and voice over Internet Protocol (VoIP). Protocol), Wi-MAX, protocols for email (e.g., Internet message access protocol (IMAP) and / or post office protocol (POP)), instant messaging (e.g., extensible messaging and presence protocol (XMPP), Session Initiation Protocol for Instant Messaging and Presence Leveraging Extensions (SIMPLE), Instant Messaging and Presence Service (IMPS)), and / or short message service (SMS), EtherCAT, OPC Unified Architecture (OPC UA), or any other suitable communication protocol.In some variations, devices herein may communicate directly with each other (eg, via NFC, Bluetooth, WiFi, RFID, etc.) without transmitting data over a network.

[0319]

[0424] In some variations, the systems, devices, and methods described herein may be in communication with other wireless devices via one or more networks, each of which may be any type of network (e.g., wired network, wireless network). This communication 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 communication 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 interface with the Internet, other carrier voice and data networks, business networks, and personal networks. Wired networks are typically carried on copper twisted pair wires, coaxial cable, and / or fiber optic cable. Many different types of wired networks exist, including wide area networks (WANs), metropolitan area networks (MANs), local area networks (LANs), Internet area networks (IANs), campus area networks (CANs), global area networks (GANs) such as the Internet, and virtual private networks (VPNs). Hereinafter, a network refers to any combination of wireless, wireline, public, and private data networks that are interconnected, typically via the Internet, to provide an integrated networking and information access system.

[0320]

[0425] Cellular communications can encompass technologies such as GSM, PCS, CDMA or GPRS, W-CDMA, EDGE or CDMA2000, LTE, WiMAX, and 5G networking standards. Some wireless network deployments combine networks from multiple cellular networks or use a mix of cellular, Wi-Fi, and satellite communications.

[0321] display

[0426] The image data may be output to a display of the cell processing system, such as display 130. In some variations, the display may include at least one of a light emitting diode (LED), a liquid crystal display (LCD), an electroluminescent display (ELD), a plasma display panel (PDP), a thin film transistor (TFT), an organic light emitting diode (OLED), an electronic paper / electronic ink display, a laser display, and / or a holographic display.

[0322] II. Method

[0427] Generally, the systems and devices described herein can perform one or more cell processing steps to produce a cell product. FIG. 28 is a flowchart of a method of cell processing 2800. Method 2800 can include concentrating a selected cell population in a solution (e.g., a fluid) (2802). For example, the solution can be delivered to a CCE module of the cartridge via a liquid transfer bus. A robot can be operated to move the cartridge to a CCE instrument and interface the CCE module with the CCE instrument. The CCE instrument can be operated to concentrate the selected cell population in the CCE module. Additionally or alternatively, the cell product can be delivered (manually or automatically) into and out of the cartridge via a sterile liquid transfer port for any of the steps described herein. In some variations, the cartridge can be sterilized (manually or automatically) at the feed-through port.

[0323]

[0428] In some variations, the selected cell population in the solution can be washed (2804). For example, the solution can be delivered to the CCE module of the cartridge via a fluid transfer bus. A robot can be operated to move the cartridge to the CCE instrument and interface the CCE module with the CCE instrument. The CCE instrument can be operated to cause the CCE module to remove medium from the solution, add medium to the solution, and / or replace medium in the solution.

[0324]

[0429] In some variations, a cell population in a solution can be selected (2806). For example, the solution can be delivered to a selection module on the cartridge via a fluid transfer bus. A robot can be operated to move the cartridge to the selection instrument, and the selection module can be interfaced with the selection instrument. The selection instrument can be operated to cause the selection module to select the selected cell population.

[0325]

[0430] In some variations, the cell population in the solution can be sorted (2808). For example, the solution can be delivered to a sorting module in the cartridge via a fluid transfer bus. A robot can be operated to move the cartridge to the sorting instrument and interface the sorting module with the sorting instrument. The sorting instrument can be operated to cause the sorting module to sort the cell population.

[0326]

[0431] In some variations, solutions can be delivered and settled in the bioreactor module of the cartridge via a fluid transfer bus (2810). For example, a robot can be operated to move the cartridge to the bioreactor instrument and interface the bioreactor module with the bioreactor instrument. The bioreactor instrument can be operated to cause the bioreactor module to maintain the cells at a predetermined set of conditions.

[0327]

[0432] In some variations, cells can be grown in a solution (2812). For example, the solution can be delivered to a bioreactor module of the cartridge via a fluid transfer bus. A robot can be operated to move the cartridge to the bioreactor instrument and interface the bioreactor module with the bioreactor instrument. The bioreactor instrument can be operated to grow cells in the bioreactor module by cell replication.

[0328]

[0433] In some variations, the tissue can be digested by delivering an enzymatic reagent via a fluid transfer bus to a module containing a solution containing the tissue, which releases selected cell populations into the solution (2814).

[0329]

[0434] In some variations, a selected cell population in solution can be activated by delivering an activation reagent via a fluid transfer bus to a module containing the solution containing the cell product (2816).

[0330]

[0435] In some variations, a fluid transfer bus can deliver the solution to an electroporation module of the cartridge and receive an electroporation signal to electroporate cells in the solution (2818). For example, a robot can be operated to move the cartridge to an electroporation instrument and interface the electroporation module with the electroporation instrument. The electroporation instrument can be operated to cause the electroporation module to electroporate a selected cell population in the presence of the genetic material.

[0331]

[0436] In some variations, transduction of a selected cell population in solution can be performed by delivering an effective amount of vectors via a fluid transfer bus to a module containing the solution containing the cellular product (2820).

[0332]

[0437] In some variations, a formulation solution can be delivered via a fluid transfer bus to a module containing the cell product to produce a final cell product (2822). For example, the final cell product can be delivered to one or more product collection bags. In some variations, completing the cell product can include one or more of washing the cells, concentrating the cells, exchanging a cell buffer for a formulation buffer, and depositing a predetermined amount of cells in a formulation buffer into one or more product collection bags and / or containers.

[0333]

[0438] In some variations, the cell product can be manually or automatically removed from the cartridge to harvest the cells (2824).

[0334]

[0439] In some variations, the cellular product may comprise one or more of immune cells, engineered chimeric antigen receptor T cells, engineered T cell receptor (TCR) cells, hematopoietic stem cells (HSCs), and tumor infiltrating lymphocytes (TILs). In some variations, the immune cells may comprise natural killer (NK) cells.

[0335]

[0440] The cell processing method may include any suitable subset of cell processing steps in any suitable order. For example, the cell processing method may include, in order, enrichment step 2802, selection step 2806, activation step 2816, transduction step 2820, expansion step 2812, and harvesting step 2824. In some variations, the cell processing method may include, in order, enrichment step 2802, selection step 2806, settling step 2810, transduction step 2820, and harvesting step 2824. In some variations, the cell processing method may include, in order, tissue digestion step 2820, washing step 2804, activation step 2816, expansion step 2812, and harvesting step 2824.

[0336]

[0441] Generally, the methods described herein can reduce the cost of cartridges (which may be consumables) by eliminating complex steps performed in cell processing operations relative to the instruction set. In some variations, the cartridge contains the cell product (e.g., a solution containing cells) throughout the manufacturing process, and various instruments can interface with the cartridge at the appropriate time to perform one or more cell processing steps. For example, a cell processing step may include transferring cells and reagents to each module within the cartridge. The instruction set interfaced with the cartridge facilitates process flexibility, allowing a work cell to be customized with a predetermined instruction set for a given cell therapy product. For example, as detailed herein in connection with Figures 35 through 55, the order of cell processing steps can be customized for each cell product.

[0337]

[0442] In some variations, the cell product may be retained within the cartridge throughout the manufacturing process (e.g., workflow). Additionally or alternatively, the cell product may be removed from the cartridge for one or more cell processing steps, either manually by an operator or automatically via a fluidic connector (e.g., SLTP) or other port on the cartridge. The cell product can then be returned to the same cartridge, transferred to another cartridge, or split among several cartridges. In some variations, one or more cell processing steps may be performed outside of the cartridge. In some variations, processing within a work cell can facilitate sterile cell processing within the cartridge.

[0338]

[0443] 29 is a flowchart of a cell processing method illustrating cell processing steps performed on cartridges (e.g., consumables) in a work cell including a CCE instrument module, a sterile liquid transfer (SLT) instrument module, and a bioreactor instrument module. The consumables can be configured to perform one or more cell processing steps in conjunction with any of the CCE instrument module, the SLT instrument module, and the bioreactor instrument module. For example, a robot (or operator) can be configured to move cartridges between any of the modules in the work cell. Pump heads within the instrument can engage consumable cartridges to transfer fluids between modules in the cartridge, into and out of various reservoirs within the cartridge, and / or through ports that allow reagents to be added or removed from the cartridge.

[0339]

[0444] In some variations, the CCE instrument module may include a pump and a centrifuge configured to interface with the cartridge (e.g., consumables). The SLT instrument module may include one or more fluid connectors configured to interface with one or more of the cartridge pouches and the bioreactor. The bioreactor instrument module may include one or more sensors, temperature regulators, pumps, agitators, etc., and may be configured to interface with the cartridge. In some variations, the cell product may be contained within the cartridge throughout cell processing.

[0340]

[0445] The cell processing method depicted in FIG. 29 may include using a pump to move fluid (e.g., cells in solution) in a product bag to a CCE module (e.g., rotor) of a cartridge (e.g., consumable) (2910). In some variations, the CCE module may be used to concentrate the fluid (2912). For example, blood components may be collected in a waste bag (2913). In some variations, the CCE module may be used to wash the fluid (2914). For example, buffer may be collected in a waste bag (2915). In some variations, the CCE module may be used to change media (2916). For example, one or more buffers (e.g., formulation buffer) and media may be collected in a waste bag (2917). In some variations, the fluid may be moved to a bioreactor of the cartridge (2918).

[0341]

[0446] In some variations, the fluidic connector can fill the bag with reagents 2920. In some variations, reagents (e.g., beads, vectors) can be added to the bioreactor of the cartridge 2922. In some variations, the fluidic connector removes waste from the bag 2924. In some variations, the fluidic connector may optionally remove sample from the bioreactor.

[0342]

[0447] In some variations, the cells can be transferred to a bioreactor (2930). In some variations, the cells can be activated or genetically modified (2932). In some variations, the cells can be cultured (2934). In some variations, the cells can be perfused using a pump (2936). For example, spent media can be collected in a waste bag (2937). In some variations, the cells can be expanded (2938). In some variations, the cells can be harvested after a media change (2940).

[0343]

[0448] Figure 30A is a flowchart of a cell processing method for autologous CAR-T cells or genetically engineered TCR cells. Method 3000 may include the steps of enrichment, selection, activation, genetic modification, expansion, harvesting / preparation, and cryopreservation. Figure 30B is a flowchart of a cell processing method for allogeneic CAR-T cells or genetically engineered TCR cells. Method 3010 may include the steps of enrichment, activation, genetic modification (e.g., transduction, transfection), alpha / beta T cell depletion, expansion, harvesting / pooling / preparation, and cryopreservation.

[0344]

[0449] 31 is a flow chart of a method for cell processing of hematopoietic stem cell (HSC) cells. Method 3100 can include enrichment, selection, settling, genetic modification, collection / preparation, and cryopreservation steps.

[0345]

[0450] 32 is a flow chart of a cell processing method for tumor infiltrating lymphocyte (TIL) cells. Method 3200 may include the steps of tissue digestion, washing, selection, activation, expansion, harvesting / preparation, and cryopreservation.

[0346]

[0451] Figure 33 is a flow chart of a cell processing method for natural killer (NK) CAR cells. Method 3300 can include enrichment, selection, activation, genetic modification, expansion, harvesting / preparation, and cryopreservation steps.

[0347]

[0452] 34A to 34C show the controllability T (T reg 34 is a flowchart of a cell processing method for (a) 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, expansion, selection (optional), harvesting / preparation, and cryopreservation. Method 3404 may include introducing a feeder cell culture for enrichment, selection, activation / expansion, and harvesting / irradiation. Another set of cells may be subjected to enrichment, selection, co-culture with treated feeder cells, harvesting, and cryopreservation.

[0348]

[0453] 98 through 101 are flowcharts of cell processing methods for cell therapy workflows that include split (e.g., parallel) processing. Method 9800 can include enrichment, selection, activation, genetic modification, expansion, formulation, and cryopreservation steps. For example, cell processing method 9800 (e.g., workflow) can include splitting a cell product into two or more portions after an enrichment step. The split portions can be processed in parallel within a single cartridge. In some variations, one or more split portions can be transferred to two or more cartridges for parallel processing. One or more cell processing parameters (e.g., timing of process steps, types of reagents added, transfection constructs, etc.) can be configured independently for each split portion of the cell product. In some variations, the split portions can be pooled after the expansion step.

[0349]

[0454] The method 9900 may include enrichment, selection, activation, genetic modification, expansion, formulation, and cryopreservation steps. For example, the cell processing method 9900 (e.g., workflow) may include dividing the cell product into two or more portions after the activation step. The divided portions may be processed in parallel within a single cartridge. In some variations, one or more divided portions may be transferred to two or more cartridges for parallel processing. One or more cell processing parameters (e.g., timing of process steps, types of reagents added, transfection constructs, etc.) may be configured independently for each divided portion of the cell product. In some variations, the divided portions may be pooled after the expansion and / or genetic modification steps.

[0350]

[0455] Method 10000 may include enrichment, selection, activation, genetic modification, expansion, formulation, and cryopreservation steps. For example, cell processing method 10000 (e.g., workflow) may include splitting the cell product into two or more portions after a selection step. The split portions may be processed in parallel within a single cartridge. In some variations, one or more split portions may be transferred to two or more cartridges for parallel processing. One or more cell processing parameters (e.g., timing of process steps, types of reagents added, transfection constructs, etc.) may be configured independently for each split portion of the cell product. In some variations, the split portions may not be pooled.

[0351]

[0456] Method 10100 can include enrichment, selection, activation, genetic modification, expansion, formulation, and cryopreservation steps. For example, cell processing method 10100 (e.g., workflow) can include splitting a cell product into two or more portions as a starting material. The separate products can be maintained separate and processed in parallel as aliquots within a single cartridge or multiple cartridges. One or more cell processing parameters (e.g., timing of process steps, types of reagents added, transfection constructs, etc.) can be configured independently for each aliquot. In some variations, aliquots can be pooled after the expansion step.

[0352]

[0457] Figure 102 is a schematic diagram of a cell processing system 10200 configured for fractionated processing within a single cartridge. For example, methods 9800-10100 described in connection with Figures 98-101 may be performed within a cartridge 10210. In some variations, the system 10200 may include a sterile fluid transfer device 10220 containing a reagent 10222, and the cartridge 10210 including multiple bioreactor modules 10230, a pump module 10240, a thermal module 10245, a pressure-driven flow module 10250, a MACS module 10255, an electroporation module 10260, a FACS module 10265, a CCE module 10270, and a blank module 10280. The cartridge 10210 may further include a reagent reservoir 10285, multiple product bags 10290, and a fluid transfer bus 10295. The liquid transfer bus 10295 can be configured to couple the components of the cartridge 10210 for fluid communication.

[0353]

[0458] In some variations, insertion and removal of the cell product from the cartridge can be performed within or outside the system. In some variations, the cartridge is placed on the patient or donor at bedside and then delivered to a cell processing system in or near the hospital, or shipped to a facility where the cell processing system is located. Similarly, the cell product can be removed from the cartridge after processing at a facility or near the recipient (patient) of the cell product. Optionally, the cell product is frozen before, during, or after the disclosed methods, optionally after adding one or more cryoprotectants to the cell product. In some variations, the system can include a freezer and / or a liquid nitrogen source. In some variations, the system can include a water bath or a heated chamber containing a controlled temperature gas to allow controlled thawing of the cell product, such as a water bath set at about 20°C to about 40°C. In some variations, the cartridge is made of a material that resists mechanical damage when refrigerated.

[0354] Automated Cell Processing

[0459] Described herein are methods for translating user-defined cell processing operations into cell processing steps using the automated cell processing systems and devices described herein. In some variations, cell processing operations are accepted and translated into cell processing steps for execution by the system, subject to a predetermined set of constraints. For example, a user may input a set of biological process steps and corresponding biological process parameters to be executed by the cell processing system. Optionally, the process parameters may be customized for each cartridge or set of cartridges.

[0355]

[0460] FIG. 35 is a flowchart generally describing a variation of an automated cell processing method. Method 3500 may include receiving (3502) an ordered input list of cell processing operations. For example, a set of two or more ordered input lists of cell processing operations may be received for execution on two or more cartridges in the automated cell processing system. For example, as shown in GUI 4900 of FIG. 49 and described in detail herein, one or more biological process inputs (e.g., available operations) may be selected for the ordered input list of cell processing operations, such as enrichment, MACS selection, activation, transduction, transfection, proliferation, and in-line analysis. Additionally, GUI 5200 of FIG. 52 shows a complete ordered input list (e.g., selected set of operations) 5220 of cell processing operations selected by a user.

[0356]

[0461] In some variations, one or more sets of cell processing parameters can be received (3504). Each set of cell processing parameters can be associated with one of the cell processing operations. Each set of cell processing parameters can define characteristics of the cell processing step performed by the instrument in that cell processing step. For example, GUI 4000 in FIG. 40 shows reagent and container parameters, GUI 4200 in FIG. 42 shows an example of process parameters, GUI 4400 in FIG. 44 shows an example of a pre-processing analysis, and GUI 4800 in FIG. 48 shows an example of an activated setting set.

[0357]

[0462] In some variations, a transformation model can be executed on the ordered input list 3506. In some variations, the transformation model can include constraints on the ordered output list determined by a predetermined configuration of the automated cell processing system. For example, the constraints can include information about the configuration of the automated cell processing system.

[0358]

[0463] In some variations, the constraints may include one or more of the type and / or number and / or condition of instruments, the type and / or number and / or condition of modules on the cartridge, the type and / or number of reservoirs on the cartridge, the type and / or number of sterile fluid transfer ports on the cartridge, and the number and location of fluid paths between modules, reservoirs, and sterile fluid transfer ports on the cartridge.

[0359]

[0464] In some variations, a set of predetermined constraints can be applied to the set of process control parameters. For example, the volume and / or type of reagents used can be constrained based on the size of the system and / or the product being manufactured. Other process parameter constraints can include, but are not limited to, one or more of the following: temperature, volume, time, pH, cell size, cell count, cell density, cell viability, dissolved oxygen, glucose level, on-board reagent storage and waste volumes, or combinations thereof. For example, GUI 4000 in FIG. 40 indicates that the reagent has a unit volume of 30 mL, the required volume is 54 mL, and the consumable container has a unit volume of 75 mL. GUI 4800 in FIG. 48 indicates that the activation concentration is 12 mg / L, the activation incubation time is 1600 seconds, the activation temperature is 18°C, and the gas mixture includes 21% oxygen, 78.06% nitrogen, and 0.04% carbon dioxide. These constraints can be applied by a transformation model to generate an ordered output list of cell processing steps that affect how one or more of the robots, instruments, and cartridges operate to produce the cellular product.

[0360]

[0465] In some variations, the order of operations can be constrained based on hardware constraints. For example, a robot may be limited to moving one cartridge at a time. Similarly, an instrument may be constrained to operating on a certain number of cartridges at a time.

[0361]

[0466] In some variations, the Insert Product operation must be the first operation performed and may be performed once in each process, as shown in GUI 4900 of Figure 49. The Fill and Complete operation is always the last operation performed before Product Completion and may be performed once in each process.

[0362]

[0467] In some variations, the system may prevent the user from performing a set of actions in an order that cannot be performed by the system.

[0363]

[0468] In some variations, a notification (e.g., a warning, alert) may be output if a user orders a set of actions in a "non-standard" way. For example, a notification may be output if the same type of action is repeated consecutively (e.g., enrichment immediately followed by enrichment). Similarly, if an action (e.g., selection, activation) is typically used only once within a given process, a notification may be output if such an action is used more than once.

[0364]

[0469] In some variations, the output of the transformation model may correspond to an ordered output list of cell processing steps that the system can perform 3508. For example, the transformation model can be executed on a set of ordered input lists to generate an ordered output list of cell processing steps. The output list of cell processing steps can control a robot, a cartridge, and one or more instruments.

[0365]

[0470] In some variations, the ordered output list is executed by the system to control the robot to move one or more cartridges, each containing a cellular product, between instruments (3510). For example, a MACS selection process selected by a user may correspond to the robot 230 of FIG. 2 moving a cartridge 250, for example, from another instrument to the cell selection instrument 216. In some variations, the ordered output list may include instructions for the robot to load a cartridge (e.g., a single-use consumable) into a cell processing system (e.g., a work cell). Further, the robot may be configured to move the cartridge to a first instrument position.

[0366]

[0471] In some variations, the ordered output list is further executed by the system to control one or more of the instruments to perform one or more cell processing steps on one or more cell products in each cartridge (3512). For example, the compute server rack 210 (e.g., controller 120) can be configured to control an electroporation module 220 configured to apply a pulsed electric field to a cell suspension in the cartridge 250. In some variations, the ordered output list can include instructions for an instrument (e.g., a bioreactor) to process a product (e.g., transfer a cell product from a small bioreactor module to a large bioreactor module). The instrument can also be configured to operate under a set of process parameters (e.g., a 9-hour period, pH 6.7, temperature 37.3°C to 37.8°C, mix mode 3). As another example, the ordered output list can include instructions for operating a sterile fluid transfer module to perform one or more of removing waste from the cartridge, adding media to the cartridge, and adding MACS reagents to the cartridge.

[0367]

[0472] In some variations, one or more electronic batch records can be generated based on the process parameters and data collected from sensors during process execution 3514. The batch records generated by the system can include process parameters, time logging, sensor readings from instruments, QC parameters determined by QC instrumentation, and other records.

[0368]

[0473] FIG. 36 is a flowchart generally describing variations of a method 3600 for executing a transformation model. In some variations, one or more biological functions can be generated and output to a user. For example, a configurable set of biological function blocks can be displayed in a graphical user interface for user selection. The GUI allows a user to select and order the biological function blocks and define biological control parameters. If desired, one or more control parameters of the biological function blocks may be modified by the user. In some variations, one or more biological function templates can be generated that include a predefined sequence of biological function blocks. If desired, one or more biological control parameters of the biological function templates may be modified by the user.

[0369]

[0474] In some variations, the cell processing system may be configured to receive and / or store one or more biological function (e.g., process) inputs from a user 3604. For example, a user may select one or more pre-defined biological function templates.

[0370]

[0475] In some variations, a biological process model (e.g., process definition) can be generated based on the biological process inputs 3606. In some variations, the biological process model can include one or more of enrichment, isolation, MACS selection, FACS selection, activation, genetic modification, gene transfer, transduction, transfection, expansion, formulation (e.g., harvesting, pooling), cryopreservation, T cell depletion, settling, tissue digestion, washing, irradiation, co-culture, combinations thereof, and the like.

[0371]

[0476] In some variations, the biological process model can be converted to an instrument-implemented process model (3608). For example, each biological function block of the biological process model may correspond to an ordered list of cell processing system operations with corresponding hardware control parameters. The instrument-implemented process model may include a sequence of hardware operations corresponding to the biological process model. As described herein, the conversion model may include one or more constraints.

[0372]

[0477] Optionally, in some variations, the cell processing system may be configured to receive and / or store one or more instrument-run process inputs from a user (3610). For example, a user may modify the converted instrument-run process model if desired. A user may select specific hardware components for execution of certain steps, change timing parameters, etc.

[0373]

[0478] In some variations, an instrument-executed process can be executed to generate a cellular product 3612. For example, a cell processing system can, at runtime, process the cellular product through the system as defined by an instrument-executed process model.

[0374]

[0479] In some variations, an instrument-executed process can be executed 3612. In some variations, the instrument-executed process model can be converted back to the biological process model 3614. The progress of this biological process can be output (e.g., displayed) to a user for monitoring. For example, the instrument-executed process model may include one or more references (e.g., pointers) that are returned to the biological process model to enable reporting of run-time execution progress against the biological process model.

[0375]

[0480] In some variations, cellular products can be monitored 3616. For example, GUIs 5300 and 5400 in Figures 53 and 54 show sensor data monitored by the system for multiple products. For example, the status of a number of viable cells and processes (e.g., as a function of percent complete) can be graphically displayed to the user.

[0376]

[0481] In some variations, electronic records can be generated 3618 based on the monitoring data, for example, one or more electronic batch records can be generated in accordance with 21 CFR regulations.

[0377]

[0482] FIG. 55 is a block diagram of an example variation of a manufacturing workflow 5500 including a processing platform 5520 (e.g., system 100, workcell 110, 200, 201) configured to produce multiple cellular products (e.g., a first product, a second product, a third product) in parallel. For example, a first workflow 5510 for the first product may include multiple biological processes 5512 performed in a predetermined sequence using corresponding elements 5522 (e.g., hardware) of the platform 5520. Concurrently, a second workflow 5530 for the second product may perform a predetermined sequence of biological processes 5530 using corresponding elements 5524 of the platform 5520. In this manner, the hardware resources of the platform 5520 can be efficiently utilized to increase throughput. In some variations, about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cellular products can be simultaneously manufactured on the platform 5520. The transformation model may include hardware constraints that eliminate scheduling overlaps, for example to ensure that the same tool is not used for different products at the same time.

[0378] Graphical User Interface

[0483] In some variations, a graphical user interface (GUI) may be configured for designing a process and monitoring the products. Figure 37 is a variation of a GUI 3700 that includes an initial process design interface. For example, GUI 3700 may be a process design home page. GUI 3700 may indicate that no process has been selected or loaded. A user may select a generate icon 3710 (e.g., "Generate Process") to begin the process design process. In some variations, one or more of the GUIs described herein may include a search bar.

[0379]

[0484] Figure 38 is a variation of a GUI 3800 related to creating a process. The GUI 3800 may be displayed after selecting the create icon 3710 of Figure 37. For example, the GUI 3800 may include a create process window 3810 that allows a user to enter and / or select one or more of a process name, a process description, and a template. In some variations, the user may select from a list of pre-defined templates. For example, a user may create a process and save it as a template for later selection.

[0380]

[0485] 39 is a variation of GUI 3900 that includes an association with an empty process. GUI 3900 may be displayed after confirming to create a process in GUI 3800. GUI 3900 shows the process name (e.g., Car T Treatment) and highlights a process setup icon 3910, allowing for the addition of process-specific parameters such as process reagents and containers, process parameters, and pre-processing analyses. GUI 3900 may further include an add process reagents and containers icon 3920, an add process parameter icon 3930, and an add pre-processing analysis icon 3940. Once the process setup is complete, one or more process elements may be specified.

[0381]

[0486] In some variations, the GUI 3900 may include one or more pre-defined templates for a set of biological processes (e.g., CAR-T, NK cell, HSC, TIL, etc.). For example, the templates may aid in process development and may be useful starting points for process development. The templates may also be modified (e.g., customized) based on user requirements.

[0382]

[0487] FIG. 40 is a variation of a GUI 4000 that includes an association for adding reagents and consumable containers. The GUI 4000 may be displayed after selecting the add process reagents and containers icon 3920 of FIG. 39. For example, the GUI 4000 may include an add reagents and containers window 4010 that allows a user to enter and / or select one or more reagents, including reagent type, manufacturer, part number, unit volume, required volume, and required reagent inputs (e.g., lot number, expiration date, container transfer required). The add reagents and containers window 4010 may include one or more input fields, selection boxes, drop-down selectors, etc. Further, the add reagents and containers window 3810 allows a user to enter and / or select one or more consumable containers, including manufacturer, part number, unit volume, and required container inputs (e.g., lot number, expiration date). In some variations, the user can select from a list of predefined templates. For example, a user can create a process and save it as a template.

[0383]

[0488] FIG. 41 is a variation of a GUI 4100 that includes a process parameter association. The GUI 4100 may be displayed after selecting the add process reagents and containers icon 3930 in FIG. 39. For example, the GUI 4100 may include an add process parameter window 4110 that allows a user to enter and / or select one or more parameters, including a name, parameter identification, description, data type, units, and parameter type. The add process parameter window 4110 may include one or more of an input field, a selection box, a drop-down selector, etc. In some variations, the user may select from a list of predefined templates. For example, the user may create a parameter and save it as a template. FIG. 42 is a variation of a GUI 4200 that includes a patient weight process parameter association. For example, the GUI 4200 may include an add process parameter window 4110 populated with parameter information, including patient weight, data type (e.g., integer), units (e.g., kilograms), and parameter type (e.g., input).

[0384]

[0489] FIG. 43 is a variation of a GUI 4300 related to a preprocessing analysis. The GUI 4300 may be displayed after selecting the add preprocessing analysis icon 3940 of FIG. 39. For example, the GUI 4300 may include an add preprocessing analysis window 4310 that allows a user to enter and / or select one or more parameters, including a name, identifier, description, data type, and display group. The add preprocessing analysis window 4310 may include one or more of an input field, a selection box, a drop-down selector, etc. In some variations, the user may select from a list of predefined templates. For example, the user may create parameters and save them as a template.

[0385]

[0490] 44 is a variation of a GUI 4400 associated with a white blood cell count preprocessing analysis. For example, the GUI 4400 may include an add preprocessing analysis window 4410 populated with preprocessing analysis information including a name (e.g., CBC white blood cell count), an identifier (e.g., CBC white blood cell count), a description (e.g., the number of white blood cells in the sample), a data type (e.g., float), and a display group (e.g., WBC).

[0386]

[0491] FIG. 45 is a variation of a GUI 4500 related to process parameter calculations. GUI 4500 may be displayed after selecting the Add Preprocessing Analysis icon 3940 of FIG. 39 and selecting the “Calculation” parameter type. For example, GUI 4500 may include an Add Preprocessing Analysis window 4510 that allows a user to enter and / or select one or more parameters, including a name, identifier, description, data type, display group, units, and parameter type. Additionally, a Calculation Builder allows a user to define a formula (e.g., algorithm, equation) to perform a given calculation. For example, the Calculation Builder may include one or more of an available parameter set (e.g., patient weight), constant values, an equation, and operands.

[0387]

[0492] 46 is a variation of a GUI 4600 associated with a completed process setup. For example, GUI 4600 may include a process setup window 4610 populated with process reagents, containers, process parameters, and pre-processing analyses. Once the process setup is complete, one or more process elements may be specified.

[0388]

[0493] Figure 47 is a variation of a GUI 4700 related to process operation activation settings. GUI 4700 may be displayed after selection of process element icon 4620 of Figure 46. For example, GUI 4700 may include an activation settings window 4710 that allows a user to enter and / or select one or more of an activation concentration (e.g., mg / L), an activation incubation time (e.g., seconds), an activation temperature (e.g., °C), and a gas mixing mode. In some variations, a user may select from a list of pre-defined templates. For example, a user may create a set of activation settings and save it as a template for later selection.

[0389]

[0494] 48 is a variation of a GUI 4800 associated with a filled-in process operation activation setting. For example, GUI 4800 may include an activation setting window 4810 with activation setting information filled in. In some variations, a set of gases (e.g., O2, N2, CO2) and corresponding concentrations may be specified.

[0390]

[0495] 49 is a variation of a GUI 4900 associated with a process operations interface. GUI 4900 may include an available operations window 4910 and a selected operations window 4920. The options available for selection may include one or more of the biological process inputs described herein, including, but not limited to, enrichment, MACS selection, activation, transduction, transfection, proliferation, and in-line analysis. One or more of these operations may be selected and dragged into the selected operations window 4920. Selected operations may be reordered within the selected operations window 4920.

[0391]

[0496] 50 is a variation of a GUI 5000 related to dragging process actions. GUI 5000 may include an available actions window 5010, a selected actions window 5020, and a selected (e.g., dragged) action 5030 that can be dragged and dropped between available actions window 5010 and selected actions window 5020. Selected actions window 5020 may include multiple selected actions.

[0392]

[0497] 51 is a variation of a GUI 5100 related to dragging process actions. GUI 5100 may include an available actions window 5110, a selected actions window 5120, and a selected (e.g., dragged) action 5130 that can be dragged and dropped between available actions window 5110 and selected actions window 5120. Selected actions window 5120 may include multiple selected actions.

[0393]

[0498] 52 is a variation of a GUI 5200 associated with completed process actions. For example, the GUI 5200 may include an available actions window 5210 and a selected actions window 5220 that includes the complete set of selected actions. In some variations, the settings (e.g., parameters) of each action can be selectively changed by the user by selecting a corresponding icon (e.g., a gear icon).

[0394]

[0499] 53 and 54 illustrate variations of GUIs 5300 and 5400 related to product monitoring. GUIs 5300 and 5400 may include respective monitoring windows 5310 and 5410. For example, GUI 5310 may monitor multiple products 5320 and output one or more product characteristics 5330. Product characteristics 5330 include, but are not limited to, summaries, process data, online analysis, imaging, process audit logs, process parameters, and process schedules. 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, identification, process identification, progress, estimated completion, current step, and messages.

[0395]

[0500] Figure 77A is a flowchart of a method 7700 of separating cells using a CCE module. Figure 77B is a flowchart of a method 7710 of concentrating cells using a CCE module. Figure 77C is a flowchart of a method 7720 of buffer exchange using a CCE module.

[0396]

[0501] FIG. 78 is a flowchart of a method 7800 for separating cells. The method 7800 for counterflow 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 a magnet relative to the rotor using a robot. The rotor may optionally be moved toward an illumination source and an optical sensor (7804). A fluid may be flowed through the rotor (7806). In some variations, flowing the fluid may include ...

Claims

1. a work cell containing equipment for cell processing; a cartridge defining a closed housing, the cartridge including a fluid transfer bus and one or more cell treatment modules, each of the one or more cell treatment modules fluidly coupled to the fluid transfer bus and including an interface configured to couple to an instrument of the work cell to perform a cell treatment operation corresponding to the cell treatment module; A controller; a robot configured to automatically move the cartridge to the tool in the work cell in response to commands from the controller; A system for cell processing comprising:

2. The system of claim 1 , wherein the instrument is configured to interface with the cartridge to perform the cell processing operation on the cartridge.

3. The system of claim 1 , further comprising a processor configured to control the robot and the instrument.

4. the cartridge is a first cartridge of a plurality of cartridges; The system of claim 1 , wherein the work cell is configured to receive two or more of the plurality of cartridges.

5. The system of claim 1 , wherein the cartridge comprises a plurality of modules.

6. 6. The system of claim 5, wherein the plurality of modules comprises two or more of a bioreactor module, a cell selection module, a magnetically activated cell selection module, a sorting module, a fluorescence activated cell sorting (FACS) module, an electroporation module, and a counterflow centrifugal elutriation (CCE) module.

7. 10. The system of claim 1, wherein the instrument comprises a bioreactor instrument, a cell selection instrument, a magnetically activated cell selection instrument, a sorting instrument, an electroporation instrument, or a counterflow centrifugal elutriation (CCE) instrument.

8. The system of claim 7 , wherein the bioreactor device includes a plurality of slots for the cartridges.

9. The system of claim 7 , wherein the sorting instrument is a fluorescence activated cell sorting (FACS) instrument.

10. The system of claim 1 , wherein the work cell further comprises a reagent reservoir.

11. The system of claim 1 , wherein the work cell is automated.

12. The system of claim 1 , wherein the work cell comprises an enclosure.

13. 13. The system of claim 12, wherein the enclosure is one of an ISO 7 clean room, an ISO 6 clean room, or an ISO 5 clean room.

14. The system of claim 12 , wherein the enclosure includes a feedthrough.

15. The system of claim 1 , wherein the system performs automated manufacturing of a cellular product.

16. A housing; a liquid transfer bath; one or more cell processing modules, each fluidly coupled to the fluid transfer bus; tubing external to the cartridge housing, the tubing being in fluid communication with the liquid transfer bus; A closed cartridge for cell processing comprising: A closed cartridge, wherein the tubing forms a pump when engaged with a pump actuator of a work cell configured to receive the closed cartridge therein for performing one or more cell processing operations.

17. 17. The cartridge of claim 16, wherein the one or more cell processing modules comprise one or more of a bioreactor module, a cell selection module, a magnetically activated cell selection module, a sorting module, a fluorescence activated cell sorting (FACS) module, an electroporation module, and a counterflow centrifugal elutriation (CCE) module.

18. 17. The cartridge of claim 16, further comprising one or more sterile fluid transfer ports within the housing.

19. The cartridge described in claim 16, wherein the liquid transfer bus has a plurality of valves configured to control fluid flow between the one or more cell processing modules.

20. The cartridge of claim 16 , wherein the pump formed by the tubing is exposed through an opening in the housing configured to receive the pump actuator.

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