Method of use in bioreactors and automated cell processing systems

Abstract

This disclosure relates to systems, devices, and methods for automated cell processing within a cell processing system. In embodiments, this disclosure relates to a cartridge comprising a bioreactor module having a bioreactor, a first thermal compartment located adjacent to the bioreactor, and a second thermal compartment located adjacent to the bioreactor, and a fluid manifold connecting the first and second thermal compartments to the bioreactor.

Description

Technical Field

[0004] , , , ,

[0001] (Cross - Reference to Related Applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 520,858, filed Aug. 21, 2023, the entire content of which is incorporated herein by reference for all purposes.

[0002] (Field of the Invention) The present disclosure relates to improved bioreactor systems, such as bioreactors for use in automated cell processing systems, for example.

Background Art

[0003] Cell therapy involves collecting cells from an individual, processing the cells, and using the processed cells to achieve a clinical response in the same or a different individual. Cell processing is typically a complex workflow that includes multiple steps, with each step typically requiring a separate cell processing device and / or system to achieve a particular step. Performing separate steps with separate cell processing devices increases operational complexity, increases the space requirements for storing the separate cell processing devices, and typically requires manual labor. That is, with separate cell processing devices, it becomes difficult to effectively manage the various workflow complexities, such as one step taking longer than another or the requirement to complete one step before another. Further, the space required to store the separate cell processing devices needed to perform these steps can be large. When performing cell processing in large quantities, these workflow complexities and storage requirements can worsen. Additionally, the environmental conditions of each cell processing device are generally difficult to control, which becomes an increasing burden as the number of cell processing devices increases. Therefore, an improved bioreactor, such as a bioreactor for use in automated cell processing, is desirable.

Summary of the Invention

[0004] This disclosure relates to systems, devices, and methods for processing cells within a bioreactor for use in a bioreactor, such as an automated cell processing system. Generally, an automated cell processing system may comprise a cartridge having a bioreactor module. The bioreactor module may comprise a bioreactor, a first thermal compartment located adjacent to the bioreactor, and a second thermal compartment located adjacent to the bioreactor. The cartridge may further comprise a fluid manifold connecting the first and second thermal compartments to the bioreactor in order to assist in the transfer of fluids between the bioreactor and various components of other cell processing modules. The bioreactor may comprise one or more of the following: an impeller for assisting in agitation of cells and reagents, an air-permeable liner, a sampling straw for removing cells from the bioreactor for sampling, and a perfusion filter for holding cells while exchanging fluids.

[0005] A first thermal compartment and a second thermal compartment, which can be configured to maintain a fluid in a stable environment, can interlock with each other. The first thermal compartment and the second thermal compartment can be configured to hold the same or different volumes. In some modifications, the first and second thermal compartments are configured to hold different volumes. For example, in some modifications, the first thermal compartment may be configured to hold a volume of about 600 mL, and the second thermal compartment may be configured to hold a volume of about 100 mL.

[0006] In some modifications, the bioreactor module may further include a mixing chamber configured to receive one or more cell processing reagents. The mixing chamber may include an impeller to assist in agitating the cells and reagents. In some modifications, the impeller of the mixing chamber and the impeller of the bioreactor may be connected to each other. One or more of the bioreactor, the first thermal compartment, the second thermal compartment, and the mixing chamber may have, for example, a window for optical detection to observe the progress of cell processing. One or more of the bioreactor, the first thermal compartment, the second thermal compartment, and the mixing chamber may be fluidically connected, for example, via a fluid manifold.

[0007] In some modifications, the cartridge may further comprise a third thermal compartment located adjacent to the bioreactor, which may be configured to maintain the fluid in a stable environment. The third thermal compartment may be configured to hold the same volume as the first and / or second thermal compartments. In some modifications, the third thermal compartment is configured to hold a volume of approximately 600 mL. The third thermal compartment may have a window configured for optical detection. The cartridge may further comprise a second bioreactor module. The bioreactor modules may be thermally coupled to each other and / or to other components of the bioreactor.

[0008] Also described herein are methods aimed at automatically processing cells, for example, by using a bioreactor to grow or culture cells. A method for cell processing may include providing cell material to a cartridge having a bioreactor module comprising a bioreactor, a first thermal compartment located adjacent to the bioreactor, and a second thermal compartment located adjacent to the bioreactor. The method may further include transferring the cell material from either the first or second thermal compartment to the bioreactor and culturing the cells within the bioreactor. The cells may be cultured for any desired time. In some modifications, the cells are cultured for, for example, about 12 hours to about 15 days by one or more cell processing steps. The bioreactor may be configured to perform one or more of the following processes: a static process, an agitation process, and a perfusion process. A static process can keep the cells in an un-agitated state, thereby promoting cell growth and / or division. An agitation process can agitate the cells, thereby promoting homogeneity and / or cell division within the cell material. The perfusion process can replace at least a portion of the cellular material, such as its culture medium. The perfusion process can also remove and replace a portion of the cellular material.

[0009] The method may further include measuring one or more of the pH and dissolved oxygen values ​​of the cell material in the bioreactor, sampling the cell material using a sampling straw in the bioreactor, and / or measuring one or more of the lactate and glucose values ​​of the cell material sample.

[0010] In some modifications, the bioreactor module may further comprise a mixing chamber. The method may further comprise performing one or more of transduction and transfection within the mixing chamber, and / or transferring cell material from either the first and second thermal compartments to the mixing chamber.

[0011] In further modifications, the cartridge may further comprise a second bioreactor module. In modifications in which the cartridge comprises a second bioreactor module, the method described herein may also include performing independent cell processing steps in each of the bioreactor modules.

[0012] Further embodiments, features, and advantages of the present invention will become apparent from the following detailed description and through the implementation of the present invention. [Brief explanation of the drawing]

[0013] [Figure 1A] This is a block diagram of an exemplary modified example of a cell processing system. [Figure 1B] Figure 1A is a block diagram of a cartridge that may be used in the cell processing system. [Figure 1C] Figure 1B is a block diagram of the bioreactor module of the cartridge. [Figure 1D] Figure 1C is a block diagram of the bioreactor in the bioreactor module. [Figure 1E] Figure 1C is a block diagram of the mixing chamber of the bioreactor module. [Figure 1F] Figure 1C is a block diagram of the port system of the bioreactor module. [Figure 1G] It is a block diagram of the first heat section of the bioreactor module in FIG. 1C. [Figure 1H] It is a block diagram of the second heat section of the bioreactor module in FIG. 1C. [Figure 1I] It is a block diagram of the third heat section of the bioreactor module in FIG. 1C. [Figure 2A] It is a front perspective view of a cartridge that can be provided to a cell processing system. [Figure 2B] It is a rear perspective view of the cartridge in FIG. 2A. [Figure 3A] It is a front perspective rendering of an exemplary modification of the bioreactor module. [Figure 3B] It is a front perspective cross-sectional rendering of the bioreactor module shown in FIG. 3A. [Figure 3C] It is a horizontal cross-sectional rendering of the bioreactor module shown in FIG. 3A. [Figure 3D] It is a bottom rendering of the bioreactor module shown in FIG. 3A. [Figure 3E] It is a top rendering of the bioreactor module shown in FIG. 3A. [Figure 4A] It is a perspective rendering of the gear system of the bioreactor module. [Figure 4B] It is a top rendering of the gear system shown in FIG. 4A. [Figure 5] It is a flowchart of an exemplary modification of the culturing of cells using the bioreactor module.

MODE FOR CARRYING OUT THE INVENTION

[0014] This specification discloses devices, systems, and methods for processing cells. Multiple cell processes or cell processing steps may be performed on cells in a cartridge, in a cell processing work cell. The cell processing work cell may be any suitable work cell. For example, the cell processing work cell described herein may have multiple bays, each bay having equipment that, when connected to a corresponding module in a cartridge, performs the cell processing step corresponding to that module. For example, the cartridge described throughout this specification includes a bioreactor module. The bioreactor module of the cartridge may be configured to perform cell processing steps, such as cell culture, when connected to the bioreactor equipment of a work cell. For example, a fluid (e.g., a cell solution containing one or more of cells, reagents, buffers, and culture media) may be supplied to the bioreactor module, which may comprise a bioreactor, a mixing chamber, and one or more additional thermal compartments. The bioreactor module may provide an environment suitable for cell growth and / or division. In other words, a bioreactor module may have a thermally stable environment to facilitate cell culture by avoiding changes in the fluid temperature as the fluid can be transported throughout the bioreactor module; otherwise, the cell culture process may be interrupted or slowed. A thermally stable environment can be facilitated by a heat exchanger. For example, the heat exchanger of a bioreactor module may be thermally connected to one or more of the bioreactor, thermal compartments, and mixing chambers. Each of the bioreactor, thermal compartments, and mixing chambers may be thermally connected, resulting in a stable thermal environment throughout the bioreactor module (e.g., substantially equal).

[0015] A bioreactor module may be configured to perform one or more processes on the fluid within the bioreactor of the bioreactor module. For example, one or more processes may include a static process, an agitation process, and a perfusion process. In some modifications, a static process can maintain an unagitated fluid, thereby keeping the cells within it undisturbed while undergoing cell growth (e.g., cellular respiration) and / or division. Cells may consume oxygen (O2) during cell growth and / or division, while a byproduct of cell growth and / or division may be carbon dioxide (CO2). A relatively low concentration of carbon dioxide (e.g., about 0 to 10 vol%) in the cell solution and / or the gaseous environment surrounding the cell solution (e.g., in the headspace above the cell solution) may be useful in maintaining a stable pH within a target range (e.g., about 7 to about 7.4), because cell growth and / or division may be inhibited by volatile pH values ​​and / or pH values ​​outside the target range. For example, carbon dioxide reacts with water to form carbonic acid, which then forms bicarbonate ions and hydrogen ions, the latter of which can determine the pH of the cell solution. However, if the concentration of carbon dioxide is too high, it may suffocate the cells in the cell solution by displacing the oxygen in the cell solution and / or the pH may drop below the target range due to the formation of many hydrogen ions. Therefore, maintaining the right balance between oxygen and carbon dioxide can be important for cell growth and / or division. Thus, the balance of oxygen and carbon dioxide can be adjusted by a stirring process. That is, a stirring process allows the fluid to be stirred (e.g., using an impeller), which mixes the fluid and makes it easily homogeneous. When the fluid is homogeneous (e.g., the components are evenly distributed), it may be easier to equally expose the cells to the oxygen or one or more reagents dissolved in the cell solution and / or local concentrations of carbon dioxide may be reduced. Furthermore, as cells in the cell solution continue to grow and / or divide, this may result in an increase in cell size and / or cell number, which may lead to increased oxygen consumption and / or carbon dioxide production.Next, there is a risk that the availability of oxygen and / or the pH of the cell solution may decrease. Therefore, a perfusion process can be used to remove a portion of the fluid (e.g., spent medium) and replace the removed portion with a new fluid (e.g., fresh medium). The perfusion process can re-establish the dissolved oxygen and / or pH of the fluid within the target range. The static, agitation, and / or perfusion processes may be performed within a single bioreactor, thereby rationalizing the cell processing methods described herein by avoiding fluid transfer to one or more separate modules. Further, the static, agitation, and / or perfusion processes may be performed according to a predetermined workflow. The predetermined workflow can be programmed into a controller within the work cell.

[0016] I. Cell Processing System The cell processing systems described herein may be configured to perform one or more cell processing steps within a working cell. The working cell may have a closed, automated environment that can be configured to maintain a sterile environment. The working cell may receive a cartridge and perform one or more cell processing steps on cells in a cell solution (e.g., cell suspension) contained within the cartridge. For example, a cell processing system may include a working cell having a plurality of bays having instruments inside, each instrument configured to independently perform one or more cell processing steps when coupled to a corresponding module in a cartridge, and a robot capable of moving cartridges within the working cell (e.g., between one or more bays). The robot and / or instruments may be configured to operate automatically so that no operator assistance is required at any point in the workflow. For example, the robot may receive a cartridge and move it between locations within the working cell (e.g., instruments, bays, storage, feedthroughs) according to a pre-programmed workflow, each location may be associated with one or more cell processing steps. After performing one or more cell processing steps of a pre-programmed workflow, the working cell may be configured to detach the cartridge from the working cell and transfer it (e.g., via a robot). Additionally, or alternatively, at least a portion of the cell solution may be transferred to a second cartridge (e.g., via a fluid device or fluid manifold).

[0017] The cell solutions (e.g., cell suspensions) described herein may contain cells that can be processed for subsequent use in cell therapy. The cell solutions may contain cells (e.g., allogeneic cells) in a fluid such as a culture medium (e.g., cell culture medium). The cell solutions may contain cells from the same or different donors. Cells from the same donor may be divided between one or more cartridges, so that separate cell processing steps are performed in each cartridge, which may increase the overall throughput of the cell processing system described herein. The cell solutions may be transferred to the cartridges, for example, by an operator, before loading the cartridges into the working cell. In some modifications, the cartridges may be empty when loaded into the working cell so that the working cell can transfer the cell solutions into the cartridges. In some modifications, cells from two or more cartridges may be combined according to a predetermined ratio that may correspond to the intended therapeutic procedure for a patient.

[0018] Figure 1A shows an exemplary cell processing system for use in automated devices, systems, and methods. A block diagram of a cell processing system 100 comprising a working cell 110 and a controller 120 is shown. The working cell 110 may comprise one or more of the following: instruments 112, a robot 116 (e.g., a robotic arm), a reagent storage unit 118, a sterile liquid transfer port 132, a sterilizer source 129, a fluid source 136, a pump 138, and a sensor 151. Cartridges 114 and fluid devices 142, located outside the working cell 110 and usable within the working cell 110, are shown by dashed lines. In some modifications, the fluid device 142 may be a sterile liquid transfer device (SLTD). However, it should be understood that the fluid device 142 may be configured to transfer any fluid (including liquids), whether sterile or not. The controller 120 may include one or more of the following: a processor 122, memory 124, communication device 126, input device 128, and display 130.

[0019] The working cell 110 may comprise a housing that is fully or at least partially sealed, within which one or more cell processing steps may be performed in a fully or at least partially automated process. The cartridge 114 may be moved using a robot 116 to reduce manual labor in the cell processing steps, and the transfer of fluids into and out of the cartridge 114 may also be carried out in a fully or partially automated process, as described in detail herein. For example, one or more fluids may be stored in a fluid device 142, and as a result, one or more fluids may be transferred to and / or removed from the cartridge 114 via the fluid device 142. In some modifications, the fluid device 114 may be moved within the system 100 by a robot 116. Thus, the working cell 110 described herein is advantageous in that it enables the transfer of fluids in an automated and metered manner for automating the manufacture of cell therapies.

[0020] The working cell 110 can facilitate fluid and / or cartridge transfer. For example, in some modifications, the robot 116 may be configured to move two or more cartridges 114 between different bays to perform a predetermined sequence (e.g., a workflow) of cell processing steps. In this way, multiple cartridges 114 can be processed in parallel, as different steps of the cell processing workflow can be performed simultaneously on different cartridges. In another example, a sterile fluid transfer port 132 may be connected between two or more cartridges 114 to transfer cell products and / or other fluids between the cartridges 114. Furthermore, the sterile fluid transfer port 132 may be connected between any set of fluid transport components of the system 100 (e.g., cartridges 114, reagent storage unit 118, fluid source 136, fluid device 142, etc.). For example, a first sterile fluid transfer port may be connected between a first cartridge and the corresponding sterile fluid transfer port of a fluid device.

[0021] Other preferred cell processing systems and embodiments thereof are provided, for example, in U.S. Patent Applications 17 / 198,134, 18 / 731,095, 18 / 759,602, and 18 / 807,699, published as U.S. Publication No. 2021 / 0283565, the entire contents of each of these are incorporated herein by reference.

[0022] A. Cartridge The cell processing systems described herein may comprise one or more cartridges having one or more modules configured to interface with or detachably connect to one or more instruments in a working cell. Some or all of the modules may, but are not required, be integrated in a fixed configuration within the cartridge. In fact, one or more of the modules may be configurable or movable within the cartridge, so that various forms of cartridges can be assembled. For example, the cartridge may be a single closed unit with fixed components for each module, or the cartridge may include configurable modules coupled by configurable fluidic, mechanical, optical, and electrical connections. In some modifications, one or more subcartridges, each containing a set of modules, may be used to perform various cell processing workflows. The modules may each be housed in a separate housing, or they may be incorporated together with other modules into a cartridge or subcartridge. While this disclosure generally presents modules as separate groups of components for simplicity, it should be noted that these modules may be arranged in any preferred configuration. For example, components for different modules may be scattered amongst themselves, such that each module may be defined by a set of connected components that collectively perform a predetermined function. However, the components of each module may or may not be physically grouped within the cartridge. In some embodiments, multiple cartridges may be used to process a single cell product by transferring the cell product from one cartridge to another cartridge of the same or different type, and / or by dividing the cell product into more cartridges, and / or by pooling multiple cell products into fewer cartridges.

[0023] Generally, each piece of equipment within a working cell is interfaced with or detachably connected to each or more modules on a cartridge in order to perform a specific cell processing step. For example, if a cartridge has a cell sorting module, the cartridge can be moved by a robot into a bay within a working cell containing the cell sorting equipment so that the cell sorting module can be connected to the cell sorting equipment to sort the cells in the cartridge. One advantage of such a segmented module / equipment design is that expensive components (e.g., motors, sensors, heaters, lasers, etc.) can be kept within the equipment of the system, while less expensive components can reside permanently in the cartridge.

[0024] As illustrated in Figure 1B, the cartridge 114 may be configured to contain (e.g., store) a cell solution (e.g., a cell suspension) for cell processing. Any number of cell processing steps may be performed on the cells in the cartridge. Thus, the cartridge 114 may comprise one or more of the following: a bioreactor 150, an electroporation module 160, an elutriation module 162, a spinoculation module 164, a cell sorting module 166, and a fluid manifold 168. If cell sorting is performed, specific reagents (e.g., magnetic particles) may be added to the cell solution in one or more of the cartridge molecules. The magnetic particles are configured to bind to specific types of cells (e.g., target cells), as described above. The elutriation module 162 may be configured to perform an elutriation process that can separate cell material according to size, shape, and / or density. The spinoculation module 164 may be configured to perform a spinoculation process that can bind different types of cells together.

[0025] The fluid manifold 168 may be configured to transfer one or more fluids between one or more modules of the cartridge 114. For example, the fluid manifold 168 may transfer a cell solution from the bioreactor module 150 to the cell sorting module 166. The cell solution may contain cellular material including target cells linked to magnetic particles. In another example, the fluid manifold 168 may transfer a cell solution from the cell sorting module 166 to any other module 114, for example, after a cell sorting process has been performed. The fluid manifold 168 may be configured to transfer sorted cells (e.g., target cells) to one module and non-target material to a different module.

[0026] Other suitable cartridges and cell processing modules that may be used with the automated cell processing work cell described herein are provided, for example, in U.S. Patent Application No. 18 / 652,602, U.S. Patent Application No. 18 / 532,621, U.S. Patent Application No. 18 / 620,826, and U.S. Patent Application No. 18 / 611,632, the entire contents of each of these are incorporated herein by reference.

[0027] Referring to Figures 2A and 2B, exemplary variations of the cartridge 200 are illustrated. The cartridge 200 may comprise a water elutriation module 210, a fluid manifold 222, a first cell sorting module 224a, a second cell sorting module 224b, an auxiliary module 226, a fluid device tray 228, a liquid container 230, a pump module 232, a first bioreactor module 240a, and a second bioreactor module 240b. Although these figures show two bioreactor modules, it should be understood that any number of bioreactor modules may be used as desired. For example, the cartridge may contain one, two, three, four, or more bioreactor modules, depending on the size of the cartridge, the presence of other cell processing modules within the cartridge, etc. The bioreactor modules 240a and 240b may perform cell culture processes, as will be described in more detail below. The electroporation module 220 may be configured to facilitate intracellular delivery of macromolecules (i.e., transfection by electroporation). Discharge from one or more capacitors or current sources may generate a sufficient current in the chamber to facilitate the transport of polynucleotides, proteins, nucleoprotein complexes, or other macromolecules into the cell in the cell product. The fluid manifold 222 may comprise at least one fluid conduit. At least one fluid conduit of the fluid manifold 222 may be configured to allow a fluid to pass through it. For example, at least one fluid may be a liquid or a gas. In some modifications, at least one fluid may contain a solution of cells of varying sizes and densities. The fluid manifold 222 may comprise at least one fluid inlet and at least one fluid outlet, and may comprise at least one valve. The fluid manifold 222 may be fluidically connected to at least one module in the cartridge 200. For example, the fluid manifold 222 may be configured to transport at least one fluid to the first and / or second bioreactor modules 240a, 240b. The fluid manifold 222 can communicate with a controller, such as the controller 120, which is described with reference to Figure 1A.For example, at least one valve of the fluid manifold 222 may open and / or close in response to a command transmitted by the controller 120 to transfer fluid between various modules of the cartridge according to a predetermined workflow.

[0028] The fluid transfer port tray 228 may have one or more ports configured to transfer fluid to or from one or more fluid devices. That is, each port of the fluid transfer port tray 228 may be configured to facilitate the transfer of sterile fluid. In some modifications, each port may be fluidically connected to a fluid conduit configured to fluidically connect to at least one module of the cartridge 114. For example, each port of the fluid transfer port tray 228 may be fluidically connected to a fluid manifold 222. In this way, the fluid may flow from the fluid devices connected to the ports of the fluid transfer port tray 228 to the fluid manifold 222, or vice versa. In some modifications, each port of the fluid transfer port tray 228 may be fluidically connected to a liquid storage container 230. The liquid storage container 230 may be configured to contain the fluid. In some modifications, the fluid may be a liquid or a gas. In some modifications, the liquid storage container 230 includes multiple liquid containers. For example, the liquid storage container 230 may include one container, two containers, or three containers. The liquid storage container 230 may be fluidically connected to at least one module of the cartridge 200. In some variations, the liquid container 230 may be fluidically connected to a fluid manifold 222. Thus, the fluid may flow between the ports of the fluid transfer port tray 228, the fluid manifold 222, and the liquid storage container 230.

[0029] The cartridge may further comprise a pump module 232 having a pump configured to pump fluid in one or more directions along at least one fluid path. For example, the pump module 232 may be configured to pump fluid to or from one or more of the following modules in the cartridge: the elutriation module 210, the fluid manifold 222, the cell sorting modules 224a, 224b, the auxiliary module 226, the fluid transfer port 228, the liquid container 230, and any other modules in the cartridge. The auxiliary module 226 may be configured to engage with at least one device and / or module. The auxiliary module 226 may comprise at least one electrical connector and / or at least one fluid connector. In some modifications, the auxiliary module 226 may be removed and replaced by any other module.

[0030] Various materials, including metal, plastic, rubber, and / or glass, or combinations thereof, may be used to construct the cartridge (including its modules) and the cartridge housing. The cartridge, its components, and its housing may be manufactured by molding, machining, extrusion, 3D printing, or any combination thereof. The cartridge may house commercially available components (e.g., tubes, valves, fittings). Commercially available components may be attached to or integrated with custom components or devices. The cartridge housing may constitute an additional encapsulation layer to further protect the sterility of the cell products.

[0031] i. Bioreactor Module The cartridge described herein may comprise one or more bioreactor modules. A bioreactor module may comprise one or more of a bioreactor, a mixing chamber, and a thermal compartment. Each of the bioreactor, mixing chamber, and thermal compartment may be configured to contain a fluid. One or more components of the bioreactor module may be fluidically connected, as a result enabling the transfer of fluid between one or more of the bioreactor, mixing chamber, and thermal compartment. A bioreactor module may provide an environment suitable for cell culture. For example, a bioreactor module may maintain a thermally stable environment using a heat exchanger. That is, one or more of the bioreactor, mixing chamber, and thermal compartment may be thermally connected so that the heat exchanger can maintain a stable temperature throughout the bioreactor module. In another example, a bioreactor module may provide a predetermined gas mixture. A predetermined gas mixture (e.g., carbon dioxide, oxygen, and / or nitrogen) may promote cell culture by providing one or more nutrients that may be useful in the cell culture process. For example, oxygen and / or nitrogen may be consumed by cells during cell growth and / or division. In another example, carbon dioxide may be useful in maintaining the pH of a fluid within a target range.

[0032] In some modifications, it may be desirable for the cartridge to have two or more bioreactor modules. The two or more bioreactor modules may have the same configuration or different configurations. For example, to increase the range of the cartridge's working volume, the cartridge may have two bioreactor modules, each with a bioreactor configured to contain a different volume of fluid. In some modifications, the second bioreactor of the second bioreactor module may have an internal volume that is more than twice, five times, or ten times larger than the internal volume of the first bioreactor of the first bioreactor module. In some embodiments, the cartridge achieves a wide range of working volumes (e.g., about 5 mL to about 1,000 mL or more) by having two, three, four, five, or more bioreactor modules of the same or different sizes connected to the same or multiple fluid manifolds.

[0033] A bioreactor module can perform one or more cell processing steps related to cell culture. For example, a bioreactor module may be configured to perform one or more of the following steps: proliferation, sorting, transduction, transfection, perfusion, depletion, and seeding. Cell culture, which can be facilitated by performing one or more cell processing steps described herein, may be carried out for a predetermined period according to a predetermined workflow. In some variations, the predetermined period may be about 6 hours to about 20 days, about 12 hours to about 15 days, or about 1 day to about 12 days (including 12 hours, 3 days, 6 days, 9 days, 12 days, or 15 days). One or more cell processing steps may be repeated throughout the predetermined period. Any suitable cells, including various immune and non-immune cells such as T cells, hematopoietic stem cells (HSCs), hematopoietic stem and progenitor cells (HSPCs), natural killer cells (NKs), B cells, pre-B cells, lymphocytes, 293 cells, HEK cells, CHO cells, bacterial cells, and yeast cells, can be processed in the systems described throughout this specification.

[0034] In the proliferation step, the number of cells in the cell solution can be increased. For example, a bioreactor may be equipped with an impeller configured to mix the cell solution contained therein. The rotation speed of the impeller can be adjusted to increase or decrease the effectiveness of mixing. For example, increasing the rotation speed can accommodate a larger increase in the number of cells. Conversely, decreasing the rotation speed can accommodate a smaller increase in the number of cells. Cell proliferation can be carried out over a predetermined period that corresponds to a target number of cells. For example, a relatively small target number may correspond to a relatively short predetermined period (e.g., about 30 minutes to about 72 hours), and a relatively large target number may correspond to a relatively long predetermined period (e.g., about 72 hours to about 14 days). The bioreactor equipment may provide the bioreactor with closed-loop control of temperature, dissolved oxygen, and / or acidity (pH) to enable the growth of cells intended to be cultured (e.g., leukocytes). Optionally, one or more reagents, such as cell type-specific activators, may be introduced into the bioreactor.

[0035] In the sorting step, cells intended for further processing (e.g., target cells) can be facilitated by binding one or more reagents to the target cells. In exemplary modifications, the cell solution may be housed in a mixing chamber of a bioreactor module, to which one or more reagents may be transferred. The reagents may be transferred to the mixing chamber, for example, via a fluid device and a robot. The reagents may be configured to adhere to specific cell types, such as CD4+ and / or CD8+ cells, according to a pre-programmed workflow. The reagents may contain magnetic particles. Thus, the cell solution may be transferred to a cell sorting module, to which target cells can be sorted by applying a magnetic array in close proximity to the flow cell of the cell sorting module as the cell solution flows through the flow cell.

[0036] In the transduction step, a transduction agent (e.g., a lentiviral vector and / or virus) may be introduced into the cell solution within the bioreactor module. The transduction agent may be configured to target a specific cell type. Thus, the amount (e.g., volume) of the transduction agent may correspond to the cell concentration value (e.g., number of cells per unit volume) in the cell solution. For example, a relatively large amount of transduction agent may be introduced into a cell solution with a relatively high cell concentration, and a relatively small amount may be introduced into a cell solution with a relatively low cell concentration. In this way, the amount of transduction agent may be approximately proportional to the concentration of target cells in the cell solution. The transduction agent may be used, for example, to introduce a chimeric antigen receptor (CAR) into cells in the cell solution. In another example, a lentiviral vector containing Lenti-CD19 CAR (scFv-41BB-CD3ζ, CTL019) may be configured to target CD19+ cells. In some modifications, a robot may move the cartridge to a sterile fluid transfer device in the working cell. Sterilization fluid transfer equipment can transport cell solutions to the bioreactor module and additional solutions containing viral or nonviral vectors. Optionally, vector solutions may be frozen (e.g., at -80°C) and thawed and / or warmed immediately before transport to the bioreactor module. Culturing cells in the presence of viral or nonviral vectors may result in transduction of cells by the viral or nonviral vectors.

[0037] In the transfection step, a transfection reagent (e.g., nucleic acid) can be introduced into the cell solution by a non-viral method. The transfection may be configured to knock out specific cell types that may subsequently be associated with inducing an immune response (e.g., graft-versus-host disease) in the patient. That is, transfection may reduce the likelihood that a cell therapy recipient will reject the cells generated by the cell treatment described herein. Transfection may be performed by adding one or more transfection reagents to a bioreactor module containing the cell solution, thereby enabling the cells to be modified by the transfection reagents. The transfection reagents may include liquid nanoparticles (LNPs).

[0038] In the perfusion step, a predetermined volume of culture medium can be replaced in the cell solution contained within the bioreactor of the bioreactor module. Perfusion can be useful in maintaining the amount of nutrients in the cell solution and / or maintaining a suitable pH level, which is relevant to the desired cell growth rate. For example, since cells can consume glucose and / or oxygen dissolved in the cell solution during the cell growth process, low levels of glucose and / or dissolved oxygen may reduce or prevent cell growth. Conversely, since cells can release (e.g., excrete) lactate and / or carbon dioxide during the cell growth process, excess lactate and / or carbon dioxide may inhibit cell growth by displacing glucose and / or dissolved oxygen. Therefore, the perfusion process can ensure that the amount of glucose and / or dissolved oxygen in the cell solution remains above a level sufficient to support the desired cell growth, and / or that the amount of lactate and / or carbon dioxide in the cell solution remains below a level sufficient to prevent inhibition of cell growth. In addition, in some modifications, the cell culture process can be optimally performed within a target pH range. For example, the target pH range may be approximately 7 to 7.6, 7.1 to 7.5, or 7.2 to 7.4. In some modifications, the target pH may be approximately 7, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6. Carbon dioxide may be produced from the cell growth and / or division process, which may form bicarbonate and hydrogen ions. The latter of these may lower the pH of the cell solution. Therefore, a certain volume of cell solution can be removed by perfusion so that new cell solution can be added without overfilling the bioreactor module. In some modifications, perfusion may be performed continuously so that a certain volume of medium is constantly removed and new (e.g., fresh) medium is constantly added. Cells in the cell solution may be retained in the bioreactor (e.g., via a filter), so that only the medium can be replaced. In some variations, a given volume of the culture medium to be replaced (e.g., removed and replaced) may be about 10% to about 100% (including about 25%, about 50%, and about 75%) of the original cell solution.In some modifications, perfusion may be performed within the first bioreactor module of the cartridge. In other modifications, at least a portion of the cell solution may be transferred to a second bioreactor module, thereby enabling perfusion to be performed within the second bioreactor module.

[0039] In the depletion step, unmodified cells can be removed from the cell solution. Cell depletion may be configured to separate cells in a cell solution using one or more depletion reagents. For example, a depletion reagent (e.g., depletion microbeads) may be added to a cell solution that may be stored in a bioreactor module, and as a result, cells can be incubated with the depletion reagent. That is, the depletion reagent may be configured to bind to specific cells (e.g., target cells), thereby marking unbound cells for later removal. In an exemplary modification, the depletion reagent may bind to CD3+ cells (which may include CD4+ and CD8+ cells) so that bound CD3+ can be separated from unmodified T cells. As a result, unmodified T cells can be removed from the cell solution.

[0040] In the seeding step, a new cell culture process can be initiated using cells that have already undergone one or more cell processing steps. For example, a portion of the cell solution from the first bioreactor module can be transferred to the second bioreactor module. The cell culture process can then be carried out within the second bioreactor module. The seeding step can facilitate a faster and / or more successful cell culture process.

[0041] The bioreactor modules described herein may be configured to facilitate one or more measurements. For example, one or more of the bioreactor, thermal compartment, and mixing chamber may have windows configured for optical detection. That is, sensors may be operably connected to one or more windows so that one or more parameters in the bioreactor module can be measured. Additionally or alternatively, the bioreactor module may include one or more fluid conduits configured to sample a portion of the fluid in the bioreactor module. For example, a cartridge may include one or more sensors configured to measure one or more of the lactate and glucose values ​​of a sample. That is, one or more sensors may include a glucose sensor and / or a lactate sensor. Advantageously, measuring one or more of the lactate and glucose values ​​in the cartridge can increase the efficiency of the cell processing described herein by reducing the time and / or steps required to obtain lactate and / or glucose measurements. That is, the sample can be measured in the cartridge and thus the additional steps associated with transferring the sample to an analytical instrument can be avoided. In another example, a portion of the fluid in one or more of the bioreactor and mixing chamber may be taken out via a fluid conduit and transferred to an analytical instrument. The analytical instrument may be located within the working cell (e.g., online instrument), or, in some modifications, outside the working cell (e.g., offline instrument). The analytical instrument may be configured to measure one or more of the lactate and glucose values ​​of a sample. The flexibility provided by the analytical instrument, which allows for the parallel execution of more than one measurement without interrupting the cell culture process, can facilitate higher throughput with the bioreactor module described herein.

[0042] Referring to Figure 1C, a block diagram of an exemplary modification of the bioreactor module 150 is shown. The bioreactor module 150 may comprise a bioreactor 1110, a mixing chamber 1130, a first thermal compartment 1140, a second thermal compartment 1150, a third thermal compartment 1160, a gear system 1170, a sensor 1173, a heat exchanger 1174, and a port system 1175. The bioreactor 1110, the mixing chamber 1130, the first thermal compartment 1140, the second thermal compartment 1150, and the third thermal compartment 1160 may be connected fluidically and / or thermally. For example, a fluid may be transferred between one or more of the bioreactor 1110, the mixing chamber 1130, the first thermal compartment 1140, the second thermal compartment 1150, and the third thermal compartment 1160. In another example, heat exchangers may be connected to one or more of the bioreactor 1110, mixing chamber 1130, first thermal compartment 1140, second thermal compartment 1150, and third thermal compartment 1160, thereby enabling the maintenance of a consistent thermal environment throughout the entire bioreactor module 150.

[0043] In some modifications, the bioreactor 1110, mixing chamber 1130, first thermal compartment 1140, second thermal compartment 1150, and third thermal compartment 1160 may be arranged within the bioreactor module 150 in any configuration. For example, the first thermal compartment 1140 may be located adjacent to the bioreactor, and / or the second thermal compartment 1150 may be located adjacent to the bioreactor. In some modifications, the first thermal compartment 1140 and the second thermal compartment 1150 may interlock with each other; that is, the first thermal compartment 1140 and the second thermal compartment 1150 may have corresponding shapes. For example, one or more side walls of the first thermal compartment 1140 may include one or more bends, and one or more side walls of the second thermal compartment 1150 may include one or more bends that closely resemble one or more bends of the first thermal compartment 1140. In some modifications, the first thermal compartment 1140 and the second thermal compartment 1150 may share a side wall, and as a result, the fluid in the first thermal compartment 1140 may be located on the first side of the shared side wall, and the fluid in the second thermal compartment 1150 may be located on the second side of the shared side wall. The configurations described herein can facilitate heat transfer, and as a result, the thermal environment within the bioreactor module can be made relatively uniform throughout. Maintaining a relatively uniform thermal environment can avoid problems associated with cells experiencing sudden temperature changes, otherwise the cell culture and / or growth may be inhibited, slowed, or stopped. That is, ideally, cells should be kept at a constant temperature when they are transferred between components of the bioreactor module.

[0044] The bioreactor 1110 may be configured to hold a certain volume of fluid and to perform one or more processes on the fluid therein. For example, the bioreactor 1110 may be configured to perform one or more of the following processes: a stirring process, a static process, and a perfusion process, as described herein. That is, the bioreactor 1110 may be advantageous in that it can perform one or more processes within the bioreactor 1110 itself, thereby facilitating high-throughput cell processing by reducing or eliminating the need to transfer the fluid to another module. One or more processes and / or fluid transfer into and out of the bioreactor 1110 may be performed according to a predetermined workflow. In some modifications, the bioreactor 1110 may have one or more sidewalls that may be impermeable to liquids and / or gases. The bioreactor 1110 may be configured to hold a fluid volume of approximately 5 mL to 2 L, approximately 50 mL to 800 mL, or approximately 100 mL to 600 mL, including approximately 50 mL, approximately 100 mL, approximately 200 mL, approximately 300 mL, approximately 400 mL, approximately 500 mL, approximately 600 mL, approximately 700 mL, approximately 800 mL, approximately 900 mL, approximately 1 L, approximately 1.5 L, or approximately 2 L. The bioreactor 1110 may have a cross-sectional shape such as circular, elliptical, rectangular, triangular, or a combination thereof. The bioreactor 1110 will be described in more detail with reference to Figure 1D.

[0045] The mixing chamber 1130 may be configured to hold a certain volume of fluid and to perform one or more processes on the fluid within it. For example, the mixing chamber 1130 may be configured to perform a stirring process. That is, the mixing chamber 1130 may accept one or more reagents, which may be combined with the fluid using an impeller in the mixing chamber 1130. The impeller may rotate so that one or more reagents are mixed (e.g., in contact with) target cells (e.g., cells intended for further processing and / or use in cell therapy). The resulting mixture may be transferred from the mixing chamber 1130 to the bioreactor 1110, thermal compartments 1140, 1150, 1160, and / or other modules of cartridge 114. Fluids and / or reagents may be transferred into and out of the mixing chamber 1130 according to a predetermined workflow. For example, the mixing chamber 1130 may have one or more side walls that may be impermeable to liquids and / or gases. The mixing chamber 1130 may be configured to hold volumes of fluid ranging from approximately 5 mL to approximately 1 L, approximately 50 mL to approximately 800 mL, or approximately 100 mL to approximately 600 mL, including approximately 50 mL, approximately 100 mL, approximately 200 mL, approximately 300 mL, approximately 400 mL, approximately 500 mL, approximately 600 mL, approximately 700 mL, approximately 800 mL, approximately 900 mL, or approximately 1 L. The mixing chamber 1130 may have a cross-sectional shape such as circular, elliptical, rectangular, triangular, or a combination thereof. The mixing chamber 1130 will be described in further detail with reference to Figure 1E.

[0046] Each of the thermal compartments 1140, 1150, and 1160 may be configured to hold a certain volume of fluid in a stable environment. For example, one or more thermal compartments 1140, 1150, and 1160 may be thermally connected to the bioreactor 1110 and / or the mixing chamber 1130, resulting in thermal equilibrium between them. A thermally balanced configuration can facilitate cell culture by mitigating problems associated with the cell solution experiencing temperature changes. For example, if the temperature of the cell solution falls below the intended value, cell growth and / or division may slow down or stop completely. In another example, if the temperature of the cell solution rises above the intended value, one or more cellular proteins may denature. Additionally or alternatively, the thermal compartments 1140, 1150, and 1160 may maintain a stable gaseous environment within them. That is, one or more thermal compartments 1140, 1150, 1160 may be fluidly connected to a port system 1175, which may be configured to provide oxygen, carbon dioxide, nitrogen, and / or sterile air as needed to maintain the cell solution at a stable pH and with sufficient oxygen and / or nitrogen dissolved therein. Thus, the thermal compartments 1140, 1150, 1160 may be configured to promote cell culture by maintaining a thermal and / or gaseous environment. In some modifications, each of the thermal compartments 1140, 1150, 1160 may have one or more side walls that may be impermeable to liquids and / or gases. Each of the thermal compartments 1140, 1150, and 1160 may be configured to hold a volume of fluid ranging from approximately 5 mL to approximately 1 L, approximately 50 mL to approximately 800 mL, or approximately 100 mL to approximately 600 mL, including approximately 50 mL, approximately 100 mL, approximately 200 mL, approximately 300 mL, approximately 400 mL, approximately 500 mL, approximately 600 mL, approximately 700 mL, approximately 800 mL, approximately 900 mL, or approximately 1 L. In some modifications, the thermal compartments 1140, 1150, and 1160 may, but are not required, be configured to hold the same volume of fluid. For example, the first thermal compartment 1140 may be configured to hold a volume of approximately 600 mL, the second thermal compartment 1150 may be configured to hold a volume of approximately 100 mL, and the third thermal compartment 1160 may be configured to hold a volume of approximately 600 mL.The volume may be determined by a predetermined workflow. For example, after the cell sorting step, the cell material may be transferred to a second thermal compartment 1150. The second thermal compartment 1150, with its relatively small volume, may correspond to a relatively small number of cells that can be obtained through the aforementioned cell sorting step. In another example, the 600 mL capacities of the first and third thermal compartments 1140 and 1160 may be suitable for storing cell material after performing a growth step and may correspond to a relatively large number of cells. Fluids may be transferred into and / or out of each of the thermal compartments 1140, 1150, and 1160 according to a predetermined workflow. Each of the thermal compartments 1140, 1150, and 1160 will be described in further detail with reference to Figures 1G to 1I, respectively.

[0047] The heat exchanger 1174 may be configured to maintain the thermal environment within the bioreactor module 150. The heat exchanger 1174 may be thermally connected to one or more of the bioreactor 1110, the mixing chamber 1130, the first thermal compartment 1140, the second thermal compartment 1150, and the third thermal compartment 1160. For example, the heat exchanger 1174 may be configured to generate a certain amount of heat and / or remove a certain amount of heat from components within the bioreactor module. Thermal connection may be facilitated by one or more thermal paths. Thermal paths may be defined by heat conduits and / or thermal pads. Thermal paths (e.g., heat conduits, thermal pads) may include thermally conductive materials. For example, the thermally conductive material may be a metal (e.g., copper, aluminum). In some variations, the thermal pad may be located beneath one or more of the bioreactor 1110, the mixing chamber 1130, the first thermal compartment 1140, the second thermal compartment 1150, and the third thermal compartment 1160. The thermal pad may be substantially flat, so as to be able to connect with the corresponding flat surfaces of the bioreactor 1110, the mixing chamber 1130, the first thermal compartment 1140, the second thermal compartment 1150, and / or the third thermal compartment 1160. Heat conduits may connect one or more of the thermal pads located beneath one or more of the bioreactor 1110, the mixing chamber 1130, the first thermal compartment 1140, the second thermal compartment 1150, and the third thermal compartment 1160. The heat exchanger 1174 may be controlled by a controller such as controller 120. For example, the controller 120 can control the amount of heat generated and / or removed by the heat exchanger 1174 by increasing and / or decreasing the current supplied to the heat exchanger 1174. Thus, the heat exchanger 1174 can control the thermal environment of the entire bioreactor module 150.

[0048] The sensor 1173 may be configured to measure one or more parameters of the bioreactor module 150. For example, the sensor 1173 may be operably connected to the bioreactor 1110, the mixing chamber 1130, and / or the thermal compartments 1140, 1150, 1160. The sensor 1173 may be configured to measure temperature, pressure, gas concentration, humidity, pH, fluid level, and / or dissolved oxygen. For example, the sensor 1173 may be operably connected to a window of the bioreactor 1110, so that the sensor 1173 can measure the pH and / or dissolved oxygen values ​​of the fluid in the bioreactor 1110. In another example, the sensor 1173 may measure the gas concentrations (e.g., units of gas per unit volume) of oxygen, nitrogen, and / or carbon dioxide in one or more bioreactors 1110, the mixing chamber 1130, and / or the thermal compartments 1140, 1150, 1160. Sensor 1173 may communicate with a controller such as controller 120. Thus, sensor 1173 can facilitate a closed-loop system by providing one or more measurements to controller 120, which can then adjust one or more parameters of the bioreactor 150 (e.g., temperature, gas concentration, fluid level) in response.

[0049] The gear system 1170 may comprise one or more gears configured to rotate one or more components within the bioreactor module 150. For example, in some modifications, one or more gears may be coupled to the impellers of the mixing chamber 1130 and / or the bioreactor 1110. Advantageously, in some modifications, the gear system 1170 may be optimized to facilitate the motion of the impellers of both the mixing chamber 1130 and the bioreactor 1110. Thus, the impellers of the mixing chamber 1130 and the bioreactor 1110 may share components, thereby reducing the number of components and / or their operational complexity in the bioreactor module 150. For example, a first gear (e.g., a drive gear) may be coupled to each of a second gear (e.g., a first driven gear) and a third gear (e.g., a second driven gear). The first gear can receive an input, and as a result, the first gear can rotate in response to the input. For example, the input may be provided via one or more electromagnets connected to the first gear, thereby allowing the first gear to rotate by supplying current to the electromagnets. In another example, the input may be provided via a motor connected to the first gear so that the first gear can be rotated by operating the motor. The rotation of the first gear can rotate each of the second and third gears. The amount of rotation may be determined by a gear ratio defined by the number of teeth of the drive gear relative to the number of teeth of the driven gear. In some variations, the gear ratio may be about 5:1 to about 1:1. The gear ratio may be optimized to minimize the amount of power required to operate the gear system 1170. For example, in some variations, the gear ratio may be 5:1, 4:1, 3:1, 2:1, or 1:1. In some variations, there may be 1 to 10 gears, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 gears. Each gear may be connected to the others, so that rotating one gear can rotate all the other gears. In a further variation, each gear may not be connected to the others, so that each gear can rotate independently in response to an input.For example, each gear may be connected to an electromagnet, and as a result, each electromagnet can be controlled by an electrical signal.

[0050] The port system 1175 may be configured to transfer one or more gases to and / or from one or more of the bioreactor 1110, the mixing chamber 1130, the first thermal compartment 1140, the second thermal compartment 1150, and the third thermal compartment 1160. The port system 1175 may be configured to facilitate a gaseous environment within the bioreactor module 150 that is suitable for cell growth and / or culture. The port system 1175 will be described in more detail with reference to Figure 1F.

[0051] Figure 1D shows a block diagram of the bioreactor 1110. The bioreactor 1110 may comprise an impeller 1112, a liner 1114, a perfusion filter 1116, a window 1118, a sampling straw 1120, and a fluid transfer straw 1122. The impeller 1112 may comprise a shaft and one or more impeller blades connected to the shaft. The shaft may be configured to rotate, so that the impeller blades rotate around an axis defined by the shaft. The impeller blades may be configured to agitate the fluid; that is, the impeller blades may have a shape configured to displace a certain volume of fluid. For example, the impeller blades may have a cross-sectional shape such as rectangular, triangular, circular, or a combination thereof. The impeller 1112 may have 1 to 10 impeller blades, including 1, 2, 3, 4, 5, 6, 8, or 10 impeller blades. The impeller 1112 may be configured to rotate at predetermined speeds such as approximately 1 rpm to approximately 2000 rpm, approximately 250 rpm to approximately 1500 rpm, approximately 500 rpm to approximately 1500 rpm, or approximately 1 rpm to approximately 1000 rpm, including approximately 100 rpm, 200 rpm, approximately 300 rpm, approximately 400 rpm, approximately 500 rpm to approximately 600 rpm, approximately 700 rpm, approximately 800 rpm, approximately 900 rpm, or approximately 1000 rpm. The predetermined speed may relate to the stirring process, which may then relate to cell proliferation. For example, the stirring process may be adjusted based on the type of cells contained in the cell solution. That is, certain cells (e.g., T cells) can withstand relatively vigorous stirring, so a relatively fast rotation speed (e.g., approximately 1000 rpm) may be used. Other cells, such as human pluripotent stem cells (hPSCs), may only survive if a relatively slow rotation speed (e.g., approximately 100 rpm) is used.

[0052] The liner 1114 may be configured to be gas permeable. For example, a gas (e.g., air) introduced through a gas port can permeate the liner 1114. The liner 1114 may be positioned along the inner surface of the sidewall of the bioreactor 1110. In some modifications, the entire surface of the bioreactor sidewall may be covered by the liner 1114. The liner 1114 may not be liquid permeable, as this would allow liquid to be retained within the bioreactor 1110. In some modifications, the liner 1114 may be formed integrally with the bioreactor 1110. Thus, in some modifications, the bioreactor 1110 may be gas permeable. The liner may be made from a gas permeable material such as polydimethylsiloxane (PDMS), superhydrophobic polyvinylidene fluoride (PVDF), or superhydrophobic polyether sulfone (PES).

[0053] The perfusion filter 1116 may be configured to filter fluid during the perfusion process. For example, the perfusion filter 1116 may comprise a substrate connected to a fluid conduit. The substrate may have a plurality of openings, which may be configured to allow fluid to flow through them while preventing cells from flowing through them. The fluid conduit may be configured to provide suction, thereby allowing fluid to be drawn from within the bioreactor 1110 into the fluid conduit through the plurality of openings in the substrate during the perfusion process. The fluid conduit may be fluidically connected to a fluid manifold. The substrate of the perfusion filter 1116 may be positioned close to the inner surface of the side wall of the bioreactor 1110. The fluid conduit may extend over the top of the bioreactor 1110. Placing the fluid conduit over the top of the bioreactor 1110 can prevent unintended fluid flow (e.g., leakage). In some modifications, multiple perfusion filters may be present. For example, there may be 1 to 4 perfusion filters, such as 1, 2, 3, or 4 perfusion filters.

[0054] The fluid transfer straw 1122 may be configured to transfer fluid into and / or out of the bioreactor 1110. For example, the fluid transfer straw 1122 may have a fluid conduit that can be fluidically connected to a fluid manifold. The fluid conduit may have a cross-sectional shape such as circular, square, triangular, or a combination thereof. The fluid transfer straw 1122 may be connected to a fluid transfer port located in conjunction with the bioreactor 1110. The fluid transfer straw 1122 may transfer fluid to the bioreactor 1110 via the fluid transfer port. For example, the fluid may be transferred to the bioreactor after being removed through the perfusion filter 1116 in a perfusion process. In another example, the fluid may be transferred to the bioreactor after being removed through the sampling straw 1120. In some variations, there may be multiple fluid transfer straws. For example, there may be one to four fluid transfer straws, such as one, two, three, or four fluid transfer straws.

[0055] The sampling straw 1120 may be configured to transfer fluid outside the bioreactor 1110 for sampling. For example, the sampling straw 1120 may have a fluid conduit that can be fluidically connected to a fluid manifold. The sampling straw 1120 can extract at least a portion of the fluid in the bioreactor 1110 (e.g., a sample) by applying suction through its opening. In some modifications, the sampling straw 1120 may be configured to perform one or more measurements on the sample. For example, the sampling straw 1120 can measure the glucose and / or lactate levels of the fluid. In further modifications, the sample may be transferred from the sampling straw to a fluid manifold, thereby, in some modifications, the sample may be further transferred to a portable fluid device and / or analytical instrument. The analytical instrument may be configured to perform one or more measurements on the sample. The fluid conduit may have a cross-sectional shape such as circular, square, triangular, or a combination thereof. The sampling straw 1120 may have an opening located inside the bioreactor 1110. In some variations, multiple sampling straws may be present. For example, there may be 1 to 4 sampling straws, such as 1, 2, 3, 4, or even more.

[0056] The window 1118 may be configured to allow one or more measurements. For example, the window 1118 may be configured for optical detection. That is, the window 1118 may include a transparent material, as a result allowing a sensor positioned close to the window 1118 to measure one or more parameters within the bioreactor 1110. One or more parameters may be fluid level, pH, temperature, and dissolved oxygen. The window may extend along a portion of the side wall of the bioreactor 1110. In some modifications, the window may be positioned along the bottom of the bioreactor 1110. The window along the bottom of the bioreactor 1110 may be operably coupled to a sensor 1175, which may be an optical sensor configured to measure the pH and / or dissolved oxygen of the fluid contained within the bioreactor 1110. In further modifications, the window may extend along the entire side wall of the bioreactor 1110. For example, the bioreactor 1110 may be integrally formed with the window portion 1118. The window portion 1118 may be made from a polymer (e.g., polyethylene terephthalate glycol, polymethyl methacrylate) or glass. In some modifications, multiple window portions may be present. For example, there may be 1 to 10 window portions, 1 to 8 window portions, or 1 to 4 window portions, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 window portions.

[0057] Sensor 1124 may be configured to measure one or more parameters within the bioreactor 1110. In some modifications, sensor 1124 may include a pressure sensor, a temperature sensor, an optical sensor, a humidity sensor, or a pH sensor. For example, sensor 1124 may include a temperature sensor (e.g., a thermistor, a thermocouple) configured to measure the temperature of the fluid within the bioreactor 1110. Sensor 1124 may be positioned close to the bottom surface of the bioreactor 1110 so that the sensor can come into contact with the liquid contained within the bioreactor. For example, sensor 1124 may be a liquid temperature sensor so that the bulk temperature of the liquid within the bioreactor 1110 can be measured by the liquid temperature sensor. In a further modification, sensor 1124 may be a gas temperature sensor so that the bulk temperature of the gas within the bioreactor 1110 can be measured by the temperature sensor. In such modifications, the sensor 1124 may be positioned close to the top surface of the bioreactor, thereby enabling measurement of the gas temperature within the bioreactor, but not the liquid temperature. In other words, in these modifications, the gas temperature sensor does not come into contact with the liquid contained within the bioreactor.

[0058] Figure 1E shows a block diagram of an exemplary mixing chamber 1130. The mixing chamber 1130 may comprise an impeller 1132, a window 1134, a sampling straw 1136, a fluid transfer straw 1138, and a sensor 1139. The impeller 1132 may correspond to the description provided for impeller 1112 with reference to Figure 1D. Similarly, the window 1134 may correspond to the description provided for window 1118, the sampling straw 1136 may correspond to the description provided for sampling straw 1120, the fluid transfer straw 1138 may correspond to the description provided for fluid transfer straw 1122, and the sensor 1139 may correspond to the description provided for sensor 1124 with reference to Figure 1D.

[0059] Figure 1F shows a block diagram of an exemplary port system 1175 configured to control the gaseous environment of the bioreactor module 150. The port system 1175 may comprise any number of preferred ports, e.g., a gas port 1182, a vent port 1176, a pressure port 1178, and a vacuum port 1180. One or more of the gas port 1182, vent port 1176, pressure port 1178, and vacuum port 1180 may have outlets connected to fluid conduits. The fluid conduits may be fluidly connected to a fluid manifold. The fluid manifold may transport one or more gases to and from the bioreactor module 150 in response to a given workflow and / or one or more measurements generated by sensors. That is, the port system 1175 may be configured to control the amount of gas above the liquid in the bioreactor, mixing chamber, and / or thermal compartment, i.e., the headspace environment. For example, the gas port 1182 may be configured to transfer one or more gases to the bioreactor module 150 (e.g., its headspace environment). That is, the gas port 1182 may transfer nitrogen, oxygen, carbon dioxide, sterile air, or a combination thereof. In some variations, multiple gas ports may be present. For example, there may be 1 to 10 gas ports, including 1, 2, 3, 4, 5, 6, 8, or 10 gas ports. The gas port 1182 may be fluidically connected to one or more of the bioreactor 1110, the mixing chamber 1130, the first thermal compartment 1140, the second thermal compartment 1150, and the third thermal compartment 1160. Thus, the gas port 1182 may be configured to provide a favorable environment for one or more cellular processes throughout the bioreactor module 150 by transferring one or more gases that can be consumed by cells and / or maintain the pH of the fluid within a target range.

[0060] The vent port 1176 may be used when performing an integrity check of the bioreactor module 150 to determine whether there is leakage. For example, the vent port 1176 may be configured to transfer a fluid (e.g., gas) into the bioreactor module at a relatively high pressure. For example, the relatively high pressure could be about 1 psi to 10 psi, including about 1 psi, 3 psi, or 5 psi, which is higher than the existing pressure in the bioreactor module. An integrity check (e.g., a leak test) may be used to determine one or more leaks in the bioreactor module. For example, the relatively high-pressure gas supplied through the vent port 1176 may be observed for a predetermined period (e.g., 1 hour). The leak rate can be determined by dividing the pressure drop by the predetermined time. A leak rate above a predetermined threshold may indicate leakage in the bioreactor module 150 (e.g., through a crack in the side wall). To verify the integrity of the bioreactor module 150, integrity checks may be performed before, during, and / or after any of the cell processes described herein.

[0061] A pressure port 1178 may be used to assist in reducing the pressure within the bioreactor module 150. The pressure port 1178 may be fluidically connected to a fluid manifold by a fluid conduit. For example, in some modifications, the pressure port 1178 may include a pressure relief valve configured to open in response to a given pressure measurement within the bioreactor module that exceeds a predetermined threshold. The predetermined threshold may correspond to pressures such as approximately 1 psi to 5 psi, including 1 psi, 2 psi, 3 psi, 4 psi, or 5 psi. In another example, a sensor such as a pressure sensor may measure a pressure value exceeding a threshold measurement within one or more of the bioreactor 1110, mixing chamber 1130, first thermal compartment 1140, second thermal compartment 1150, and third thermal compartment 1160. In response, the controller may send an electrical signal to the pressure port 1178 to open, thereby allowing fluid (e.g., gas) to be transferred out of the bioreactor module 150 to reduce the pressure within it.

[0062] When one or more of the bioreactor, mixing chamber, and thermal compartments do not contain cell material, the vacuum port 1180 may be used to assist in the transfer of fluid (e.g., gas) out of the bioreactor module 150. The vacuum port 1180 may be fluidically connected to a fluid manifold by a fluid conduit. In some modifications, the fluid manifold may provide suction to the vacuum port 1180, which can then be applied to one or more of the bioreactor 1110, mixing chamber 1130, first thermal compartment 1140, second thermal compartment 1150, and third thermal compartment 1160. Thus, suction can remove substantially all gas from any part of the bioreactor module 150. Suction can establish an empty environment within the bioreactor module 150, as a result allowing one or more gases to be reintroduced through the gas port 1182.

[0063] Figures 1G to 1I are illustrative block diagrams of the first thermal compartment 1140, the second thermal compartment 1150, and the third thermal compartment 1160, respectively. In some modifications, the thermal compartments 1140, 1150, and 1160 may be referred to as intermediate cell vessels, cell hotels, or compartments. The thermal compartments 1140, 1150, and 1160 may be configured to contain a fluid (e.g., cell solution) in a stable environment. Each of the thermal compartments 1140, 1150, and 1160 may be provided with fluid ports 1142, 1152, and 1162 and windows 1144, 1154, and 1164. The windows 1144, 1154, and 1164 may correspond to the description provided for window 1118 with reference to Figure 1D. Fluid ports 1142, 1152, and 1162 may be configured to transfer fluid into and / or out of the fluid thermal compartments 1140, 1150, and 1160. Fluid ports 1142, 1152, and 1162 may be connected to fluid conduits, which in turn may be fluidically connected to a fluid manifold. Thus, the flow of fluid into and / or out of the thermal compartments 1140, 1150, and 1160 may be controlled via the fluid manifold. For example, fluid may be transferred between the first thermal compartment 1140 and the bioreactor 1110. In another example, fluid may be transferred between the first thermal compartment 1140 and the second thermal compartment 1150. In some modifications, each of the thermal compartments 1140, 1150, and 1160, the bioreactor 1110, and the mixing chamber 1130 may be fluidically connected.

[0064] Each of the thermal compartments 1140, 1150, and 1160 can be thermally connected to each other or to other components of the bioreactor module. That is, in some modifications, the thermal compartments 1140, 1150, and 1160 are maintained at the same temperature relative to each other and to the bioreactor 1110 and / or mixing chamber 1130. For example, heat can be transferred to and from the thermal compartments 1140, 1150, and 1160 via the heat exchanger 1174. In another example, heat can be transferred through one or more side walls of the thermal compartments 1140, 1150, and 1160. The compartments 1140, 1150, and 1160 can also be thermally connected to one or more of the bioreactor and mixing chambers. A stable thermal environment within the bioreactor module can help facilitate cell culture by avoiding thermal shocks to the cell solution (e.g., rapid temperature changes that could harm or interrupt the cell culture process) when moving the cell solution between one or more of the thermal compartments 1140, 1150, 1160, bioreactor 1110, and mixing chamber 1130.

[0065] Figures 3A to 3E show exemplary variations of the bioreactor module 300. The bioreactor module 300 may comprise a bioreactor 310, a mixing chamber 320, a first thermal compartment 340, a second thermal compartment 350, a third thermal compartment 360, a first plurality of fluid ports 370a, and a second plurality of fluid ports 370b. The plurality of fluid ports 370a, 370b may be located on the upper surface of the bioreactor module 300. The plurality of fluid ports 370a, 370b may be configured to transfer fluid to one or more of the bioreactor 310, the mixing chamber 320, the first thermal compartment 340, the second thermal compartment 350, and the third thermal compartment 360. In other words, the multiple fluid ports 370a, 370b can be fluidically connected to the fluid manifold, and as a result, fluid can be transferred from the fluid manifold to the bioreactor module and / or from the fluid manifold to the bioreactor module via the multiple fluid ports 370a, 370b. The multiple fluid ports 370a, 370b will be described in more detail with reference to Figure 3E.

[0066] As illustrated, the bioreactor 310 may have side walls configured to contain fluid. The side walls of the bioreactor 310 may be connected to a gas (e.g., air) permeable liner (not shown). Thus, the bioreactor 310 may be configured to hold liquid while allowing gas to flow through the side walls. In some modifications, the side walls and / or gas permeable liner may be bidirectional permeable. In this way, gas can be permeated into and around the bioreactor 310 so that equilibrium can be established between the bioreactor and adjacent components (e.g., one or more of the thermal compartment and mixing chamber). Additionally or alternatively, the gas permeability of the side walls may prevent a pressure increase within the bioreactor 310. For example, relatively high pressure can permeate through the gas permeable liner to areas with lower pressure. The bioreactor may further have a concave bottom. The concave shape of the bottom may help prevent cells or other materials from sticking to its inner surface. Furthermore, the concave shape of the bottom surface may provide a cavity for receiving the impeller blade, which can help allow the impeller blade to agitate substantially all of the cells contained within the bioreactor 310.

[0067] As shown in Figures 3B to 3C, the bioreactor 310 may comprise an impeller 312 having an impeller shaft 312b connected to a plurality of impeller blades 312a, a perfusion filter 314a connected to a fluid conduit 314b, a fluid transfer straw 313, and a sampling straw 316. The impeller shaft 312b may be positioned along a central axis defined by the bioreactor 310. The position of the impeller 312 may help facilitate the uniform mixing of the fluid contained therein. That is, if the position of the impeller blades 312b is close to the bottom surface of the bioreactor 310, it can promote the formation of vortices as the impeller 312 rotates, and as a result the vortices can easily homogenize substantially all of the fluid by mixing or swirling it. The position of the impeller blades 312a may be at the distal end of the impeller shaft 312b. That is, the proximal end of the shaft 312b may be operably connected to the upper surface of the bioreactor 310, and the distal end may be close to the bottom surface of the bioreactor 310. The impeller 312 may comprise any number of impeller blades as desired, having any preferred shape and / or configuration. In some modifications, the impeller comprises three blades 312a, as illustrated in Figure 3C. The impeller blades 312a may be pitched blades that can help gently agitate the fluid without damaging the cells inside. That is, the impeller blades 312a may be angled with respect to the impeller shaft 312b. The angle of the impeller blades 312a may range from about 5 degrees to about 60 degrees, including about 45 degrees.

[0068] The fluid transfer straw 313 may be configured to transfer fluid to and / or from the bioreactor 310 to assist in facilitating one or more cell processing steps. For example, the fluid transfer straw 313 may be fluidically connected to a fluid manifold, as a result enabling the fluid manifold to supply fluid to the bioreactor 310 via the fluid transfer straw. Fluid may be provided to carry out a seeding process, to transfer fresh medium during a perfusion process (e.g., after removing the medium via the perfusion filter 314a), and / or to carry out the transfer of fluid from another part of the bioreactor module (e.g., a mixing chamber 320, thermal compartments 340, 350, 360). The fluid transfer straw 313 may be coaxial with the impeller shaft 312b. In some modifications, the fluid transfer straw 313 may remain stationary while the impeller shaft 312b rotates around the fluid transfer straw 313.

[0069] The perfusion filter 314a may be configured to facilitate the perfusion process by retaining solid particles (e.g., cells) within the bioreactor 310 while allowing liquid to flow through it. For example, the perfusion filter 314a may have multiple openings on one or more of its inner and outer surfaces configured to allow liquid to flow through while preventing fine particles from flowing through it. That is, the inner surface may define the inner diameter of the outer periphery of the perfusion filter 314a, and the outer surface may define its outer diameter. Thus, the perfusion filter 314a can receive fluid (e.g., liquid) from either side. The perfusion filter 314a may extend circumferentially around the inner surface of the bioreactor 310. The perfusion filter 314a may be positioned around the impeller shaft 312b so that the impeller blades 312a can rotate freely. The perfusion filter 314a may have a height lower than the height of the bioreactor 310. The height of the perfusion filter 314a relative to the bioreactor 310 can facilitate the perfusion process by keeping the perfusion filter 314a submerged in the fluid (e.g., liquid) that can be contained within the bioreactor 310. For example, the height of the perfusion filter 314a may be about 1 / 6 to 1 / 2 of the height of the bioreactor 310. The perfusion filter 314a can be connected to a fluid conduit 314b. The fluid conduit 314b can be fluidically connected to a fluid manifold. Thus, the fluid removed from the bioreactor 310 via the perfusion filter 314a can be transferred to the fluid manifold for further processing (e.g., additional filtration, waste).

[0070] The sampling straw 316 may be configured to sample at least a portion of the fluid contained within the bioreactor 310. For example, the sampling straw may have an opening close to the bottom surface of the bioreactor 310 so that the opening can be exposed to the liquid. The sampling straw 316 may be further configured to measure one or more parameters of the sample. For example, the sampling straw 316 may include a lactate sensor, a glucose sensor, a pH sensor, a temperature sensor, and / or a dissolved oxygen sensor. The heat exchanger 316 may be electrically connected to a controller such as the controller 120. Thus, one or more measurements obtained via the sampling straw 316 can be transmitted to the controller 120. In some modifications, a gap may exist between the opening of the sampling straw and the bottom surface of the bioreactor 310 to avoid clogging of the opening. The sampling straw 316 may be positioned between the perfusion filter 314a and the side wall of the bioreactor 310. In this way, the sampling straw 316 cannot interfere with the rotation of the impeller 312 and / or the functionality of the perfusion filter 314a. That is, a gap can be maintained between the sampling straw 316 and each of the impeller 312 and the perfusion filter 314a. In this way, the vortex formed by the impeller 312 cannot be blocked by the sampling straw 316.

[0071] The mixing chamber 320 may comprise an impeller 322 having an impeller shaft 322c connected to a plurality of impeller blades 322a, 322b, a fluid transfer straw 323, and a sampling straw 326. The impeller shaft 322c may be positioned along a central axis defined by the mixing chamber 320. The position of the impeller 322 can facilitate the uniform mixing of the fluid contained within it. That is, if the impeller blades 322b are positioned close to the bottom surface of the mixing chamber 320, the formation of vortices can be facilitated when the impeller 322 rotates, and as a result, the vortices can easily homogenize substantially all of the fluid by mixing or swirling it. Mixing can be further facilitated by additional impeller blades 322b positioned further away from the bottom surface of the mixing chamber 320 along the impeller shaft 322c. That is, additional impeller blades 322b can facilitate a more effective mixing process by extending the vortices vertically within the mixing chamber 320. Any number of impeller blades may be used, and the impeller blades may have any preferred shape and / or configuration. In some modifications, each impeller may have three impeller blades, and as a result, the mixing chamber 320 may have a total of six impeller blades. In some modifications, the impeller blades 322a, 322b are flat blades (e.g., Rushton impeller design), which may be configured to vigorously agitate the fluid. The impeller blade 312a may be coaxial with the impeller shaft 322c, or, in some modifications, may be angled with respect to the impeller shaft 322c.

[0072] The impellers described herein can be coupled to one another to optimize spatial constraints within the bioreactor module 300. For example, impellers 312 and 322 can each be coupled to one or more gears. As shown in Figure 3B, impeller 312 can be coupled to gear 342b, and impeller 322 can be coupled to gear 342c. Gears 342b and 342c can be coupled to one another by gear 342a. Thus, the rotation of gear 342a can rotate both gears 342b and 342c. In this way, impellers 312 and 322 can rotate in tandem in response to a single input; that is, both impellers 312 and 322 can be rotated using a single actuator. Advantageously, this reduces operational complexity by minimizing the number of inputs required to rotate the impellers 312 and 322 and / or reducing the number of actuators required, thereby reducing the overall size of the bioreactor module 300.

[0073] The fluid transfer straw 323 may be coaxial with the impeller shaft 322c. The fluid transfer straw 323 may be configured to transfer fluid to and / or from the mixing chamber 320. For example, the fluid transfer straw 323 may be fluidically connected to a fluid manifold, so that the fluid manifold can supply fluid to the mixing chamber 320 via the fluid transfer straw. The fluid supplied by the fluid manifold may contain one or more reagents, which can be combined with the cell solution in the mixing chamber 320. That is, one or more reagents can be mixed with the cell solution using the impeller 322. In some modifications, the mixed solution can then be transferred to one or more of the thermal compartments 340, 350, 360 and the bioreactor 310. In some modifications, the fluid transfer straw 323 may remain stationary while the impeller shaft 322c rotates around the fluid transfer straw 323.

[0074] The sampling straw 326 may be configured to sample at least a portion of the fluid contained within the mixing chamber 320. For example, the sampling straw 326 may have an opening close to the bottom of the mixing chamber 320 so that the opening can be exposed to the liquid. Similar to the description provided for the sampling straw 316 of the bioreactor 310, the sampling straw 326 may be configured to measure one or more parameters of the fluid contained therein. To avoid clogging of the opening, a gap may exist between the opening of the sampling straw 326 and the bottom of the mixing chamber 320. The sampling straw 326 may be positioned between the impeller 322 and the side wall of the mixing chamber 320. In this way, the sampling straw 326 cannot obstruct the rotation of the impeller 322 and / or the vortices formed by the impeller 322.

[0075] As illustrated in Figure 3C, the bioreactor 310 and / or mixing chamber 320 may further include sensors. For example, the bioreactor 310 may include sensor 380a, and the mixing chamber may include sensor 380b. Sensors 380a and 380b may be configured to measure one or more parameters within the bioreactor 310 and mixing chamber 320, respectively. For example, sensors 380a and 380b may include temperature sensors (e.g., thermistors) configured to measure the liquid temperature of the liquid contained within their respective components. Temperature measurements generated via sensors 380a and 380b may be transmitted to a controller. In response to the temperature measurements, the controller may adjust the temperature of the bioreactor 310 and / or mixing chamber 320 via a heat exchanger. For example, the controller may adjust the temperature to facilitate one or more cell processing steps. That is, in some modifications, a given cell processing can be optimally performed at a predetermined temperature according to a predetermined workflow. For example, cell proliferation and / or division can be optimally carried out at approximately 15°C to 25°C, for example, approximately 20°C. Therefore, the controller may use a heat exchanger to adjust the temperature of the bioreactor 310 and / or mixing chamber 320 so that the respective sensors 380a and 380b measure the temperature corresponding to a predetermined temperature.

[0076] Figure 3D shows a bottom view of the bioreactor module 300. The bioreactor 310 may comprise optical detection windows 382a, 382b, thermal pads 390, 392, and a magnetic base 384a. For example, as shown, the bioreactor 310 may comprise a first optical detection window 382a and a second optical detection window 382b. The optical detection windows 382a, 382b may be configured to facilitate the measurement of one or more parameters. That is, the optical detection windows 382a, 382b may be positioned below the bottom of the bioreactor 310 so as to measure parameters associated with the fluid (e.g., liquid) contained therein. For example, a sensor such as the sensor 1173 described with reference to Figure 1C may be operably coupled to the optical detection windows 382a, 382b. In some modifications, one or more parameters may be pH and dissolved oxygen. For example, the optical detection window 382a may include a pH detection window, and the optical detection window 382b may include a dissolved oxygen window. In some modifications, the pH and / or dissolved oxygen measurements may correspond to a response by the bioreactor module. That is, when the pH measurement falls below or exceeds a predetermined value, a perfusion process (e.g., medium exchange) may be initiated to bring the pH of the cell solution to a predetermined value. In another example, when the dissolved oxygen value falls below or exceeds a predetermined value, a gas transfer process may be initiated. The gas transfer process may include removing one or more gases from the bioreactor module via a port system (e.g., port system 1175 as described with reference to Figure 1F) and transferring a new gas (e.g., oxygen) to the bioreactor module. Measurements facilitated by the optical detection windows 382a and 382b may be evaluated in real time by a controller, which may automatically adjust one or more environmental conditions associated with the bioreactor module (e.g., in a closed-loop system).

[0077] The bioreactor module 300 may have a thermal network configured to maintain its thermal environment. For example, the bioreactor module 300 may include thermal pads 390, 392 which can be thermally connected to a heat exchanger, such as the heat exchanger 378 illustrated in Figure 1E. The heat exchanger 378 may be configured to generate a certain amount of heat and / or remove a certain amount of heat from the bioreactor module 310 via the thermal pads 390, 392. For example, the heat exchanger 378 may include an electric heat exchanger, which can then provide the heat exchanger 378 with electrical signals to control the amount of heat supplied to or removed from the bioreactor module 310 via the thermal pads 390, 392. The heat exchanger 378 may be thermally connected to the thermal pads 390, 392 by thermal paths. The thermal paths and thermal pads 390, 392 may include thermally conductive metals such as copper, aluminum, or alloys thereof. The thermal pads 390 and 392 may be configured to transfer heat to components within the bioreactor module 300. For example, thermal pad 390 may be positioned beneath the bottom surface of the bioreactor 310. That is, thermal pad 390 may form a thermal conduction path with the bottom surface of the bioreactor 310. Thermal pad 390 may have a shape corresponding to the bottom surface of the bioreactor 310. That is, thermal pad 390 may cover substantially the entire bottom surface of the bioreactor 310. Thus, thermal pad 390 can transfer heat to or receive heat from the bioreactor 310. In another example, thermal pad 392 may be located beneath the bottom surface of the mixing chamber 320. That is, thermal pad 392 may form a thermal conduction path with the bottom surface of the mixing chamber 320. Thermal pad 392 may have a shape corresponding to the bottom surface of the mixing chamber 320. That is, thermal pad 390 may cover substantially the entire bottom surface of the mixing chamber 320. Therefore, the heat pad 392 can transfer heat to or receive heat from the mixing chamber 320.

[0078] The bioreactor module 300 may further include a magnetic coupling configured to move one or more components of the bioreactor module 300. For example, the magnetic coupling may be configured to actuate (e.g., rotate) one or more gears and / or impellers. In some modifications, the magnetic coupling may include a base 384a connected to a shaft 384b as shown in Figure 3E. The base 384a may be isolated from the fluid via the bottom surface of the thermal compartment 340. In this way, the base 384a can avoid interaction with the fluid. The base 384a may receive electrical signals, such as electrical signals transmitted by a controller. The base 384a may include one or a magnet, such as an electromagnet. For example, the electromagnet may be configured to rotate in response to an electrical signal. The rotation of the base 384a can cause the shaft 384b to rotate. The shaft 384b may be routed through the thermal compartment 340, resulting in it extending parallel to the vertical dimensions of the bioreactor module 300. The shaft 384b may be connected to a gear (not shown) located above the thermal compartment 340. Exemplary variations of the gear are described with reference to Figures 4A-4B.

[0079] Figure 3E shows a top view of the bioreactor module 300. As shown, the bioreactor module 300 may further include a pressure port 374, a vacuum port 375, a vent port 376, a gas port 377, and a heat exchanger 378. Each of the pressure port 374, vacuum port 375, vent port 376, and gas port 377 may be fluidically connected to a fluid manifold, thereby allowing the fluid manifold to control the transfer of fluid (e.g., gas) to and from the bioreactor module 300. In some modifications, ports 374-377 may be connected to two or more bioreactor modules. For example, ports 374-377 may be connected to a first bioreactor module and a second bioreactor module. In such modifications, the environments of the first and second bioreactor modules may be maintained independently. In other words, ports 374-377 can be used to transfer gas to and / or from the first and second bioreactor modules without affecting the environment of other bioreactor modules.

[0080] In some modifications, the pressure port 374, vacuum port 375, vent port 376, and gas port 377 can each be used to help maintain a stable environment within the bioreactor module suitable for cell culture. That is, a stable environment may contain one or more nutrients necessary for cell culture. For example, the gas port 377 may be configured to introduce one or more of nitrogen, oxygen, carbon dioxide, and sterile air into any part of the bioreactor module. For example, oxygen, nitrogen, and / or carbon dioxide can be introduced into the mixing chamber and / or bioreactor. Some of the oxygen, nitrogen, and / or carbon dioxide may dissolve in the fluid contained therein. In some modifications, nitrogen and / or oxygen may be consumed by the cells in the fluid during the cell culture process. Carbon dioxide may be useful in maintaining the pH of the fluid within a target range (e.g., about 7.2 to about 7.4) by forming bicarbonate and hydrogen ions. In further modifications, sterile air may be provided to avoid the introduction of any gas particles, otherwise the cell culture process may be inhibited. If any undesirable gaseous particles are introduced into the bioreactor module 300, the environment can be altered by removing at least some of the gas within it via the vacuum port 375.

[0081] The ventilation port 376 may be used to facilitate a stable environment by assisting in ensuring the integrity of the bioreactor module. Specifically, pressurized gas can be introduced through the pressure port 374, which can be used to perform integrity tests (e.g., leak tests). For example, pressurized gas may be introduced at a known pressure, the gas may be maintained in the bioreactor module for a known period of time, and then the gas pressure may be measured. The leak rate can be determined by dividing the difference between the initial known pressure and the measured pressure by the known period of time. An acceptablely small leak rate (e.g., about zero) may indicate that the bioreactor module 300 is suitable for cell culture. In some modifications, the pressurized gas may then be removed from the bioreactor module via the pressure port 374. In further modifications, any gas above a given pressure may be removed from the bioreactor module via the pressure port 374.

[0082] As illustrated in Figure 3E, the top surface of the bioreactor module 300 may be connected to a plurality of fluid ports 370a, 370b, which may be configured to transfer fluid into and / or out of the bioreactor module 300. For example, a plurality of fluid ports 370a may be fluidically connected to fluid transfer ports 372a to 372d. Each of the fluid transfer ports 372a to 372c may be fluidically connected to one or more components via a fluid conduit. For example, fluid transfer port 372a may be fluidically connected to the sampling straw 316 of the bioreactor 310. Fluid transfer port 372b may be fluidically connected to the fluid transfer straw 313 of the bioreactor 310. Fluid transfer port 372c may be fluidically connected to the fluid conduit 314b and the perfusion filter 314a. In another example, a plurality of fluid ports 370b may be fluidically connected to fluid transfer ports 372d to 372g. Each of the fluid transfer ports 372d to 372h can be fluidically connected to one or more components. For example, fluid transfer port 372d can be fluidically connected to the second thermal compartment 350. Fluid transfer port 372e can be fluidically connected to the fluid transfer straw 323 of the mixing chamber. Fluid transfer port 372f can be fluidically connected to the sampling straw 326 of the mixing chamber. Fluid transfer port 372g can be fluidically connected to the third thermal compartment 360.

[0083] Figures 4A and 4B show exemplary modifications of a gear system for a bioreactor module 400 connected to its intermediate surface (e.g., below the upper surface, which has a plurality of fluid ports and conduits as described with reference to Figure 3E). The gear system may be configured to move (e.g., rotate) one or more impellers of the bioreactor module 400. The gear system may have any number of gears as desired. In the modifications illustrated in Figures 4A and 4B, there are a first gear 410, a second gear 412, a third gear 414, and a fourth gear 416. The first gear 410 may be connected to an actuator. For example, the actuator may include an electromagnet. The electromagnet may be configured to receive an electrical signal corresponding to the rotational speed. The electrical signal may be provided by a controller. In some modifications, the first gear 410 may be connected to a shaft 384b as described with reference to Figure 3C. That is, the first gear 410 can be actuated by one or more electromagnets on a magnetic base 384a connected to the shaft 384b. The first gear 410 is sometimes called the driving gear, and each of the second gear 412, the third gear 414, and the fourth gear 416 is sometimes called the driven gear. That is, the rotation of the first gear 410 can cause one or more of the second gear 412, the third gear 414, and the fourth gear 416 to rotate. Thus, the gears 410, 412, 414, and 416 can be magnetically coupled. For example, the teeth of the first gear 410 can engage with the teeth of the second gear 412. The teeth of the second gear 412 can engage with the teeth of the third gear 414 and the fourth gear 416, respectively. The ratio of the first gear 410 to the second gear 412 may be defined by the number of teeth of the first gear 410 relative to the number of teeth of the second gear 412. This ratio may be 5:1 to 1:1, for example, 5:1, 4:1, 3:1, 2:1, or 1:1. This ratio may be determined to minimize the power required to rotate the gears and / or impeller while still facilitating the stirring process described herein. A third gear 414 may be coupled to an impeller of a mixing chamber, such as an impeller 322, as described with reference to Figure 3B.The fourth gear 416 may be coupled to the impeller of a bioreactor, such as the impeller 312 described with reference to Figure 3B. In some modifications, the third gear 414 and the fourth gear 416 may, but do not have to be, equal in size. The ratio of the second gear 412 to the third gear 414 and / or the fourth gear 416 may be 1:1 to 1:5, for example, 1:1, 1:2, 1:3, 1:4, or 1:5.

[0084] II. Methods of cell processing In general, the systems and devices described herein may be used, for example, in an automated cell processing work cell to perform one or more methods of cell processing. The cell processing methods described herein may increase operational efficiency (e.g., reduce delays), reduce and / or eliminate manual intervention (e.g., labor), optimize workspace utilization, and / or increase the throughput of cell processing byproducts. In methods in which cell processing includes cell culture, these methods may be performed automatically, thereby enabling the supply of fluids (e.g., cell suspension, cell solution) to a bioreactor module in a cartridge, and the cell culture process may be performed when the cartridge is connected to the corresponding instrument in the cell processing work cell. The cultured cells may then be transferred out of the bioreactor module into another module of the cartridge without human intervention, as described above.

[0085] Figure 5 provides a flowchart of an exemplary method of cell processing in an automated cell processing work cell. As illustrated therein, method 501 may include providing cell material to a cartridge having a bioreactor module 510. The cell material may include a cell solution and may include one or more of cells (e.g., allogeneic cells), culture medium, buffer, and reagents. The cell material may be liquid, or in some modifications, frozen. In some modifications, the cell material may be supplied to the cartridge by a fluid manifold. In further modifications, the cell material may be supplied by a portable fluid device, such as a sterile fluid transfer device connected to a fluid transfer port on a fluid transfer port tray of the cartridge. The cell material may be transferred to the bioreactor, thermal compartment, and / or mixing chamber of the bioreactor module. Method 501 may further include transferring the cell material from either a first thermal compartment or a second thermal compartment of the bioreactor module to the bioreactor of the bioreactor module 520. For example, the cell material in liquid form may be transferred via the fluid ports of the respective thermal compartments. A fluid port may be fluidically connected to a fluid conduit, which in turn may be fluidically connected to a fluid manifold.

[0086] Method 501 may further include culturing cells in a bioreactor 530. Cell culture may be facilitated by performing one or more cell treatment steps. For example, a bioreactor module may be configured to perform one or more of the following: a growth step, a transduction step, a transfection step, a perfusion step, a depletion step, a seeding step, etc. Cells may be cultured for a predetermined period of time through one or more cell treatment steps according to a predetermined workflow. In some modifications, the predetermined period may be about 6 hours to about 20 days, about 12 hours to about 15 days, or about 1 day to about 12 days (including 12 hours, 3 days, 6 days, 9 days, 12 days, or 15 days). One or more cell treatment steps may be repeated throughout the predetermined period. The cell culture steps may be performed in the bioreactor, mixing chamber, and / or thermal compartment of the bioreactor module.

[0087] Method 501 may further include measuring one or more of the pH and dissolved oxygen values ​​of the cell material in the bioreactor 540. For example, the pH and / or dissolved oxygen values ​​may be measured by a sensor operably connected to a window of the bioreactor module. The window may be transparent. In some modifications, the window may be located below the bioreactor, so that the sensor can measure one or more parameters of the cell material contained therein. In further modifications, the window may be located on the side wall of the bioreactor. The sensor may be located inside the bioreactor module. In some modifications, the sensor may be connected to the bioreactor equipment of the working cell. The sensor described herein may communicate with a controller so that the controller can adjust one or more conditions of the bioreactor module in response to one or more measurements. For example, in response to a dissolved oxygen measurement below a target value, oxygen may be transferred to the bioreactor to increase the dissolved oxygen level of the fluid in the bioreactor. In another example, carbon dioxide may be transferred into the bioreactor to increase the carbon dioxide level in the fluid within the bioreactor in response to a pH reading above the target value. In yet another example, carbon dioxide may be transferred out of the bioreactor to decrease the carbon dioxide level in the fluid within the bioreactor in response to a pH reading below the target value.

[0088] Method 501 may further include sampling cell material using a sampling straw of a bioreactor 550. For example, a sample of cell material can be taken from a bioreactor module via a sampling straw. In some modifications, one or more of the bioreactor and mixing chamber may be equipped with a sampling straw. The sampling straw can take a portion of the cell material. For example, the sample may have a volume of about 0.5 mL to about 5 mL, containing about 1 mL, 2 mL, 3 mL, 4 mL, or 5 mL. The sampling straw may be fluidly connected to a fluid manifold. Method 501 may further include measuring one or more of the lactate and glucose values ​​of a sample of cell material 560. For example, a cartridge may be equipped with one or more sensors configured to measure one or more of the lactate and glucose values ​​of a sample. That is, one or more sensors may include a glucose sensor and / or a lactate sensor. In some modifications, the sampling straw may be configured to measure glucose and / or lactate. Advantageously, measuring one or more of the lactate and glucose values ​​in the cartridge can increase the efficiency of the cell processing described herein by reducing the time and / or steps required to obtain the lactate and / or glucose measurements. That is, sampling, measurement, and / or evaluation of the measurements can be performed automatically (e.g., in a closed-loop system). The lactate and / or glucose measurements can be used to determine the response by the bioreactor module. For example, a perfusion process may be performed in response to lactate measurements above a threshold and / or glucose measurements below a threshold. That is, the culture medium may be perfused (e.g., exchanged) so that the cells in the bioreactor have sufficient levels of glucose while minimizing the level of lactate. In another example, a fluid manifold may transfer a sample of cell material to an analytical instrument. The analytical instrument may be located within the working cell (e.g., an online instrument), or in some modifications, outside the working cell (e.g., an offline instrument).The analytical instrument may be configured to measure one or more of the following values ​​from a sample: lactate and glucose. The measured values ​​may be transmitted to a controller for evaluation (e.g., comparison with a given condition).

[0089] Method 501 may further include performing one or more of transduction and transfection in a mixing chamber of a bioreactor module 570. For example, transduction may be performed by introducing a transduction reagent (e.g., a lentiviral vector and / or virus) into a cell solution in a mixing chamber. The transduction reagent may be configured to target a specific cell type. Thus, the amount (e.g., volume) of the transduction reagent may correspond to a cell concentration value (e.g., number of cells per unit volume) in the cell solution. The transduction reagent may be used, for example, to introduce a chimeric antigen receptor (CAR) into cells in the cell solution. In another example, a lentiviral vector containing a Lenti-CD19 CAR (scFv-41BB-CD3ζ, CTL019) may be configured to target CD19+ cells. Optionally, the vector solution may be frozen (e.g., at -80°C) and thawed and / or warmed immediately before being transported to the bioreactor module. When cells are cultured in the presence of a virus or non-viral vector, transduction of the cells by the virus or non-viral vector may occur. In another example, transfection may be carried out by introducing a transfection reagent (e.g., nucleic acid) into a cell solution in a mixing chamber by a non-viral method. Transfection may be configured to knock out specific cell types that may subsequently be associated with inducing an immune response (e.g., graft-versus-host disease) in a patient. The transfection reagent may include liquid nanoparticles (LNPs). The transduction and / or transfection reagent may be mixed with the cell material using an impeller in the mixing chamber. The impeller may be controlled by a controller configured to send an electrical signal to an actuator, such as an electromagnet, connected to the impeller. In some modifications, transduction and / or transfection may be carried out in a bioreactor. For example, the bioreactor may also have an impeller, which may, but is not required, be magnetically connected to the impeller of the mixing chamber.

[0090] Method 501 may further include transferring cell material from either a first thermal compartment or a second thermal compartment to a mixing chamber 580. For example, cell material may be removed from each thermal compartment via its fluid port. The fluid port may be fluidically connected to a fluid conduit, which may then be fluidically connected to a fluid manifold. Thus, the fluid manifold can control the transfer of cell material. The fluid manifold may be equipped with one or more valves for controlling the flow of fluid through it. One or more valves may be individually actuated by a controller. In some modifications, cell material may be transferred to a second bioreactor module of the cartridge. Thus, Method 501 may further include performing independent cell processing steps in each of the bioreactor modules 590. That is, for example, different reagents may be provided to each bioreactor module to facilitate one or more cell processing steps. In another example, bioreactor modules may be independently controllable so that any impellers, heat exchangers, and / or fluid transfers within a bioreactor module do not affect other bioreactor modules. In some modifications, bioreactor modules may be thermally coupled, resulting in identical thermal environments for two bioreactor modules. Thermally coupling bioreactor modules is advantageous in that it reduces the operational complexity and / or components required to perform the cell processing described herein.

[0091] Throughout this application, the term “approximately” is used to indicate that a value includes inherent variations in error in the device or method employed to determine that value, or variations present between samples being measured. Unless otherwise stated or evident from the context, “approximately” means within plus or minus 10 percent of a reported number (except where such a number is greater than 100% of a possible value or less than 0%). When used in conjunction with a range or set of values, the term “approximately” applies to each of the endpoints of the range or the values ​​listed in the set of values, unless otherwise indicated. As used herein, “approximately” and “about” are used synonymously.

[0092] While embodiments of the present invention have been shown and described herein, those skilled in the art will understand that such embodiments are provided only as examples. Those skilled in the art will recall numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various substitutes for the embodiments of the present invention described herein may be used in carrying out the invention. The following claims define the scope of the present invention, and methods and structures within these claims, as well as their equivalents, are intended to be encompassed thereby.

Claims

1. A cartridge for automated cell processing, A bioreactor module comprising a bioreactor, a first thermal compartment arranged adjacent to the bioreactor, and a second thermal compartment arranged adjacent to the bioreactor, A fluid manifold connecting the first thermal compartment and the second thermal compartment to the bioreactor, A cartridge equipped with these features.

2. The cartridge according to claim 1, wherein the first thermal compartment and the second thermal compartment are interlocked with each other.

3. The cartridge according to claim 1, wherein the first thermal compartment and the second thermal compartment are configured to hold different volumes.

4. The first thermal compartment is configured to hold a volume of approximately 600 mL. The cartridge according to claim 1, wherein the second thermal compartment is configured to hold a volume of approximately 100 mL.

5. The cartridge according to claim 1, wherein the bioreactor comprises one or more of the following: an impeller, an air-permeable liner, a sampling straw, and a perfusion filter.

6. The system further comprises a mixing chamber configured to accept one or more cell processing reagents, The cartridge according to claim 1, wherein the mixing chamber comprises an impeller.

7. The cartridge according to claim 6, wherein one or more of the bioreactor, the first thermal compartment, the second thermal compartment, and the mixing chamber are fluidly connected.

8. The bioreactor comprises an impeller, The cartridge according to claim 6, wherein the impeller of the mixing chamber and the impeller of the bioreactor are connected to each other.

9. The cartridge according to claim 6, wherein one or more of the bioreactor, the first thermal compartment, the second thermal compartment, and the mixing chamber have a window for optical detection.

10. The cartridge according to claim 1, further comprising a third thermal compartment arranged adjacent to the bioreactor.

11. The cartridge according to claim 10, wherein the third thermal compartment is configured to hold a volume of approximately 600 mL.

12. The cartridge according to claim 10, wherein the third thermal compartment has a window portion configured for optical detection.

13. The cartridge according to claim 1, further comprising a second bioreactor module.

14. The cartridge according to claim 13, wherein the bioreactor module is thermally connected.

15. A method of cell processing, To provide cell material to a cartridge having a bioreactor module comprising a bioreactor, a first thermal compartment arranged adjacent to the bioreactor, and a second thermal compartment arranged adjacent to the bioreactor, Transferring the cell material from either the first thermal compartment or the second thermal compartment to the bioreactor, The process involves culturing cells in the bioreactor, Methods that include...

16. The method according to claim 15, wherein the cells are cultured for about 12 hours to about 15 days.

17. The method according to claim 15, wherein the bioreactor is configured to perform one or more of the following processes: a static process, a stirring process, and a perfusion process.

18. The method according to claim 15, further comprising measuring one or more of the pH value and dissolved oxygen value of the cell material in the bioreactor.

19. The method according to claim 15, further comprising sampling the cell material using the sampling straw of the bioreactor.

20. The method according to claim 19, further comprising measuring one or more of the lactate and glucose values ​​of the sample of the cell material.

21. The method according to claim 15, wherein the bioreactor module further comprises a mixing chamber.

22. The method according to claim 21, further comprising performing one or more of transfection and transfection in the mixing chamber.

23. The method according to claim 21, further comprising transferring the cell material from either the first thermal compartment or the second thermal compartment to the mixing chamber.

24. The method according to claim 15, wherein the cartridge further comprises a second bioreactor module.

25. The method according to claim 24, further comprising performing an independent cell processing step in each of the bioreactor modules.

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