Systems and methods for cell culturing

Abstract

To provide systems and methods for cell culturing.SOLUTION: Cell culture systems and methods provide improved immunotherapeutic product manufacturing with greater scalability, flexibility, and automation. Cell culture systems are configured with interchangeable cartridges, allowing versatility and scalability. Systems are configured to have multiple connected cell culture chambers, which allows parallel processing of different types of cells. Gas-impermeable cell culture chambers and methods for generating cells in closed systems prevent contamination and user error. Methods for recycling cell culture medium provide additional efficiencies.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 923,963, filed October 21, 2019, U.S. Provisional Application No. 62 / 923,967, filed October 21, 2019, U.S. Provisional Application No. 62 / 923,973, filed October 21, 2019, U.S. Provisional Application No. 62 / 923,975, filed October 21, 2019, U.S. Provisional Application No. 62 / 923,978, filed October 21, 2019, and U.S. Provisional Application No. 62 / 923,982, filed October 21, 2019, the contents of which are incorporated herein by reference in their entireties.

[0002] The present disclosure relates generally to systems and methods for cell culture. [Background technology]

[0003] Cancer is a leading cause of mortality and morbidity worldwide, and despite years of extraordinary research efforts, treatments remain elusive. The diversity of tumor types presents a challenge in cancer therapy, as treatments tailored to one tumor may not be effective against another. Personalized treatments are being pursued, but many challenges exist in developing them.

[0004] One promising area is T cell therapy, in which a patient's T cells are engineered to target a particular cancer. This includes chimeric antigen receptor T cell (CAR-T) therapy, T cell receptor (TCR) therapy, and neoantigen-based T cell therapy. Neoantigen-based therapy offers the ability to identify antigens from tumor sequencing data and design highly personalized, patient-specific immunotherapies.

[0005] Unfortunately, many challenges exist in the development and manufacture of T cell therapies. Existing processes for the isolation, preparation, and expansion of cancer antigen-specific T cells are limited. Conventional protocols for stimulating human T cells with autologous antigen-presenting dendritic cells (DCs) involve several manual steps, including transferring cells between culture vessels, changing culture media, and replenishing cytokines and cell culture media. These processes are labor-intensive and not easily scalable. The number of manual steps required to execute the protocols is prohibitive. In addition, these protocols involve the use of flasks or other containers that are opened and closed between uses, increasing the risk of contamination that can compromise the quality and safety of the cell product. Such methods do not comply with current Good Manufacturing Practices (cGMP) and are not useful for large-scale production of T cell therapies. Additional challenges also exist, such as cell preparation time, maintaining optimal phenotype, expansion to sufficient cell numbers, and the quality and safety of the cell product. Summary of the Invention [Means for solving the problem]

[0006] The present invention recognizes that automation of T cell therapy processing and manufacturing has not been successful due to the complex biological processes involved and the bioprocessing and regulatory requirements associated with autologous cell processing. The few systems that exist are overly complex and expensive, and therefore not useful for preclinical assays. The inventive cell culture system and related methods of the present invention provide solutions to many of the problems in cell culture, offering numerous features to reduce contamination and user error, and improve efficiency, scalability, and ease of use. The systems and methods of the present invention provide the capacity for robust T cell generation while minimizing cost and improving simplicity and ease of use, making the disclosed systems and methods useful for both preclinical research and routine cell culture, while being able to meet current good manufacturing practice requirements for clinical manufacturing.

[0007] In certain aspects, the disclosed systems and methods provide improved automated techniques for generating antigen-specific T cells. Automation of manual processes dramatically reduces the potential for user error and decreases the risk of contamination. For example, the present disclosure provides systems and methods for generating CAR-T and TCR-transduced T cells and neoantigen-targeted T cells in a closed system. This avoids the need to open and close T-flasks, as is common in the prior art, thereby simplifying the process and avoiding sources of contamination. As another example, a cell culture system configured with easily interchangeable cell culture chambers is disclosed, allowing users to scale up or down cell populations. Various chambers and vessels are connectable via sterile tubing welds so that the system can remain closed throughout use. The present disclosure also provides a gas-impermeable cartridge for cell culture, which offers a solid polystyrene surface for optimal cell attachment and a rigid cartridge structure that is easy to manufacture and less susceptible to contamination when operated with welded tubing connections. The disclosed system also enables parallel processing of dendritic cells and T cells in the process to generate stimulated T cells. The system architecture streamlines the process of T cell culture, providing time and material savings. Another way the system saves material is by recirculating cell culture medium to ensure that cells can be cultured using minimal amounts of expensive culture medium and supplements.

[0008] In addition to the features described above, other features will be apparent to those skilled in the art, as the disclosed systems and methods offer numerous opportunities for process optimization in immunotherapy product manufacturing.

[0009] Aspects of the present disclosure provide a cell culture system with an exchangeable cartridge. The cell culture system includes a first area configured to receive a fluid reservoir containing cell culture medium and a second area configured to receive a waste reservoir. The cell culture system also has one or more pumps fluidly connectable to the fluid reservoir and a substrate configured to receive and retain cell culture chambers of different shapes and / or sizes.

[0010] In embodiments, the substrate has a plurality of different openings arranged such that the substrate is configured to receive and retain cell culture chambers of different shapes and / or sizes. The substrate can be configured to simultaneously receive and retain multiple cell culture chambers. A first portion of the substrate may be configured to receive a first cell culture chamber of a first size, while a second portion of the substrate is configured to receive a second cell culture chamber of a second shape different from the first size. In embodiments, the first portion of the substrate is configured to receive a first cell culture chamber of a first shape, and the second portion of the substrate is configured to receive a second cell culture chamber of a second shape different from the first shape. In embodiments, the first portion of the substrate is configured to receive a first cell culture chamber of a first size and shape, and the second portion of the substrate is configured to receive a second cell culture chamber of a second size and shape different from the first size and shape.

[0011] In embodiments, a fluid reservoir is positioned in the first area and a waste reservoir is positioned in the waste area. One or more tubes can be included fluidly connecting the fluid reservoir to one or more pumps, and / or the one or more pumps to the cell culture chambers, and / or the cell culture chambers to the waste reservoir. Each cell culture chamber can be fluidly coupled to a separate pump. In some embodiments, a processor is operably connected to the one or more pumps and one or more sensors operable to measure properties of the fluid in the cell culture system, and the processor operates the one or more pumps based on the measured properties.

[0012] In a related aspect, the present disclosure provides a method for culturing cells. The method includes providing a cell culture system having a first area configured to receive a fluid reservoir containing cell culture medium, a second area configured to receive a waste reservoir, one or more pumps fluidly connectable to the fluid reservoir, and a substrate configured to receive and retain cell culture chambers of different shapes and / or sizes. The method further includes loading the fluid reservoir into the first area and the waste reservoir into the second area, loading a first cell culture chamber of a first size and / or shape onto a first portion of the substrate, and loading a second cell culture chamber of a second size and / or shape onto a second portion of the substrate. The method further includes connecting the fluid reservoir, the one or more pumps, the first and second cell culture chambers, and the waste reservoir with tubing. The method further involves operating the system to culture cells in the first and second cell culture chambers.

[0013] In embodiments, the substrate has a plurality of different openings arranged such that the substrate is configured to receive and retain cell culture chambers of different shapes and / or sizes. The first cell culture chamber can be a first size, and the second cell culture chamber can be a second size. The first cell culture chamber can be a first shape, and the second cell culture chamber can be a second shape. The first cell culture chamber can be both a first size and shape, and the second cell culture chamber can be both a second size and shape.

[0014] In embodiments, the first and second cell culture chambers are each fluidly coupled to a separate pump. The system can also include a processor operably connected to the one or more pumps and one or more sensors operable to measure properties of fluid within the cell culture system, the processor operating the one or more pumps based on the measured properties.

[0015] In an embodiment, after cell culture is completed in the first and second cell culture chambers, the cultured cells in each of the first and second cell culture chambers are collected. The cultured cells in each of the first and second cell culture chambers can be collected in the same collection vessel or in different collection vessels.

[0016] In another aspect, the present disclosure provides a method for generating transduced T cells with a CAR or TCR in a closed system. The method involves providing a cell culture device having first and second culture chambers and flowing a cell-containing suspension into the first culture chamber. The method further involves perfusing the T cells in the first culture chamber with an appropriate transduction and expansion reagent to generate transduced T cells that expand in the first culture chamber. The method further involves flowing the transduced and expanded T cells from the first culture chamber into the second culture chamber. The method further involves flowing cell culture medium into the second culture chamber to further expand the transduced and expanded T cells, the method being performed on a single device in a closed manner so that sterile conditions are maintained throughout the method.

[0017] In some embodiments, the second culture chamber is larger than the first culture chamber. One or both culture chambers can be made of polystyrene. The culture chambers can be connected via sterile tubing. The first culture chamber can contain an activation reagent and / or a cell transduction reagent, which can be an inactive virus expressing a CAR or TCR. Alternatively, the second culture chamber can be a separate cell culture device that is not part of the first cell culture device.

[0018] In some embodiments, the cell culture medium is provided in a sterile container and connected to the closed system by a sterile tubing weld. The step of flowing the cell culture medium into the first culture chamber may involve eliminating a headspace within the first culture chamber. The cell culture medium may include Aim V with interleukin-2.

[0019] The method may further involve activating the T cells in the first culture chamber, which may be done by contacting them with one or more activating antibodies or soluble activating antibody-containing reagents and magnetic or non-magnetic beads comprising a transduction reagent. The method may further involve draining fluid from the second culture chamber, washing the transduced and expanded T cells with a buffer, and flowing cryopreservation medium into the second culture chamber to resuspend the transduced and expanded T cells. The method may further involve flowing the transduced and expanded T cells into a collection container in a closed manner.

[0020] In embodiments, each of the flowing steps may be performed through sterile tubing, which may be connected by a sterile tubing weld.

[0021] In another aspect, the present disclosure provides a method for generating neoantigen-targeted T cells in a closed system. The method includes providing a cell culture device having first and second culture chambers and flowing a cell culture medium containing monocytes into the first culture chamber. The method also involves perfusing purified monocytes in the first culture chamber to generate dendritic cells in the first culture chamber, and contacting the dendritic cells with an antigenic substance, which may include a tumor-specific peptide, in the first culture chamber to generate mature dendritic cells. The method further involves flowing the mature dendritic cells from the first culture chamber into a second culture chamber containing purified T cells, and co-culturing the mature dendritic cells and the purified T cells, thereby generating neoantigen-targeted T cells. The method is performed on a single device in a closed manner so that sterile conditions are maintained throughout the method.

[0022] In embodiments, the method also includes flowing a second batch of monocytes into a second culture chamber to perform a second co-culture with purified T cells, differentiating them into dendritic cells, and maturing the dendritic cells. The first and second culture chambers can be made of polystyrene. The first and second culture chambers can be connected via sterile tubing. The cell culture medium can be provided in a sterile container and can be connected to the closed system by a sterile tubing weld. Flowing the cell culture medium into the first culture chamber can involve eliminating the headspace within the first culture chamber. Each flowing step can be performed through sterile tubing, which can be connected by a sterile tubing weld.

[0023] In embodiments, the method also includes activating the T cells in the second culture chamber. In embodiments, the method also includes draining fluid from the second culture chamber, washing the neoantigen-targeted T cells with a buffer, and flowing cryopreservation medium into the second culture chamber to resuspend the neoantigen-targeted T cells. The method may also involve flowing the neoantigen-targeted T cells into a collection container in a closed manner.

[0024] In another aspect, the present disclosure provides a method for parallel processing to generate dendritic cells and stimulate T cells in parallel. The method includes providing a cell culture device with first and second culture chambers and flowing a cell culture medium containing monocytes into the first culture chamber. The method further includes perfusing the monocytes in the first culture chamber to generate dendritic cells in the first culture chamber. The method further includes flowing T cells cultured in the second culture chamber from the second culture chamber into the first culture chamber with the dendritic cells and further culturing the T cells in the first culture chamber. In embodiments, sterile conditions are maintained throughout the method.

[0025] The method may also include collecting the cultured T cells from the first culture chamber by flowing the cultured T cells into a collection container. The method may also include maturing the dendritic cells in the first culture chamber by contacting the dendritic cells with an antigenic substance, which may include a tumor-specific peptide. In embodiments, the method also includes activating the T cells in the second culture chamber, which may be performed using an activation reagent. In embodiments, the method also includes washing the stimulated T cells with a buffer solution and, optionally, transferring the stimulated T cells to a cryopreservation medium. The method may also include flowing the neoantigen-targeted T cells into the collection container in a closed manner. Each flowing step may be performed through sterile tubing, optionally connected by a sterile tubing weld.

[0026] The cell culture medium may be provided in a sterile container and connected to the closed system by a sterile tubing weld. Flowing the cell culture medium into the first culture chamber may involve eliminating a headspace within the first culture chamber. In embodiments, the first and second culture chambers may be made of polystyrene and may optionally be connected via sterile tubing. In some embodiments, one or both of the first and second cell culture chambers from the cell culture apparatus may be replaced, and the method may be repeated.

[0027] In another aspect, the present disclosure provides a gas-impermeable cell culture chamber, wherein the top, bottom, and both sidewalls are made of a gas-impermeable material. The gas-impermeable material may also be a material to which cells adhere. The gas-impermeable material may be polystyrene.

[0028] In embodiments, the cell culture chamber has an inlet. The cell culture chamber may also have an outlet. The inlet and outlet can be located on the cell culture chamber, and optionally, the inlet and outlet are each configured to fluidly and sealably couple with tubing. The cell culture chamber can be integrally formed, which can be sized and configured to fit within an incubator. The cell culture chamber can be sized and configured to couple to a substrate of a cell culture apparatus.

[0029] In a related aspect, the present disclosure provides a method for culturing cells that involves providing a cell culture chamber having an inlet and an outlet, where the top, bottom, and both sidewalls are made of a gas-impermeable material. The method also involves loading cells into the cell culture chamber and flowing cell culture medium into the cell culture chamber via the inlet and out of the cell culture chamber via the outlet to culture the cells in the culture chamber, where the flow of cell culture medium through the cell culture chamber via the inlet and outlet causes continuous flow of the cell culture medium through the cell culture chamber, allowing gas exchange to occur between the cells and the cell culture medium in the cell culture chamber.

[0030] In embodiments, the gas-impermeable material is also a material to which cells adhere, such as polystyrene. In embodiments, the inlet and outlet are located on the cell culture chamber and are optionally configured to fluidly and sealably couple with tubing. The tubing can be high-permeability tubing that allows gas exchange while the cell culture medium is within the high-permeability tubing. In embodiments, the cell culture chamber is integrally formed. The cell culture chamber can be sized and configured to fit within the incubator, and optionally, it can be sized and configured to couple to the substrate of the cell culture apparatus.

[0031] In another aspect, the present disclosure provides a method for culturing cells that involves culturing cells in a cell culture chamber on a cell culture apparatus by flowing cell culture medium through the cell culture chamber, wherein a portion of the cell culture medium that has been flowed through the cell culture chamber is recirculated back into the cell culture chamber during the cell culture process.

[0032] In embodiments, the method also involves measuring one or more parameters of the spent medium prior to recycling. The parameters can be the concentration of one or more compounds in the spent medium, such as glucose, lactate, dissolved oxygen, or cellular metabolites. The parameters can also be pH or cell count.

[0033] In embodiments, the method involves using a processor operably connected to the cell culture chamber to determine whether at least one of one or more parameters of the spent medium meets a predetermined threshold prior to the recirculating step. The measuring step can be performed by one or more sensors operably associated with the cell culture chamber. The one or more sensors can be operably associated with a waste reservoir in fluid communication with the cell culture chamber. The cell culture chamber can be operably connected to one or more pumps and may have an inlet and an outlet. The recirculating step can involve redirecting a portion of the spent medium from the waste reservoir back into the cell culture chamber. In embodiments, the portion of the spent medium is combined with a bolus of fresh medium.

[0034] In a related aspect, the present disclosure provides a method for culturing cells, comprising providing a cell culture chamber containing cells, flowing cell culture medium into the cell culture chamber, removing spent cell culture medium from the cell culture chamber, assessing a parameter of the spent cell culture medium, and returning the spent cell culture medium to the cell culture chamber if the parameter meets a predetermined threshold.

[0035] In embodiments, the method also involves combining the spent cell culture medium with a bolus of fresh cell culture medium prior to the returning step. The assessing step may involve measuring a parameter using a sensor operably coupled to the cell culture chamber. The assessing step may involve determining, using a processor, whether the parameter meets a predetermined threshold. The parameter may be a measured concentration of one or more compounds in the spent cell culture medium, such as glucose, lactate, or a cellular metabolite. In embodiments, the returning step includes redirecting the spent cell culture medium from a waste reservoir into the cell culture chamber. In other embodiments, the method also involves discarding the spent cell culture medium and flowing fresh cell culture medium into the cell culture chamber if the parameter does not meet a predetermined threshold.

[0036] In another aspect, the present disclosure provides a cell culture system including a plurality of shelves for receiving fluid reservoirs. The shelves may be stacked with a first shelf on top of a second shelf, and each of the first and second shelves may be configured to receive a fluid reservoir. Each of the shelves may include a retaining mechanism for retaining the fluid reservoir on each of the first and second shelves.

[0037] The system may further include at least one pump, a processor operably coupled to the at least one pump, and a substrate sized and configured to simultaneously hold multiple cell culture chambers. Preferably, the system further includes at least one sensor, and the processor may be connected to the at least one sensor and configured to operate the at least one sensor based on a characteristic measured by the sensor. For example, the sensor may measure the concentration of one or more compounds in the cell culture medium, such as glucose, lactate, or cell metabolites. The processor may adjust the pump (e.g., turn the pump on or off) based on measurements made from the one or more sensors. For example, one sensor may be attached to the cell culture chamber. The sensor may measure, for example, the glucose level in the medium in the cell culture chamber. When the glucose level falls below a predetermined threshold, the processor may trigger the pump to exchange the medium in the cell culture chamber.

[0038] In some embodiments, the processor is configured to receive and execute instructions for culturing a cell type, hi other cases, the processor may be configured to receive and execute instructions for transducing T cells.

[0039] In some embodiments, the substrate is configured to receive cell culture chambers of different sizes and / or shapes. This configuration is advantageous because it allows the system to be customized to culture different qualities of cells or different cell types depending on the specific needs of the user. The system may further include multiple pumps. For example, the system may include a separate pump for each cell culture chamber included within the system, allowing cells in each cell culture chamber to be cultured separately.

[0040] In some embodiments, the system further includes a plurality of tubes fluidly connecting a first fluid reservoir on the first shelf to the plurality of pumps, from the plurality of pumps to the plurality of cell culture chambers, and from the plurality of cell culture chambers to a second fluid reservoir on the second shelf. Preferably, the system is sized for insertion into an incubator.

[0041] In a related aspect, the present disclosure provides a method for sterile culturing of cells, the method including a plurality of shelves stacked with a first shelf above a second shelf, each of the first and second shelves configured to receive a fluid reservoir, at least one pump, and a processor operably coupled to the at least one pump;

[0042] and a substrate sized and configured to simultaneously hold multiple cell culture chambers, the method further including loading a first fluid reservoir on a first shelf, loading a second fluid reservoir on a second shelf, loading the first cell culture chamber and a second cell culture chamber on the substrate, connecting the first and second fluid reservoirs, at least one pump, and the first and second cell culture chambers with tubing, and operating the system to culture cells inside the first and second cell culture chambers.

[0043] In some embodiments, the first and second cell culture chambers comprise different sizes or shapes. In some embodiments, the system includes at least one sensor. In some embodiments, the processor is coupled to the at least one sensor and configured to operate the pump based on a characteristic measured by the sensor. The present invention provides, for example, the following. (Item 1) 1. A cell culture system comprising: a first area configured to receive a fluid reservoir comprising a cell culture medium; a second area configured to receive a waste reservoir; one or more pumps fluidly connectable to the fluid reservoir; a substrate configured to receive and retain cell culture chambers of different shapes and / or sizes; A cell culture system comprising: (Item 2) Item 10. The cell culture system of item 1, wherein the substrate comprises a plurality of different openings arranged therein such that the substrate is configured to receive and retain cell culture chambers of different shapes and / or sizes. (Item 3) Item 10. The cell culture system of item 1, wherein the substrate is configured to simultaneously receive and retain multiple cell culture chambers. (Item 4) 2. The cell culture system of claim 1, further comprising the fluid reservoir positioned within the first area and the waste reservoir positioned within the waste area. (Item 5) 5. The cell culture system of item 4, further comprising one or more tubes fluidly connecting a fluid reservoir to the one or more pumps, from the one or more pumps to the cell culture chamber, and from the cell culture chamber to the waste reservoir. (Item 6) A first portion of the substrate is configured to receive a first cell culture chamber of a first size. 2. The cell culture system of claim 1, wherein a second portion of the substrate is configured to receive a second cell culture chamber of a second shape different from the first size. (Item 7) 2. The cell culture system of claim 1, wherein a first portion of the substrate is configured to receive a first cell culture chamber of a first shape and a second portion of the substrate is configured to receive a second cell culture chamber of a second shape different from the first shape. (Item 8) 2. The cell culture system of claim 1, wherein a first portion of the substrate is configured to receive a first cell culture chamber of a first size and shape, and a second portion of the substrate is configured to receive a second cell culture chamber of a second size and shape different from the first size and shape. (Item 9) 2. The cell culture system of item 1, wherein each cell culture chamber is fluidly coupled to a separate pump. (Item 10) 2. The cell culture system of claim 1, further comprising a processor operably connected to the one or more pumps and one or more sensors operable to measure properties of fluid within the cell culture system, the processor operating the one or more pumps based on the measured properties. (Item 11) 1. A method for culturing cells, the method comprising: providing a cell culture system comprising: a first area configured to receive a fluid reservoir comprising cell culture medium; a second area configured to receive a waste reservoir; one or more pumps fluidly connectable to the fluid reservoirs; and a substrate configured to receive and retain cell culture chambers of different shapes and / or sizes; loading the fluid reservoir into the first area and the waste reservoir into the second area; loading a first cell culture chamber of a first size and / or shape onto a first portion of the substrate; loading a second cell culture chamber of a second size and / or shape onto a second portion of the substrate; connecting the fluid reservoir, the one or more pumps, the first and second cell culture chambers, and the waste reservoir with tubing; operating the system to culture cells in the first and second cell culture chambers; A method comprising: (Item 12) Item 12. The method of item 11, wherein the substrate comprises a plurality of different openings arranged therein such that the substrate is configured to receive and retain cell culture chambers of different shapes and / or sizes. (Item 13) 12. The method of claim 11, wherein the first cell culture chamber is a first size and the second cell culture chamber is a second size. (Item 14) Item 12. The method of item 11, wherein the first cell culture chamber is a first shape and the second cell culture chamber is a second shape. (Item 15) 12. The method of claim 11, wherein the first cell culture chamber is a first size and shape and the second cell culture chamber is a second size and shape. (Item 16) 12. The method of claim 11, wherein the first and second cell culture chambers are each fluidly coupled to a separate pump. (Item 17) 12. The method of claim 11, wherein the cell culture system further comprises a processor operably connected to the one or more pumps and one or more sensors operable to measure properties of fluid within the cell culture system, the processor operating the one or more pumps based on the measured properties. (Item 18) Item 12. The method according to item 11, wherein after cell culture is completed in the first and second cell culture chambers, the cultured cells in each of the first and second cell culture chambers are harvested. (Item 19) Item 19. The method of item 18, wherein the cultured cells in each of the first and second cell culture chambers are collected in the same collection vessel. (Item 20) Item 19. The method of item 18, wherein the cultured cells in each of the first and second cell culture chambers are collected in different collection vessels. (Item 21) 1. A method for generating transduced T cells, the method comprising: providing a cell culture apparatus comprising first and second culture chambers; flowing a suspension containing T cells into the first culture chamber; perfusing the T cells within the first culture chamber to generate transduced T cells that expand within the first culture chamber; flowing the transduced and expanded T cells from the first culture chamber into the second culture chamber; flowing cell culture medium into the second culture chamber to further expand the transduced and expanded T cells, wherein the method is performed on a single instrument in a closed manner such that sterile conditions are maintained throughout the method; A method comprising: (Item 22) 22. The method of claim 21, wherein the second culture chamber is larger than the first culture chamber. (Item 23) 22. The method of claim 21, wherein the first and second culture chambers are made of polystyrene. (Item 24) 22. The method of claim 21, wherein the first and second culture chambers are connected via sterile tubing. (Item 25) 22. The method of claim 21, wherein the first culture chamber further comprises an activation reagent and / or a cell transduction reagent. (Item 26) 26. The method of claim 25, wherein the cell transduction reagent comprises an inactivated virus expressing a CAR or TCR. (Item 27) 22. The method of claim 21, wherein the cell culture medium is provided in a sterile container and connected to the closed system by a sterile tubing weld. (Item 28) The step of flowing the cell culture medium into the first culture chamber comprises: 22. The method of claim 21, comprising eliminating the headspace within the culture chamber. (Item 29) 22. The method of claim 21, further comprising activating the T cells in the first culture chamber. (Item 30) 30. The method of claim 29, wherein the activating step comprises contacting with a reagent comprising an antibody. (Item 31) 31. The method of claim 30, wherein the antibody is attached to a bead. (Item 32) draining fluid from the second culture chamber; washing the transduced and expanded T cells with a buffer; flowing cryopreservation medium into the second culture chamber to resuspend the transduced and expanded T cells; 22. The method of claim 21, further comprising: (Item 33) 22. The method of claim 21, further comprising flowing the transduced and expanded T cells into a collection vessel in a closed fashion. (Item 34) 22. The method of claim 21, wherein each of the flowing steps is performed through a sterile tube. (Item 35) 35. The method of claim 34, wherein the sterile tubing is connected by a sterile tubing weld. (Item 36) 22. The method of claim 21, wherein the cell culture medium comprises Aim V with interleukin-2. (Item 37) 1. A method for generating neoantigen-transduced T cells, said method comprising: providing a cell culture apparatus comprising first and second culture chambers; flowing cell culture medium containing monocytes into the first culture chamber; perfusing the purified monocytes in the first culture chamber to generate dendritic cells in the first culture chamber; contacting the dendritic cells with an antigenic substance in the first culture chamber to generate mature dendritic cells; flowing a cell suspension containing T cells into the first culture chamber and co-culturing the mature dendritic cells and the T cells, thereby generating expanded neoantigen-targeting T cells; Including, The method is performed on a single instrument in a closed manner such that sterile conditions are maintained throughout the method. (Item 38) generating a second batch of mature dendritic cells in the second culture chamber; transferring the expanded T cells from the first culture chamber into the second culture chamber and co-culturing the expanded T cells with a second batch of the mature dendritic cells; Item 38. The method of item 37, further comprising: (Item 39) 39. The method of claim 38, wherein the second batch of mature dendritic cells has been stimulated with a different set of peptides than the mature dendritic cells from the first culture chamber. (Item 40) 39. The method of claim 38, further comprising transferring the expanded T cells co-cultured with the second batch of mature dendritic cells back to the first culture chamber, wherein the first culture chamber comprises a third batch of mature dendritic cells. (Item 41) 38. The method of claim 37, wherein the antigenic substance comprises a tumor-specific peptide. (Item 42) 38. The method of claim 37, wherein the first and second culture chambers are made of polystyrene. (Item 43) Item 38. The method of item 37, wherein the first and second culture chambers are connected via sterile tubing. (Item 44) 38. The method of claim 37, wherein the cell culture medium is provided in a sterile container and connected to the closed system by a sterile tubing weld. (Item 45) 38. The method of claim 37, wherein flowing the cell culture medium into the first culture chamber comprises eliminating a headspace within the first culture chamber. (Item 46) draining fluid from the first culture chamber; washing the neoantigen-transduced T cells with a buffer; flowing cryopreservation medium into the first culture chamber to resuspend the neoantigen-transduced T cells; Item 38. The method of item 37, further comprising: (Item 47) 38. The method of claim 37, further comprising flowing the neoantigen-transduced T cells into a collection vessel in a closed fashion. (Item 48) 38. The method of claim 37, wherein each of the flowing steps is performed through a sterile tube. (Item 49) Item 49. The method of item 48, wherein the sterile tubing is connected by a sterile tubing weld. (Item 50) 1. A method for generating dendritic cells and stimulating T cells in parallel, the method comprising: providing a cell culture apparatus comprising first and second culture chambers; flowing a cell culture medium containing monocytes into the first culture chamber; perfusing the monocytes in the first culture chamber to generate dendritic cells in the first culture chamber; flowing a cell suspension containing T cells into the first culture chamber and co-culturing the dendritic cells and the T cells, thereby generating activated expanded T cells; flowing the activated expanded T cells from the first culture chamber into the second culture chamber containing dendritic cells generated from a second separate batch of monocytes and further culturing the T cells; A method comprising: (Item 51) 51. The method of claim 50, wherein sterile conditions are maintained throughout the method. (Item 52) 51. The method of claim 50, further comprising collecting the cultured T cells from the first culture chamber by flowing the cultured T cells into a collection vessel. (Item 53) 51. The method of claim 50, further comprising maturing the dendritic cells in the first culture chamber by contacting the dendritic cells with an antigenic substance. (Item 54) 54. The method of claim 53, wherein the antigenic substance comprises a tumor-specific peptide. (Item 55) 51. The method of claim 50, wherein the first and second culture chambers are made of polystyrene. (Item 56) 51. The method of claim 50, wherein the first and second culture chambers are connected via sterile tubing. (Item 57) 51. The method of claim 50, wherein the cell culture medium is provided in a sterile container and connected to the closed system by a sterile tubing weld. (Item 58) 51. The method of claim 50, wherein flowing the cell culture medium into the first culture chamber comprises eliminating a headspace within the first culture chamber. (Item 59) 51. The method of claim 50, further comprising activating the T cells in the second culture chamber. (Item 60) 60. The method of claim 59, wherein the activating step comprises contacting with a reagent comprising an antibody. (Item 61) 51. The method of claim 50, further comprising washing the stimulated T cells with a buffer. (Item 62) 62. The method of claim 61, further comprising transferring the stimulated T cells to a cryopreservation medium. (Item 63) 51. The method of claim 50, further comprising flowing the neoantigen-transduced T cells into a collection vessel in a closed fashion. (Item 64) 51. The method of claim 50, wherein each of the flowing steps is performed through a sterile tube. (Item 65) Item 65. The method of item 64, wherein the sterile tubing is connected by a sterile tubing weld. (Item 66) 51. The method of claim 50, wherein one or both of the first and second cell culture chambers from the cell culture apparatus are replaced and the method is repeated. (Item 67) A cell culture chamber, the top, bottom, and both side walls of which are made of a gas impermeable material. (Item 68) Item 68. The cell culture chamber of item 67, wherein the gas impermeable material is also a material to which cells adhere. (Item 69) Item 69. The cell culture chamber of item 68, wherein the gas impermeable material comprises polystyrene. (Item 70) Item 68. The cell culture chamber of item 67, wherein the cell culture chamber further comprises an inlet. (Item 71) Item 71. The cell culture chamber of item 70, wherein the cell culture chamber further comprises an outlet. (Item 72) Item 72. The cell culture chamber of item 71, wherein the inlet and the outlet are located on the cell culture chamber. (Item 73) Item 73. The cell culture chamber of item 72, wherein the inlet and the outlet are each configured to fluidly and sealably couple with tubing. (Item 74) Item 68. The cell culture chamber of item 67, wherein the cell culture chamber is integrally formed. (Item 75) Item 68. The cell culture chamber of item 67, wherein the cell culture chamber is sized and configured to fit within an incubator. (Item 76) Item 68. The cell culture chamber of item 67, wherein the cell culture chamber is sized and configured to couple to a substrate of a cell culture apparatus. (Item 77) 1. A method for culturing cells, the method comprising: providing a cell culture chamber with an inlet and an outlet, wherein the top, bottom, and both sidewalls are made of a gas impermeable material; loading cells into the cell culture chamber; flowing cell culture medium into the cell culture chamber via the inlet and out of the cell culture chamber via the outlet to culture the cells in the culture chamber, wherein the flow of the cell culture medium through the cell culture chamber via the inlet and the outlet causes a continuous flow of cell culture medium through the cell culture chamber, allowing gas exchange to occur between the cells in the cell culture chamber and the cell culture medium; A method comprising: (Item 78) Item 78. The method of item 77, wherein the gas impermeable material is also a material to which cells adhere. (Item 79) Item 79. The method of item 78, wherein the gas impermeable material comprises polystyrene. (Item 80) Item 78. The method of item 77, wherein the inlet and the outlet are located above the cell culture chamber. (Item 81) Item 81. The method of item 80, wherein the inlet and the outlet are fluidly and sealably coupled to tubing. (Item 82) 78. The method of claim 77, wherein the cell culture chamber is integrally formed. (Item 83) 78. The method of claim 77, wherein the cell culture chamber is sized and configured to fit within an incubator. (Item 84) Item 78. The method of item 77, wherein the cell culture chamber is sized and configured to couple to a substrate of a cell culture apparatus. (Item 85) 82. The method of claim 81, wherein the tubing is high permeability tubing. (Item 86) 86. The method of claim 85, wherein the cell culture medium exchanges gases while in the high permeability tubing. (Item 87) 1. A method for culturing cells, the method comprising: Culturing cells in a cell culture chamber on a cell culture apparatus by flowing cell culture medium through the cell culture chamber, wherein a portion of the cell culture medium already flowed through the cell culture chamber is recirculated back into the cell culture chamber during the cell culture process. A method comprising: (Item 88) 88. The method of claim 87, further comprising measuring one or more parameters of the spent medium prior to said recycling. (Item 89) 89. The method of claim 88, wherein at least one of the one or more parameters comprises a concentration of one or more compounds in the spent medium. (Item 90) 90. The method of claim 89, wherein the one or more compounds comprise glucose, lactate, dissolved oxygen, or a cellular metabolite. (Item 91) Item 89. The method of item 88, wherein the parameter comprises pH. (Item 92) Item 89. The method of item 88, further comprising using a processor operably connected to the cell culture chamber to determine whether at least one of the one or more parameters of the spent medium meets a predetermined threshold prior to the recirculating step. (Item 93) Item 89. The method of item 88, wherein the measuring step is performed by one or more sensors operably associated with the cell culture chamber. (Item 94) Item 94. The method of item 93, wherein the one or more sensors are operably associated with a waste reservoir in fluid communication with the cell culture chamber. (Item 95) 95. The method of claim 94, wherein the recirculating step comprises redirecting a portion of the spent medium from the waste reservoir back into the cell culture chamber. (Item 96) 88. The method of claim 87, further comprising combining a portion of the spent medium with a bolus of fresh medium. (Item 97) Item 88. The method of item 87, wherein the cell culture chamber is operably connected to one or more pumps. (Item 98) Item 88. The method of item 87, wherein the cell culture chamber comprises an inlet and an outlet. (Item 99) 1. A method for culturing cells, the method comprising: providing a cell culture chamber with cells; flowing cell culture medium into the cell culture chamber; removing spent cell culture medium from the cell culture chamber; assessing a parameter of the spent cell culture medium; returning the spent cell culture medium to the cell culture chamber if the parameter meets a predetermined threshold. A method comprising: (Item 100) 100. The method of claim 99, further comprising the step of combining the spent cell culture medium with a bolus of fresh cell culture medium prior to the returning step. (Item 101) 100. The method of claim 99, wherein the assessing step comprises measuring the parameter using a sensor operably coupled to the cell culture chamber. (Item 102) 99. The method of claim 99, wherein the assessing step includes using a processor to determine whether the parameter satisfies the predetermined threshold. (Item 103) 103. The method of claim 102, wherein the parameter comprises a measured concentration of one or more compounds in the spent cell culture medium. (Item 104) 104. The method of claim 103, wherein the one or more compounds comprise glucose, lactate, or a cellular metabolite. (Item 105) Item 100. The method of item 99, wherein the returning step comprises redirecting the spent cell culture medium from a waste reservoir into the cell culture chamber. (Item 106) discarding the spent cell culture medium if the parameter does not meet the predetermined threshold; flowing fresh cell culture medium into the cell culture chamber; Item 99. The method of item 99, further comprising: (Item 107) 1. A cell culture system, comprising: a plurality of shelves stacked with a first shelf on top of a second shelf, each of the first and second shelves configured to receive a fluid reservoir; at least one pump; a processor operably coupled to the at least one pump; a substrate sized and configured to simultaneously hold multiple cell culture chambers; A system comprising: (Item 108) Item 108. The system of item 107, wherein the system comprises at least one sensor. (Item 109) Item 109. The system of item 108, wherein the processor is connectable to at least one sensor and configured to operate the at least one pump based on a characteristic measured by the sensor. (Item 110) Item 108. The system of item 107, wherein the substrate is configured to receive cell culture chambers of different sizes and / or shapes. (Item 111) Item 108. The system of item 107, wherein the at least one pump is a plurality of pumps. (Item 112) Item 112. The system of item 111, further comprising a plurality of tubes fluidly connecting a first fluid reservoir on the first shelf to the plurality of pumps, from the plurality of pumps to a plurality of cell culture chambers, and from the plurality of cell culture chambers to a second fluid reservoir on the second shelf. (Item 113) Item 108. The system of item 107, wherein the first and second shelves each include a retaining mechanism for retaining the fluid reservoir on the first and second shelves, respectively. (Item 114) Item 108. The system of item 107, wherein the system is sized for insertion into an incubator. (Item 115) Item 108. The system of Item 107, wherein the processor is configured to receive and execute instructions for culturing a cell type. (Item 116) Item 108. The system of item 107, wherein the processor is configured to receive and execute instructions for transducing T cells. (Item 117) 1. A cell culture method, the method comprising: providing a cell culture system comprising a plurality of shelves stacked with a first shelf on top of a second shelf, each of the first and second shelves configured to receive a fluid reservoir; at least one pump; a processor operably coupled to the at least one pump; and a substrate sized and configured to simultaneously hold a plurality of cell culture chambers; loading a first fluid reservoir onto the first shelf; loading a second fluid reservoir onto the second shelf; loading a first cell culture chamber and a second cell culture chamber onto the substrate; connecting the first and second fluid reservoirs, the at least one pump, and the first and second cell culture chambers with tubing; operating the system to culture cells inside the first and second cell culture chambers; A method comprising: (Item 118) Item 118. The method of item 117, wherein the first and second cell culture chambers comprise different sizes or shapes. (Item 119) Item 118. The method of item 117, wherein the system comprises at least one sensor. (Item 120) Item 120. The method of item 119, wherein the processor is connected to at least one sensor and configured to operate the pump based on a characteristic measured by the sensor. [Brief explanation of the drawings]

[0044] [Figure 1] FIG. 1 shows an example of a multiple bioreactor system.

[0045] [Figure 2] FIG. 2 shows an embodiment of a bioreactor.

[0046] [Figure 3] FIG. 3 shows a multiple bioreactor system.

[0047] [Figure 4] Figure 4 shows the CAR-T / TCR workflow.

[0048] [Figure 5] Figures 5-8 show a comparison of T cell proliferation using different systems. [Figure 6] Figures 5-8 show a comparison of T cell proliferation using different systems. [Figure 7-1] Figures 5-8 show a comparison of T cell proliferation using different systems. [Figure 7-2] Figures 5-8 show a comparison of T cell proliferation using different systems. [Figure 7-3] Figures 5-8 show a comparison of T cell proliferation using different systems. [Figure 8] Figures 5-8 show a comparison of T cell proliferation using different systems.

[0049] [Figure 9] Figures 9-10 show the methods and results for co-culturing freshly cultured dendritic cells and PBMCs or T cells. [Figure 10] Figures 9-10 show the methods and results for co-culturing freshly cultured dendritic cells and PBMCs or T cells.

[0050] [Figure 11] FIG. 11 illustrates a method for forming a cell-based immunotherapy product.

[0051] [Figure 12] FIG. 12 illustrates a system architecture according to some embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0052] Detailed Description The cell culture system of the present invention significantly improves immunotherapy product manufacturing, providing a flow-based immunotherapy production technology with unparalleled consistency, quality, safety, economy, scalability, flexibility, and portability. Generally, cells are grown in single-use cell culture chambers, sometimes referred to as cartridges, which are perfused at low flow rates to achieve high proliferation without the need for filters. The system supports one or more cell culture chambers that are fluidly coupled to each other to carry out processing of a patient's cellular material to generate an immunotherapy product, as described herein. It should be understood that a bioreactor is provided in one embodiment in a closed environment. Scaling up of this exemplary embodiment would be within the knowledge of one skilled in the art by adding modules (e.g., bioreactors) to enable serial and / or parallel processing. One skilled in the art would also understand that different or alternative arrangements may be desired based on the product to be produced.

[0053] 1 shows an example of a multiple bioreactor system 900. System 900 includes a first cell culture chamber 820 and a second cell culture chamber 920 having inlets 845 and 945 connected to tubing 940 in fluid communication with a fluid reservoir 980. The cell culture chambers have outlets 835 and 935 in fluid communication with a waste reservoir 984. Pumps 910a and 910b facilitate the pumping of fluid from fluid reservoir 980 to cell culture chambers 820 and 920. The pumps are controlled by a processor 999 to perform the functions described herein.

[0054] Another embodiment of the bioreactor 110 is shown in FIG. 2 , which provides a more detailed schematic view of a portion of the cell culture chamber 120. It is important to note that the cell culture platform described herein is configured to allow cell culture chambers of different volumes, shapes, and physical characteristics to be used. The chamber shown in FIG. 2 is exemplary only, and other embodiments will be apparent to those skilled in the art. As shown in FIG. 2 , the cell culture chamber 120 includes a bottom surface 122 and at least one additional surface 124. The bottom surface 122 is made of a first material to which cells adhere. In some embodiments, the at least one additional surface 124 is made of a second material that is gas permeable. In other embodiments, which will be described in more detail below, the entire cell culture chamber 120, including the surface 124, is made of a first material that is gas impermeable. The cell culture chamber also includes one or more inlets 126, 136 and one or more outlets 128, 138. In certain embodiments, the biological reactor also includes at least one perfusion fluid reservoir 132, at least one waste fluid reservoir 134, at least one pump 140 for moving perfusion fluid through the chamber 120, and associated inlets 136 and outlets 138 for transporting fluid to and from the reservoirs 132, 134 and through the chamber 120.

[0055] With respect to cell culture chamber 120, the first material can be any material that is biocompatible and to which antigen-presenting cells (APCs) or their precursors, such as dendritic cells (DCs) or monocytes, respectively, will adhere. During the T cell stimulation and expansion process that occurs within cell culture chamber 120, mature APCs will develop and preferably adhere to bottom surface 122, while T cells will remain in the supernatant above the bottom surface, making it easier to separately obtain the expanded T cells.

[0056] In one exemplary embodiment, the first material comprises polystyrene. One benefit of using polystyrene for the bottom surface on which culture will occur is the useful role this material plays in the process of generating dendritic cells from PBMCs. Specifically, polystyrene surfaces can be used to enrich monocytes from a heterogeneous suspension of PBMCs. This is the first step in the culture process utilized to generate DCs, for example, by differentiation of monocytes via culture in a medium containing IL4 and GM-CSF. The use of the same polystyrene surface for dendritic cell generation throughout a complete cycle of T cell stimulation is highly beneficial from a bioprocessing perspective, as it eliminates multiple transfer steps that would otherwise be required, thereby enabling a closed system for DC-stimulated therapeutic T cell manufacturing.

[0057] The bottom is 9.5 cm for 6- and 24-well plates, respectively. 2 and 3.8 cm 2 ) or the like. 2 ~about 200cm 2 surface areas of, for example, about 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 100.0, 125.0, 150.0, 175.0, and 200.0 cm 2 It should be understood that the surface area may be smaller than a conventional well plate, or even much larger (e.g., having a surface area comparable to standard cell culture dishes and flasks), such as having a surface area of 1000 sq. mm or 1000 sq. mm, and any surface area in between.

[0058] The at least one additional surface 124 can have any configuration, such as one or more side walls and a top wall. In one embodiment, the side walls can be arranged at a 90-degree angle relative to each other such that a box shape is formed in conjunction with the bottom surface 122, as shown in FIG. 2. In another embodiment, the at least one additional surface 124 forms a curved side wall such that the chamber 120, or a cross-section thereof, forms a cylinder, an elliptical cylinder, a cone, a dome-like shape, or a triangular shape. It should be understood that the above exemplary configurations are non-limiting and that the at least one additional surface can have other configurations not provided in the aforementioned exemplary configurations.

[0059] An exemplary configuration of a multiple bioreactor system is found in FIG. 3 , and additional details regarding processes carried out using this configuration can be provided below. As shown in panel B of FIG. 3 , in cases involving a second bioreactor 210, a second cell culture chamber 220 is positioned to connect with the first cell culture chamber 120 via the first chamber's outlet and the second chamber's inlet. The connection is preferably a sterile connection. The connection allows for the injection of sterile air into the first cell culture chamber 120, transferring the supernatant containing the expanded T cells into the second cell culture chamber 220. Alternative techniques known in the art of fluid flow may be employed to transfer the supernatant from the first cell culture chamber 120 to the second cell culture chamber 220. Also, as shown, each bioreactor includes its own fluid and waste collection reservoirs, pumps, and associated tubing. However, it should be understood that reservoirs and pumps may be shared between bioreactors.

[0060] The present system is configured to allow perfusion of cells within the cell culture chamber with culture medium, as required for the various cell culture methods described herein. Perfusion ensures uniform nutrient and cytokine supply to the cell mixture, along with sufficient gas exchange and waste removal to support the formation of cell-based immunotherapy products. Maintaining consistent local concentration profiles of culture medium and cytokines ensures higher yields and the potential to accelerate the process of monocyte differentiation into DCs compared to prior art plate-based protocols. However, the combination of adherent (DC) and nonadherent (T cell) types, along with the high sensitivity of DCs to mechanical forces, poses challenges for the stimulation and proliferation of antigen-specific T cells, particularly with regard to fluid flow through the cell culture chamber. Therefore, in those embodiments in which culture medium and cytokines are provided via perfusion, the inventive system must be capable of supplying nutrients and cytokines to cells without removing them from the bioreactor, while also taking into account the shear sensitivity of certain antigen-presenting cells, such as DCs. The inventive system and method aim to refresh growth factors and optimize the retention of autocrine / paracrine signals that favor T cell proliferation, while maintaining minimal physical stimulation of DCs. To account for this, both the direction and rate of perfusion flow through the cell culture chamber must be considered.

[0061] In one aspect, the fluid flow rate is maintained below the sedimentation rate of the antigen-presenting cells. Thus, the antigen-presenting cells will remain within the culture chamber due to their mass. In other words, the antigen-presenting cells will sink toward the bottom of the cell culture chamber and therefore remain within the cell culture chamber. The flow rate, which is less than the settling rate, can be calculated according to Equation 1 below. [ka]

[0062] where v_max is the liquid velocity above which the cell will be lifted upward, ψ is the cell shape factor (the ratio of the surface area of the cell to the surface area of a sphere of equal volume; note that cells are not perfectly spherical and this factor is expected to be less than 1), d_p is the diameter of a spherical particle of volume equal to that of the cell, μ is the viscosity of the liquid containing the cell, g is the gravitational constant, p_cell is the density of the cell, p_liquid is the density of the liquid containing the cell, and e is the fraction of the volume of interest not occupied by the cell.

[0063] In the methods described below involving perfusion of medium, it should be understood that perfusion can be performed continuously during culture, or at specific time points throughout the time period that cells are cultured in any one cell culture chamber, such as, for example, once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more times each day or week. Continuous perfusion helps maintain a nearly constant culture volume throughout the process. Similarly, cytokines can be infused at one or more time points during culture, such as, for example, once, two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more times, or continuously. In these embodiments, continuous perfusion helps maintain a consistent local concentration profile of cytokines, which can help ensure higher yields compared to static cell culture methods and has the potential to increase the rate at which T cells are stimulated and expanded.

[0064] Perfusion parameters can be varied at any time during the culture cycle. Exemplary parameters include, but are not limited to, median flow rate, cytokine concentration, and duration of the culture cycle. Each of these parameters can affect the efficacy of T stimulation. For example, as described in International Patent Applications PCT / US2016 / 040042 and PCT / US2016 / 60701, recent studies designing culture chambers for monocyte diffusion to DCs used a perfusion rate of 0.1 dyn / cm. 2It has been determined that medium perfusion rates corresponding to wall shear stress levels of 0.05% are capable of generating DCs that are phenotypically identical to those generated using conventional 6- or 24-well plate-based protocols. Thus, by measuring one or more of the phenotypic and functional measurements described above during the culture cycle, the effect of one or more perfusion parameters on efficacy can be monitored and appropriate adjustments can be made.

[0065] To facilitate perfusion, the system includes one or more pumps 140. The pumps can be operably coupled to the cell culture chamber 120 for perfusing perfusion medium into the cell culture chamber. The bioreactor 110 can also include one or more fluid reservoirs 132. The fluid reservoirs 132 are in fluid communication with the cell culture chamber 110 and can be operably coupled to the one or more pumps 140. One or more tubes for connecting the fluid reservoirs to the pumps and the cell culture chambers are also provided. In certain aspects, the one or more pumps are configured to pump fluid from the fluid reservoirs, through the cell culture chambers, and into a waste collection reservoir. In the exemplary embodiment shown in FIG. 2, fluid travels from fluid reservoir 132 through tubing 152 to pump 140 and into cell culture chamber 120 via inlet 136, returns from cell culture chamber 120 via outlet 138, and travels through tubing 154 into waste collection reservoir 134.

[0066] In some embodiments, the fluid reservoirs and / or waste collection reservoirs can each be provided as one or more sealed bags or containers fluidly coupled to the chambers. Each reservoir contains an inlet port and an outlet port, or an outlet port and an outlet that are fluidly coupled to the inlets of one or more cell culture chambers. In some embodiments, a luer connector and a silicone gasket cut to fit around the luer connector can be used to prevent leakage through one or both of the inlets or outlets. In some embodiments, the sealed reservoirs can be connected to the cell culture chambers using a sterile tubing welding device, which creates a fluid connection that maintains sterility without exposing either container to the external environment. As will be discussed in more detail below, this allows the methods of the present invention to be performed in a closed system.

[0067] Due to the small size and portability of the disclosed cell culture systems, they can be easily used in conjunction with tubing welding devices. The systems of the present invention can be easily lifted and transported into close proximity with a tubing welder to make the necessary sterile connections. The size and configuration of the cell culture systems also make them compatible with standard incubators. The cell culture systems are sized and configured to fit on a single shelf inside a conventional incubator so that the disclosed processes can be performed therein. Multiple instruments can fit within a single incubator, depending on the configuration. Conditions within the incubator include a sustained temperature of 37°C and 95-100% humidity. Therefore, the materials selected must have the integrity to withstand these conditions, given that materials (including fluids and biologics) tend to expand under such conditions. Furthermore, in some situations, conditions within the incubator remain stable, allowing for automated recording of temperature to have knowledge of temperature fluctuations to correlate with any anomalies in reactions performed within the incubator.

[0068] Therefore, the supply of any power should not alter the environment within the incubator. For example, some pumps generate heat. Thus, in one embodiment, the pump is housed separately from the bioreactor but is still in fluid and operative communication with the reactor. In another embodiment, the pump is attached directly to the bioreactor and located within the incubator, but does not contain heat or is operatively connected to a heat sink and / or fan to dissipate heat. In another embodiment, the pump operates on a duty cycle to reduce the amount of heat generated. Regardless of the configuration, the pump is operatively coupled to the bioreactor, which in turn is connected to the cell culture chamber. In some embodiments, the system also includes a cell culture reservoir and, optionally, a heater to control the temperature of the fluid reservoir. In such a configuration, no incubator is required, and the system can operate autonomously using only a source of power. If the system lacks a heater, it can be operated inside the cell incubator.

[0069] Additional details regarding perfusion-based automated cell culture systems, such as a small-scale culture system for endothelial cell culture with on-board reagent storage and perfusion enabled by an on-board disposable peristaltic pump and a larger-scale culture system for dendritic cell generation from monocytes using chambers with polystyrene bottoms, can be found in International Patent Publication Nos. WO 2017 / 004169, WO 2017 / 079674, and WO 2018 / 005521, and U.S. Patent Application No. 16 / 539,916, each of which is incorporated herein by reference in its entirety.

[0070] The systems or devices of the present invention are modular and may be configured to allow fluid connection to other similar devices in series (i.e., fluid flows from one device into another) and / or in parallel, and to allow physical stacking with each other or physical arrangement within related devices such as incubators. The modular design of the systems allows for modules to be flexibly switched in and out, particularly depending on the desired processes to be included within the system.

[0071] Fluidic devices of the present invention, including bioreactors with cell culture chambers, can be provided in either or both microfluidic embodiments (i.e., one or more channels or chambers therein have dimensions in the range of about 1 μm to about 999 μm) or macrofluidic embodiments (all channels or chambers therein have dimensions of about 1 mm or greater).

[0072] Fluidic devices can further include additional fluid channels or compartments, gaskets or seals, mixing zones, valves, pumps, outlets, channels for pressurized gas, electrical conductors, reagents, ports, and tubing as required by the particular design. They may also contain one or more control modules, transmitters, receivers, processors, memory chips, batteries, displays, buttons, controls, motors, pneumatic actuators, antennas, electrical connectors, and the like. The devices preferably contain only materials that are non-toxic to mammalian cells and compatible with sterilization by the use of alcohol and / or other means such as heat or gamma irradiation or exposure to ethylene oxide gas.

[0073] The materials of the equipment are selected with appropriate chemical compatibility under the different temperature and pressure ratings specific to each process. Additionally, the pump options implemented in this device, including syringe, peristaltic, pressure, and rotary pumps, range from nL to mL in flow rates and from 10 to 10,000 psi in pressures depending on the flow and pressure requirements for different functions.

[0074] The systems of the present invention can also include one or more sample solution reservoirs or wells or other devices for introducing samples into the device at various inlets of the modules in fluid communication with the inlet channels. Reservoirs and wells used to load one or more samples onto the fluidic devices of the present invention include, but are not limited to, syringes, cartridges, vials, Eppendorf tubes, and cell culture materials (e.g., 96-well plates).

[0075] Where useful, the surfaces of the devices can be made more hydrophilic, such as by exposure to plasma, or can be coated with one or more gels, chemically functionalized coatings, proteins, antibodies, proteoglycans, glycosaminoglycans, cytokines, or cells. Fluidic devices of the invention are preferably fluid-leak-free under operating conditions and capable of sterile operation over periods of days to weeks. Fluidic devices of the invention also include a sampling mechanism that allows fluid to be removed from the system for testing without introducing new materials or contaminants into the system.

[0076] In certain aspects, at least a portion of the cell culture system comprises disposable components, some or all of which can be housed within a non-disposable frame. In other aspects, all components of the system are disposable. Additionally, in some embodiments, the cell culture system includes a sample tracking component for tracking and documenting patient material.

[0077] At least one step, and sometimes multiple or all steps during the manufacturing process, are monitored for product characteristics (e.g., purity and polymorphism) using various in-line process analytical tools (PAT) or miniaturized micro total analytical systems (microTAS).

[0078] As described above, the cell culture system of the present invention is capable of controlling the direction and flow of fluids and substances within the system. The system of the present invention can use pressure-driven flow control, for example, utilizing valves and pumps, to manipulate the flow of cells, reagents, etc. in one or more directions and / or within one or more channels of a fluidic device. However, other methods, such as electroosmotic flow control, electrophoresis, and dielectrophoresis (Fulwyer, Science 156, 910 (1974); Li and Harrison, Analytical Chemistry 69, 1564 (1997); Fiedler, et al. Analytical Chemistry 70, 1909-1915 (1998); U.S. Patent No. 5,656,155), can also be used alone or in combination with pumps and valves.

[0079] The systems of the present invention can also include or be operatively coupled to one or more control systems for controlling the movement of fluids through the system, monitoring and controlling various parameters within the system, such as temperature, as well as detecting the presence of cell-based immunotherapy product, product quantity (directly or indirectly), conversion rate, etc. The systems may also be equipped with numerous classes of software, such as advanced real-time process monitoring and control processes that allow feedback control, and processes that allow integration and scale-up given reaction and purification results obtained using the systems.

[0080] In certain embodiments, the system includes a combination of micro-, milli-, or macrofluidic modules and tubing that are interchangeable in terms of channel dimensions, flow geometries, and interconnections between the different modules of the device. Each module and tubing may be designed for a specific function. In one embodiment, all modules in the system are designed for cell culture and T cell stimulation. In other embodiments, modules in the system are designed for different functions, such as tissue processing, dendritic cell generation, cell culture, enrichment, and / or purification, all integrated for continuous manufacturing of immunotherapy products. Both homogeneous and heterogeneous processes suitable for flow applications are considered. These processes are designed and optimized with respect to starting materials and operating conditions, such as temperature, pressure, and flow rate, so as not to easily clog the system during flow processes. (Gas-impermeable cell culture chamber)

[0081] In some embodiments, the cell culture chamber of the disclosed system is made from a gas-impermeable material. A gas-impermeable material is a material that is biocompatible and to which dendritic cells will adhere. In one exemplary embodiment, the gas-impermeable material is made from polystyrene, which is useful for enriching monocytes from a heterogeneous suspension of PBMCs, as described above. Making the entire cell culture chamber from a gas-impermeable material provides a larger surface area to which cells can adhere, improving the sterility of the system.

[0082] The gas-impermeable cell culture chamber can be substantially identical to chamber 120 shown in FIG. 2, except that additional surface 124 and all other surfaces of chamber 120 are made from the same material as bottom surface 122, and can have any volume, shape, size, and physical characteristics described above. In some embodiments, the top, bottom, and all sidewalls of chamber 120 are gas-impermeable. The bottom of the gas-impermeable cell culture chamber is designed to fit 6- and 24-well plates (9.5 cm, respectively). 2 and 3.8 cm 2) or the like. 2 ~about 200cm 2 surface areas of, for example, about 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 100.0, 125.0, 150.0, 175.0, and 200.0 cm 2 It should be understood that the surface area may be smaller than a conventional well plate, or even much larger (e.g., having a surface area comparable to standard cell culture dishes and flasks), such as having a surface area of 1000 sq. mm or 1000 sq. mm, and any surface area in between.

[0083] When the chamber is not permeable to gas, certain modifications must be made to the system described above. For example, in such embodiments where gas does not flow through one of the surfaces of the cell culture chamber, gas exchange between the cells and the medium must be facilitated in another way. The cell culture chamber includes one or more inlets 126 and 136 and one or more outlets 128 and 138. The inlet and outlet openings can be fluidly coupled to tubing that is sealed with the respective openings. The inlets and outlets are thereby connectable to perfusion fluid reservoirs and waste fluid reservoirs with corresponding pumps for moving the perfusion fluid through the chamber. The inlets and outlets can be located on any surface of the chamber. In some embodiments, they are located at the top of the chamber. The tubing is preferably high-permeability tubing. To effect gas exchange, the medium can exchange gases through the high-permeability tubing prior to entering the chamber and after leaving the chamber. Gas is thus effectively brought into the chamber through one or more inlets and removed through one or more outlets. The inlets or tubing connected thereto can include a filter, such as a 0.2 micron filter, to filter liquid or air entering the cell culture chamber.

[0084] Gas flow is affected by the perfusion rate, a parameter that can be controlled as described above. By exchanging gases through highly permeable tubing, the system maintains the ability to achieve desired levels of gas exchange without requiring the chamber to be gas permeable. Cell culture methods can be performed in a completely gas-impermeable chamber with inlets 126 and 136 and outlets 128 and 138 for perfusion and gas flow. In method embodiments, a gas-impermeable cell culture chamber can be used to culture cells by loading them into the chamber and perfusing them by flowing cell culture medium in and out of the chamber via the inlets and outlets. The perfusion flow provides nutrients and gas exchange to the cell culture. Because flow through the chamber is laminar, some methods may require additional shaking, such as for cell harvesting. However, given the size and configuration of the disclosed system, the entire system can be placed on an orbital shaker, if desired.

[0085] Another advantage of gas-impermeable embodiments is that they are easier to manufacture because they have fewer different parts and materials. They can be made as large or small as needed. The gas-impermeable chamber can be integrally formed, or it can be formed from multiple parts. For example, it can be formed from a single piece of material by conventional manufacturing processes or additive manufacturing processes such as 3D printing. In embodiments, multiple components, each made from a gas-impermeable material, are joined together using methods known in the art, such as mechanical fastening, adhesive and solvent bonding, and welding, such as ultrasonic welding. (Interchangeable cell culture chamber)

[0086] The cell culture system of the present invention is configured to be capable of connecting to cell culture chambers of various sizes and shapes. The cell culture system can include a fluid reservoir, a waste reservoir, and one or more pumps for controlling fluid flow to and from the reservoir. The cell culture system also has an area configured to receive one or more cell culture chambers of different shapes and sizes. One or more tubes fluidly connect the fluid reservoir, the cell culture chamber, and the waste reservoir. The pump is configured to move the fluid through the tubes.

[0087] Different cell culture chamber sizes can be used for different purposes or in combination with each other. Sizes can be selected based on desired cell yields or different proportions of reagents required. For example, a small cartridge size may be useful for research, preclinical use, or process development. Larger cartridges are higher-capacity versions with the same architecture.

[0088] The height of one or more cell culture chambers can vary. For example, without limitation, exemplary ranges of cell culture chamber height include heights of anywhere from 0.5 mm to 100 mm, such as 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0 mm, or greater, or any height therebetween. In certain embodiments, the chamber height can be comparable to the liquid height in cultures typically performed in 6- and 24-well plates, such as 2-6 mm, with volumetric capacity of about 0.8 mL to 6 mL. In other embodiments, the cell culture chamber is about 50 cm 2The cell culture chamber may be larger, such as 10 mm to 50 mm, with a culture surface of about 100 mL. In some embodiments, the cell culture chamber has a volume capacity of about 1 mL to about 100 mL, and may be about 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, or 90 mL, or anything in between. In other embodiments, the cell culture chamber has a volume capacity of about 100 mL to about 1,000 mL, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 mL. In a specific embodiment, the cell culture chamber has a capacity of 210 mL.

[0089] The interchangeability of cartridges associated with the present invention allows for scale-up during cell growth procedures using the same system. This avoids the need to switch to a different cell culture system to continue growing cells. For example, to produce a batch size of approximately 2 billion cells, the system can start with a small cartridge with a capacity of approximately 25 mL and then scale up to a larger cartridge with a capacity of approximately 210 mL. This interchangeability means that more parts of the process, from activation to fill and finish, can be performed in the same system. Depending on the needs of a particular protocol, the system can operate with, for example, two large (approximately 210 mL) cell culture chambers, two small (approximately 25 mL) cell culture chambers, or one of each. Because each inlet and outlet can be connected to a chamber of any size, the system can be scaled up or down as needed.

[0090] Referring again to FIG. 1 , system 900 includes platform 950 configured to support one or more cell culture chambers 820 and 920. Regardless of shape or size, the cell culture chambers can be connected via tubing 940. In this manner, cell culture chambers of different shapes and sizes are compatible with the system. The cell culture chambers can be arranged in different configurations on the platform, such as side-by-side or stacked. In an embodiment, tubing 940 is integrally formed with the chamber. The tubing and chamber body can be joined together using the same manufacturing methods discussed above. In one embodiment, the cell culture chamber is manufactured with at least a portion of the material in the sidewall and / or top wall cut out to allow for the formation of one or more inlets or outlets. In an exemplary arrangement, tubing can be separately inserted into the opening to form a seal with the container. It should be understood that the aforementioned configurations are merely examples, and that other configurations for joining the chamber and one or more tubing are also contemplated embodiments of the present invention.

[0091] Interchangeability is facilitated, in part, by the use of sterile tubing connections to join the various containers and chambers of the system. The sterile tubing is preferably connected using a sterile tubing welder. Generally, a sterile tubing welder is a device that can receive two tubes, secure them in place, then cut them using a high-temperature blade, realign them so that the first and second tubes are aligned, and melt the two cut ends together when the blade is retracted. A sterile tubing welder for use with the present invention is the SCD® IIB, Vante from Terumo BCT, Inc. (Lakewood, CO). Vante® 3690 from BioPharm (Tucson, AZ), and Genesis BPS TM Any TCD® from (Ramsey, NJ) It may be a commercially available sterile tubing welder.

[0092] As will be explained in more detail below, in certain embodiments, the system is functionally closed. The closed system is maintained by all transfers being performed using sterile tubing welds. For example, cells can be stored in a sterile bag, which is then connected to a sterile cell culture chamber. The cells can be flowed into the cell culture chamber while maintaining sterility. The tubing connectivity allows cell seeding, washing, and harvesting to all be performed on the same device under sterile conditions. By using a sterile tubing welder, there is no need for one bag to be disconnected before connecting another.

[0093] The above description focuses on system components and various possible configurations. The following description focuses on certain processes that may be performed using the system of the present invention. (CAR-T and TCR processing in a closed system)

[0094] The cell culture system described above is useful for methods of generating CAR-T and TCR-transduced T cells. A visualization of the CAR-T / TCR workflow is shown in FIG. 4. The disclosed system can be used to perform several of the workflow steps in a closed system, including genetic modification, cell expansion, cell washing and concentration, and cell formulation. Prior art methods used in the industry to generate CAR-T and TCR-transduced T cells are not performed in a closed system. Commonly used polystyrene T-flasks need to be opened and closed to perform transfers and are therefore subject to potential contamination. The present invention uses closed, replaceable cartridges made from solid polystyrene, which allows protocols designed for T-flasks to be easily reformatted to be performed on the disclosed sterile system.

[0095] In some embodiments, the methods involve flowing cell culture medium into a culture chamber with T cells, perfusing the T cells, and transducing them with a transduction reagent, such as an inactivated virus expressing a CAR or TCR. The perfusion fluid may also include an activation reagent for expanding the cells. In some embodiments, the transduction and / or activation reagents are premixed with the cells; in other embodiments, they are from separate sterile bags or containers. The bags can be connected through a sterile welding means as described above, and the reagents can be flowed into the bags by gravity or by a pump. In some embodiments, the cell culture chamber is completely filled with little or no headspace.

[0096] The cells are grown for up to approximately three days, during which time they are allowed to take on the CAR and TCR and are sustained by perfusion medium. The cells form a precipitate within the chamber, and the perfusion rate is kept low enough to prevent the cells from flowing out of the cartridge. The transduced cells are expanded within the culture chamber and then transferred to a larger culture chamber connected via sterile tubing for further expansion. Using the method of the present invention, seven days of expansion in a small cartridge (25 mL volume) can yield approximately 500 to 1 billion T cells, while a larger cartridge (210 mL volume) can yield approximately 1 to 3 billion T cells.

[0097] The connection to the perfusion bag is detached and a collection bag is attached. The cells are then drained into the collection bag. In embodiments, a buffer bag can be connected in the same manner and used to perform one or more washes of the cells before they are removed into the collection bag. The volume of liquid in which the cells reside can be increased or decreased by draining one liquid, adding another, and resuspending the cells in the new volume of liquid. In some embodiments, the system can be drained to remove accumulated lactic acid. As cell cultures grow, lactic acid accumulates and may not be removed quickly enough through perfusion alone. A solution is to drain the medium to reduce the total volume (perhaps by 90-95%) without removing the cells, and then perfuse with fresh medium. In some embodiments, the medium can be replaced with a different cell culture medium or cryopreservation medium.

[0098] The cell culture chambers are connected in a closed system so that the entire process is carried out in a closed environment without the need to expose the medium to air by opening any of the containers when transferring. As mentioned, all connections and disconnections in this process can be done using a sterile tubing welder.

[0099] Unlike the prior art, the disclosed activation, transduction, and expansion methods are performed in a closed, sterile system. Using the interchangeability described above, users can scale up to batches of 10-20 billion expanded cells, all in a closed environment. In some embodiments, the methods may be used to prepare batches for manufacturing on larger bioreactor systems. In traditional CAR-T manufacturing, batches of 10 billion or more cells are required. The disclosed systems and methods are available from GE Healthcare (Chicago, IL) for 1 billion or more cells. TMThe upstream steps of activation, transduction, and initial propagation can be performed prior to transfer to a larger propagation system such as a magnetically activated spore system. This capability makes the system highly compatible with non-magnetic activation reagents.

[0100] Figure 5-6 shows the BATON from Flaskworks, LLC (Boston, MA). TM Figure 5 shows an example of a comparison between T cell expansion performed using the disclosed system known as the G-REX® and another commercially available T cell expansion platform from WilsonWolf (St. Paul, MN). A 9-day B12 study was performed in a 100% RT-PCR cartridge with PBMCs stimulated with DYNABEADS® from Thermo Fisher (Waltham, MA). ATON TM A graph of doubling growth using BATON is shown. TM The robust doubling expansion achieved with is comparable to that of G-REX®.

[0101] Figure 6 shows the cell counts on days 0 and 7 using both systems. TM In this study, approximately 1 billion cells were obtained from 40 million T cells in 7 days in a 210 mL cartridge according to the present disclosure. The phenotype is primarily central memory. As further shown in Figure 7, the phenotypic profile is consistent with that of the BATON TM The figures are also comparable between Corning Inc. (Corning, NY ) available from BATON. TM and G-REX® both generated comparable CD4 / CD8 ratios at day 9.

[0102] As shown in Figure 8, BATON TM The cytotoxicity of T cells generated using G-REX® is comparable to that of G-REX®. Effector cells were expanded for 9 days, and target cells were The effector and target cells were Jurkat T cells. Cells were mixed at a 10:1 effector to target ratio and incubated at 37°C in 5% CO for 24 hours. The culture medium was RPMI 1640 (ATCC) + 15% FBS. Day 5 and day 7 cells from all three groups were comparable in cytotoxicity. (Neoantigen process in a closed system)

[0103] The disclosed system is also useful in a workflow for generating neoantigen-targeting T cells. This class of therapy involves co-culture of antigen-presenting cells and autologous T cells stimulated with a library of tumor-specific peptides. The production of neoantigen-presenting cells is more complex than CAR-T. Unlike CAR / TCR, this method can pursue a library of targets, rather than just one. This requires certain capabilities provided by the disclosed system that are not available from prior art systems.

[0104] The system generates fresh dendritic cells from the patient's monocytes. The system is also configured to co-culture the dendritic cells with the patient's PBMCs or T cells and deliver the stimulated T cells. The system can perform multiple cycles of co-culture with the newly generated dendritic cells to avoid competitive effects between different antigens. Parallel processing of dendritic cells and T cells to facilitate this process will be described in more detail below. A typical dose for neoantigen therapy is approximately 200 million T cells, which can be handled by the disclosed system in either a small (approximately 25 mL) or large (approximately 210 mL) cartridge.

[0105] Unlike prior art methods, the system of the present invention facilitates all of the above in a closed environment. The method utilizes the system's interconnected cell culture chambers. Purified monocytes are introduced into one of the cell culture chambers, and purified T cells are introduced into the other. The monocytes are perfused with cell culture medium to generate dendritic cells, which are then contacted with an antigenic substance containing a tumor-specific peptide. This generates mature dendritic cells that present the tumor-specific peptide. T cells are transferred into the chamber containing the mature dendritic cells and co-cultured with the T cells. The co-culture generates neoantigen-targeting T cells. After one cycle of stimulation via co-culture, the T cells can be removed and optionally flowed into a second chamber containing newly generated mature dendritic cells, and a second co-culture can be performed. A second batch of dendritic cells can be generated asynchronously from the dendritic cells generated in the first chamber.

[0106] In embodiments, mature dendritic cells are developed in a first chamber, and then T cells are added to the first chamber and co-cultured. The T cells from the first chamber are then transferred to a second chamber where they are co-cultured with newly developed dendritic cells stimulated with either the same or a different set of peptides. The expanded T cells can then be transferred back to the first chamber where another batch of fresh mature dendritic cells stimulated with either the same or a different set of peptides awaits.

[0107] T cells can be transferred back and forth between the two connected chambers any number of times as desired. This can be particularly useful for stimulating DCs with several different peptides. For example, if one has a library of 20 or so peptides with which DCs need to be stimulated, attempting to stimulate DCs with all of the peptides at once would result in insufficient stimulation due to competitive effects between the peptides. Some peptides have greater efficacy and / or affinity than others. Stimulation can be performed in a stepwise manner, for example, stimulating a batch of DCs with five peptides in a first co-culture, then stimulating the next batch of DCs with five different peptides in a second co-culture, and so on. In other embodiments, multiple stimulation cycles can be performed using a single small group of peptides. In non-limiting embodiments, T cells can be transferred two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, or even twenty-five times. Each co-culture can involve DCs stimulated with the same or different peptides.

[0108] The entire method is performed within a closed system of the present disclosure, thereby maintaining sterility throughout the method. As with other disclosed methods, culture chamber connections can be achieved using sterile tubing connected using a tubing welder. This maintains the sterility of the cell culture medium provided in the sterile container. In various embodiments, T cells can be transferred between chambers on the same instrument or between chambers on separate instruments.

[0109] After co-culture is complete, the fluid is drained from the chamber and the neoantigen-targeting T cells can be washed with buffer, resuspended in cryopreservation solution, and / or collected in a collection bag also connected in a closed fashion.

[0110] Figure 9 shows a schematic flowchart of a method for co-culturing freshly cultured dendritic cells and PBMCs or T cells on a closed system of the present invention. Using sterile welded connections, the disclosed cell culture system provides automated seeding, culture, and cell harvest. As shown in the workflow, monocytes are seeded on day 0 and differentiated into dendritic cells over days 0-6. On day 6, allogeneic PBMCs or T cells are added. The dendritic cells allow for T cell expansion from the PBMCs. The expanded T cells are harvested on day 13.

[0111] Expanded T cells generated using the disclosed method exhibit robust cytotoxicity. The results of an example performed using the workflow are shown in Figure 10. The target ratio of CD8+ T cells to Jurkat cells was 1:1, and experiments were performed using 1-3 million harvested cells. Jurkat cells were stained with PKH67 prior to the assay. Cells were incubated for one day, and all cells were stained with CD3 and Annexin V after the assay. The resulting dead Jurkat cells are shown in Figure 10. (Parallel processing of dendritic cells and T cells)

[0112] The process for forming a cell-based immunotherapy product requires the co-culturing of two types of cells. In the disclosed system, these cells can be generated in parallel for more efficient generation of antigen-specific T cells. Briefly, dendritic cells are generated from monocytes and matured by contact with antigenic material, and T cells are activated and then co-cultured with DCs. In prior art methods, this method would require several manual steps. However, the present system can generate two batches of cells in parallel in a closed system. Using the system disclosed herein, dendritic cells are generated in one chamber, and in parallel, T cells are stimulated in another connected chamber. Monocytes are perfused in the first chamber to generate DCs, which are contacted with antigenic material to mature them. Activated T cells from the other connected chamber are flowed into the first chamber to contact the DCs and further culture the T cells. As in other methods, the chambers are connected via sterile tubing so that the method is performed in a substantially closed system. The T cells can be collected by flowing them into a collection vessel and / or transferred to a cryopreservation medium while still in a closed system configuration.

[0113] Reference is made to FIG. 11 , which shows a general overview of a process for forming a cell-based immunotherapy product. According to one embodiment of the present invention, a step in producing a cell therapy product involves co-culturing stimulated antigen-presenting cells (e.g., DCs) with T cell-containing cells in a biological reactor containing a cell culture chamber. A supernatant containing an expanded therapeutic T cell product is generated during the culture. In some aspects, to generate a sufficient number of antigen-specific T cells to induce a therapeutic response in a patient, the T cells must undergo additional culture in one or more additional cell culture chambers. To effect this additional culture, the supernatant must be transferred from the culture chamber in which it was generated to a subsequent cell culture chamber containing a fresh supply of antigen-presenting cells. The transfer of the supernatant between cell culture chambers may involve the introduction of a gas flow into the first cell culture chamber, which transfers the supernatant containing the first cell product through a fluidic connector into the new cell culture chamber. Additionally, during each of the culture steps, a perfusion fluid containing, for example, medium and cytokines, can be perfused through the chamber. In one aspect, a perfusion fluid flows through the chamber along a vertical flow path to ensure that the cells remain within the chamber during culture. One or more subsequent cell culture chambers are connected to the system, each chamber capable of containing a new batch of antigenic peptide-pulsed autologous antigen-presenting cells.

[0114] To stimulate and expand antigen-specific T cells, the process begins with co-culturing T cell-containing cells with antigen-presenting cells (APCs) obtained from the same individual in a cell culture chamber. In certain embodiments, the T cell-containing cells comprise peripheral blood mononuclear cells (PBMCs), and the APCs comprise DCs. The T cell-containing cells and APCs may be co-cultured at ratios of, for example, but not limited to, about 1000:1, 900:1, 800:1, 700:1, 600:1, 500:1, 400:1, 300:1, 200:1, 100:1, 75:1, 50:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, They can be provided to the cell culture chamber at a ratio of about 1000:1 to 1:1000 (T cell-containing cells:APCs), such as 1:8, 1:9, 1:10, 1:15, 1:20, 1:50, 1:75, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, or any ratio in between. In one aspect, a 10:1 ratio is preferred.

[0115] To initiate T cell stimulation and proliferation from the interaction between APCs and T cell-containing cells, the APCs must be stimulated. This can be done through the use of one or more stimulatory molecules. In some embodiments, the stimulatory molecules are non-tumor specific. In other embodiments, the stimulatory molecules are tumor specific. For example, the stimulatory molecules can be selected from one or more characteristics of an individual's tumor, such as different antigen peptides. In some embodiments, the stimulatory molecules are preferably added only at the beginning of the culture cycle. The stimulatory molecules can be added only for a period of about several minutes, an hour, several hours, or longer. In one preferred embodiment, the stimulatory molecules are added for a time period of about one hour.

[0116] Co-culture of APCs and T cells is carried out in a culture medium. Exemplary culture media include, but are not limited to, RPMI medium and Cellgenix® medium. Any culture medium known in the art may be used. Any other suitable culture medium can be used in accordance with embodiments of the present invention. Cytokines, such as IL-4 and GM-CSF, can also be added to the culture medium.

[0117] Typically, performing only one co-culture is not sufficient. The disclosed system allows T cells to be drawn into suspension. Here, T cells can be restimulated with fresh dendritic cells, and multiple co-cultures can be performed in a closed system. In the parallel processing method of the present invention, cartridges can be connected in a chain, and cells can be pushed from one cartridge to another. One reactor containing mature, adherent DCs will be loaded with PBMCs and undergo stimulation cycles using medium and cytokine perfusion. In parallel processing, expanded T cells can be continuously exposed to fresh DCs generated in the first cell culture chamber by pulsing with antigenic peptides. The stimulation process can continue for as long as necessary to generate a sufficiently large number of cells for a therapeutic dose of T cells.

[0118] Connecting multiple chambers together via a sterile connection can involve a multiple chamber configuration as discussed above with respect to Figure 3. The connection allows for the injection of sterile air into a first cell culture chamber and transfer of the supernatant containing the expanded T cells into a second cell culture chamber. In certain embodiments, one or more bioreactors can be provided in a system containing modules for effecting various other processes prior to, in parallel with, or subsequent to the processes occurring in the cell culture chambers of the bioreactor.

[0119] One cartridge can be transferred via sterile welding and sealing into a collection bag or into a new cartridge. In some embodiments, dendritic cells can be generated, then harvested and cryopreserved for use on demand. Meanwhile, the system can independently activate a second cartridge for fresh dendritic cell generation.

[0120] In one aspect, a computational modeling approach is used to optimize the interaction between T cells and antigen-presenting cells. The computational model according to the present invention considers the effects of perfusion and the optimal time required for stimulation, and incorporates both particle interaction-based and kinetic parameter-based approaches. Exemplary particle interaction-based and kinetic parameter-based approaches are known in the art, some of which are described herein. For example, with respect to the particle interaction-based approach, Day and Lythe use the following formula to describe the time required for a T cell to find an APC on the surface of a lymph node, where D is the diffusion rate of the T cell and b is the radius of the APC centrally located within a spherical lymph node of radius R. See Day et al., Mathematical Models and Immune Cell Biology; 2011. [ka]

[0121] Regarding kinetic parameter-based approaches, Valitutti developed a model of interactions between T cells and antigen-presenting cells, as shown in Figure 9. See Valitutti et al., FEBS Lett. 2010. However, such interactions were not modeled within the context of a culture chamber or bioreactor.

[0122] By incorporating both particle interaction-based approaches as well as kinetic parameter-based approaches into the computational model of the present invention, automated determination and monitoring of the optimal perfusion rate of the perfusion fluid (e.g., cytokine infusion medium) to maximize the probability that two cell types will contact each other within the cell culture chamber can be achieved.

[0123] For example, in one embodiment, a cell culture system is provided that includes a cell culture chamber and a central processing unit with a memory containing instructions executable by the central processing unit. In one aspect, the instructions cause the system to receive, as a first input, data comprising the size of the cell culture chamber, receive, as a second input, data comprising a first concentration of a first cell type and a second concentration of a second cell type in one or more fluids to be introduced into the cell culture chamber, and calculate, based on the first and second inputs, a perfusion rate of a perfusion fluid to be introduced into the cell culture chamber that maximizes the probability that the first cell type and the second cell type will contact each other in the cell culture chamber. Additional details regarding computer systems for implementing the methods of the invention in cell culture systems are provided below.

[0124] In some aspects, the system also includes one or more pumps operably coupled to the one or more perfusion fluid reservoirs and operably coupled to the central processing unit, such that the central processing unit also controls the perfusion rate of the perfusion fluid by controlling the one or more pumps. (recirculation of medium)

[0125] Cell culture media and supplements (such as cytokines) are expensive, and spent media often has residual nutrients therein that are discarded. Recognizing this, the disclosed system provides a method for recapturing and recirculating a portion of the partially spent media. In one method of the invention, cells are cultured in one of the disclosed cell culture chambers, and cell culture media is flowed through the cell culture chamber. Generally, the fluid flows into an inlet and out an outlet. A portion of the cell culture media that has already flowed through the cell culture chamber and out the outlet is recirculated back into the cell culture chamber during the cell culture process.

[0126] To determine the amount and / or portion of spent medium that should be returned to the cell culture chamber, the present invention measures one or more parameters, such as the nutrient content or pH, of the spent medium prior to recirculation. The measured nutrients can be glucose, lactate, dissolved oxygen, or cellular metabolites. The parameter of interest is measured, and a processor determines whether the parameter meets a predetermined threshold indicating it can be recirculated. If so, the medium is pumped back into the cell culture chamber.

[0127] The spent medium can be recirculated as is, or it can be combined with a bolus of fresh medium. On the other hand, if the spent medium does not meet a predetermined threshold, it is discarded. In some embodiments, a valve operates to either direct the spent medium to a waste reservoir or back into the cell culture chamber.

[0128] In various embodiments, recirculation can be performed at any frequency. For example, one or more sensors can check the spent medium at regular intervals, such as every second, every minute, every 10 minutes, every hour, etc. In other embodiments, one or more sensors can operate continuously by measuring the medium as it progresses through the waste line. In some embodiments, recirculation is controlled through feedback from an external filter or sensor that monitors the waste medium to determine whether it can be reused or whether it is exhausted. In some embodiments, an in-line sensor is embedded within the system to monitor the waste medium and determine whether it can be recirculated.

[0129] Recirculation achieves the goal of allowing gas exchange between the exterior of the cell culture chamber and the cells contained therein while reducing the amount of medium that would be consumed by the process relative to a process in which the medium was simply perfused without recirculation.

[0130] The rate of recirculation can be modulated based on the required gas exchange and nutrient supply. For example, in a cell culture process in which T cells are expanded from a small number to a much larger number, the initial stages of the culture can be performed at a low-flow rate perfusion with 100% recirculation. This is because the nutrients in the closed perfusion loop are adequate for the small number of cells, and the flow rate is set sufficient to ensure adequate gas exchange (oxygen intake, CO2 exhaust). Then, as the cells begin to proliferate, their need for nutrients and gas exchange increases. Growth can be monitored and form the basis for decisions related to increasing the perfusion flow rate (to increase gas exchange) and changing the extent of recirculation (recirculating only a portion of the medium and adding new medium at a gradually increasing rate). In principle, this can be done dynamically and automatically.

[0131] In embodiments, the cell culture chamber includes one or more sensors operably coupled to the cell culture chamber. The system can be configured with various sensor configurations to monitor different parameters and integrate with a control system. The sensors may be capable of measuring one or more parameters in the cell culture chamber, such as pH, dissolved oxygen, total biomass, cell diameter, glucose concentration, lactate concentration, and cellular metabolite concentration. The system can be customized with off-the-shelf, single-use sensors for glucose and lactate that sample the perfusion effluent fluid and transmit data. In some embodiments, the cartridge is optically transparent and can be interfaced with sensing modalities such as optical density or Raman. In some embodiments, the sensor may be operably coupled to a waste line or waste reservoir and configured to measure one or more parameters of the fluid flowing therein. In certain aspects, the one or more sensors are operably coupled to a computer system having a central processing unit for executing instructions, such that automatic monitoring and adjustment of parameters is possible. The system may be configured to automatically redirect the fluid back into the chamber via the inlet if the fluid meets certain parameters. In some embodiments, the instrumentation interfaces with a control system architecture using computers, networks, and graphical user interfaces for process management and other peripheral devices for interfacing with the process plant machinery. Waste pipes may have valves that can direct fluids to one location or another depending, for example, on whether the fluid has sufficient levels of nutrients. Additional details regarding computer systems for implementing the methods of the invention using cell culture chambers are provided below. (System Architecture)

[0132] Aspects of the disclosure described herein, such as controlling the movement of fluids through the system and monitoring and controlling various parameters as described above, can be implemented using any type of computing device, such as a computer or programmable logic controller (PLC), including a processor, e.g., a central processing unit, or any combination of computing devices, each device performing at least a portion of the process or method. In some embodiments, the systems and methods described herein may be implemented using a handheld device, e.g., a smart tablet, a smartphone, or a specialized device produced for the system.

[0133] The methods of the present disclosure can be implemented using software, hardware, firmware, hardwiring, or any combination thereof. The features that implement the functionality can also be physically located in various locations, including being distributed such that some of the functionality is implemented in different physical locations (e.g., an imaging device in one room and a host workstation in another room or in a separate building, with wireless or wired connections).

[0134] Processors suitable for executing a computer program include, by way of example, both general-purpose and special-purpose microprocessors, and any one or more processors of any kind of digital computer. Generally, a processor will receive instructions and data from a read-only memory or a random-access memory, or both. The elements of a computer are a processor for executing instructions and one or more memory devices for storing instructions and data. Generally, a computer will also include one or more non-transitory mass storage devices, such as magnetic, magneto-optical, or optical disks, for storing data, or be operatively coupled to receive data from or transfer data to them, or both. Information carriers suitable for embodying computer program instructions and data include all forms of non-volatile memory, including, by way of example, semiconductor memory devices (e.g., EPROM, EEPROM, solid-state drives (SSD), and flash memory devices), magnetic disks (e.g., internal hard disks or removable disks), magneto-optical disks, and optical disks (e.g., CD and DVD disks). The processor and memory can be supplemented by, or incorporated in, special-purpose logic circuitry.

[0135] To provide for interaction with a user, the subject matter described herein can be implemented on a computer having I / O devices, e.g., CRT, LCD, LED, or projection devices, for displaying information to a user, and input or output devices, such as a keyboard and pointing device (e.g., a mouse or trackball), by which a user may provide input to the computer. Other types of devices can be used to provide for interaction with a user as well. For example, feedback provided to the user can be any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback), and input from the user can be received in any form, including acoustic, speech, or tactile input.

[0136] The subject matter described herein can be implemented in a computing system that includes a back-end component (e.g., a data server), a middleware component (e.g., an application server), or a front-end component (e.g., a client computer having a graphical user interface or web browser through which a user may interact with an implementation of the subject matter described herein), or any combination of such back-end, middleware, and front-end components. The components of the system can be interconnected through a network, e.g., a communications network, by any form or medium of digital data communication. Examples of communications networks include a cellular network (e.g., 3G or 4G), a local area network (LAN), and a wide area network (WAN), e.g., the Internet.

[0137] The subject matter described herein can be implemented as one or more computer program products, such as one or more computer programs tangibly embodied in an information carrier (e.g., in a non-transitory computer-readable medium) for execution by or to control the operation of a data processing device (e.g., a programmable processor, a computer, or multiple computers). The computer programs (also known as programs, software, software applications, apps, macros, or code) can be written in any form of programming language, including compiled or interpreted languages (e.g., C, C++, Perl), and can be deployed in any form, including as a standalone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. The systems and methods of the present invention can include instructions written in any suitable programming language known in the art, including, but not limited to, C, C++, Perl, Java, ActiveX, HTML5, Visual Basic, or JavaScript.

[0138] A computer program does not necessarily correspond to a file. A program can be stored among files or portions of files that hold other programs or data, in a single file dedicated to the program, or in multiple associated files (e.g., files storing one or more modules, subprograms, or portions of code). A computer program can be deployed to be executed on one computer or multiple computers at one site, or distributed across multiple sites and interconnected by a communications network.

[0139] The file may be a digital file, for example, stored on a hard drive, SSD, CD, or other tangible non-transitory medium. The file may be transmitted from one device to another over a network (e.g., from a server to a client, e.g., as packets transmitted through a network interface card, modem, wireless card, or the like).

[0140] Writing a file according to embodiments of the present invention involves transforming a tangible, non-transitory computer-readable medium, for example, by adding, removing, or rearranging particles (e.g., involving net charge or dipole moments into a pattern of magnetization by a read / write head), which then represents a new collocation of information about an objective physical phenomenon desired by and useful to a user. In some embodiments, writing involves a physical transformation of matter in the tangible, non-transitory computer-readable medium (e.g., involving certain optical properties so that an optical read / write device can then read the new, useful collocation of information, e.g., burning a CD-ROM). In some embodiments, writing a file involves transforming a physical flash memory device, such as a NAND flash memory device, to store information by transforming physical elements in an array of memory cells made from floating-gate transistors. Methods of writing files are well known in the art and can be invoked manually, programmatically, or automatically, for example, by a save command from software or a write command from a programming language.

[0141] Suitable computing devices typically include mass memory, at least one graphical user interface, at least one display device, and typically include communication between devices. Mass memory illustrates a type of computer-readable medium, i.e., computer storage media. Computer storage media may include volatile, nonvolatile, removable, and non-removable media implemented in any method or technology for storage of information such as computer-readable instructions, data structures, program modules, or other data. Examples of computer storage media include RAM, ROM, EEPROM, flash memory, or other memory technology, CD-ROM, digital versatile disks (DVDs), or other optical storage devices, magnetic cassettes, magnetic tape, magnetic disk storage, or other magnetic storage devices, radio frequency identification tags or chips, or any other medium that can be used to store desired information and that can be accessed by a computing device.

[0142] As those skilled in the art will recognize as necessary or optimal for the practice of the methods of the present invention, a computer system or machine employed in embodiments of the present invention may include one or more processors (e.g., a central processing unit (CPU), a graphics processing unit (GPU), or both), a main memory, and a static memory, communicating with each other via a bus.

[0143] In the exemplary embodiment shown in FIG. 12 , system 600 may include a computer 649 (e.g., a laptop, desktop, or tablet). Computer 649 may be configured to communicate across network 609. Computer 649 includes one or more processors 659 and memory 663, as well as input / output mechanisms 654. Where the methods of the present invention employ a client / server architecture, operations of the methods of the present invention may be performed using a server 613, including one or more processors 621 and memory 629, which may retrieve data, instructions, etc., or provide results via interface module 625 or as file 617. Server 613 may be engaged via network 609 through computer 649 or terminal 667, or server 613 may be directly connected to terminal 667, which includes one or more processors 675 and memory 679, as well as input / output mechanisms 671.

[0144] System 600 or a machine according to an exemplary embodiment of the invention may further include a video display unit (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)) for any of I / O 649, 637, or 671. A computer system or machine according to some embodiments may also include an alphanumeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse), a disk drive unit, a signal generation device (e.g., a speaker), a touch screen, an accelerometer, a microphone, a cellular radio frequency antenna, and a network interface device, which may be, for example, a network interface card (NIC), a Wi-Fi card, or a cellular modem.

[0145] Memory 663, 679, or 629 according to exemplary embodiments of the present invention may include a machine-readable medium on which one or more sets of instructions (e.g., software) are stored that embody any one or more of the methodologies or functions described herein. The software may also reside, completely or at least partially, within the primary memory and / or processor during its execution by the computer system, with the primary memory and processor also constituting machine-readable media. The software may also be transmitted or received over a network via a network interface device. (Incorporated by reference)

[0146] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, articles, web content, etc., have been made throughout this disclosure. All such documents are incorporated herein by reference in their entirety for all purposes. (Equivalent)

[0147] While the present invention has been described in conjunction with certain embodiments, those skilled in the art, after perusing the foregoing specification, will be able to effect various modifications of the compositions and methods described herein, substitutions of equivalents thereof, and other alterations.

Claims

[Claim 1] The invention described in this specification.

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