Methods for generating immunotherapeutic products

Abstract

To provide methods for generating an immunotherapeutic product.SOLUTION: The methods include: culturing peripheral blood mononuclear cells and dendritic cells in a first cell culture chamber so as to produce a supernatant comprising T-cells; and transfer the supernatant comprising the T-cells from the first cell culture chamber through a fluidic connector into a second cell culture chamber. A first cell culture chamber comprises: a bottom surface comprising a first material to which cells adhere; at least one additional surface comprising a second material which is gas permeable; at least two inlets arranged to direct one or more fluids into the first cell culture chamber as symmetric inflows substantially parallel to the bottom surface; and one or more outlets arranged to direct one or more fluids out of the first cell culture chamber as outflows substantially vertical to the bottom surface.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 356,504, filed June 29, 2016, and U.S. Provisional Application No. 62 / 357,937, filed July 1, 2016, both of which are incorporated herein by reference in their entireties.

[0002] (Government support) This invention was made with government support under Grant No. 1645205 awarded by the National Science Foundation. The government has certain rights in this invention.

[0003] The present invention relates generally to cell culture chambers and methods of using the same. [Background technology]

[0004] Cell-based cancer immunotherapy has attracted significant attention due to the exceptionally promising treatments for specific cancers based on chimeric antigen receptor T cell (CAR-T) therapy, T cell receptor (TCR) therapy, dendritic cell vaccines, and neoantigen-based T cell therapy. For the latter, the ability to predict candidate neoantigens from tumor sequencing data and monitor neoantigen-specific T cell responses in patients provides the basis for designing highly personalized immunotherapies.

[0005] T cell therapy for cancer holds great promise, but existing processes for the isolation, preparation, and expansion of cancer antigen-specific T cells have limitations. Currently, T cell therapies are prepared using labor-intensive, manual, multi-step processes, which pose significant challenges for scaling the manufacturing of such T cell therapies. Due to the complex biological processes involved in T cell therapy production and the bioprocessing and regulatory requirements associated with autologous cell processing, attempts to automate this process have not been successful. Additional challenges exist, including cell preparation time, maintaining optimal phenotype, expanding to sufficient cell numbers, and ensuring the quality and safety of the cell product.

[0006] As shown in Figure 1, a conventional prior art protocol for stimulating human T cells with autologous antigen-presenting dendritic cells (DCs) involves several manual steps, including transferring supernatant between culture plates, changing medium, and adding cytokines and cell culture media. T cells are placed in contact with dendritic cells (DCs) for seven days, during which time they are stimulated and proliferate. This process is typically repeated four times, with each stimulation cycle requiring extraction of T cells from the supernatant and plating of fresh DCs. It also typically requires manual replacement of medium and growth factors (cytokines) twice during each seven-day period. The number of manual steps required to implement this protocol is prohibitive. For example, using this conventional four-week protocol requires a total of approximately 4,650 manual steps, all of which increase the risk of contamination and potentially compromise the quality and safety of the cell product. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Patent Application No. PCT / US2016 / 040042 [Patent Document 2] Specification of International Patent Application No. PCT / US2016 / 60701 [Patent Document 3] U.S. Patent No. 5,656,155 [Non-Patent Document]

[0008] [Non-Patent Document 1] Day et al., Mathematical Models and Immune Cell Biology; 2011 [Non-Patent Document 2] Valitutti et al., FEBS Lett. 2010 [Non-Patent Document 3] Fulwyer, Science 156, 910 (1974) [Non-Patent Document 4][[ID=*25]] Li and Harrison, Analytical Chemistry 69, 1564 (1997) [Non-Patent Document 5] Fiedler, et al. Analytical Chemistry 70, 1909 - 1915 (1998) [Summary of the Invention]

[0009] Note: There seems to be a formatting issue with the tag which might be a simple typo in the original text. I've left it as is in the translation for consistency with the provided input. If it's an important tag that should be corrected, please review the original text for accuracy.The present invention recognizes the need for developing new technologies to automate the production of antigen-specific T cells. The cell culture chamber of exemplary embodiments of the present invention includes various technical features that enable automation of the manual process described above, dramatically reducing user intervention during the process and thereby significantly reducing the risk of contamination. For example, the cell culture chamber of the present embodiment can be fabricated to include a bottom surface made of a cell-adherent material and at least one additional surface, such as a side wall and / or top wall, made of a gas-permeable material. In this manner, a higher level of gas exchange is achieved without having to sacrifice the adhesive properties of the bottom surface when compared to prior art culture systems. Additionally, the cell culture chamber of the present embodiment is configured to allow culture medium and cytokines to be perfused into the chamber, so that more consistent levels can be maintained. To ensure that antigen-specific T cells and other cells involved in the culture process remain within the chamber during perfusion, one or more inlet and outlet ports of the cell culture chamber are arranged to move fluid within the cell culture chamber at least partially along a vertical flow path as it exits the chamber. The cell culture chambers are also configured to be fluidly connected to one another so that antigen-specific T cells can be automatically transferred between the chambers, allowing for further cultivation and expansion of the T cells in the new cell culture chamber. In some embodiments, the transfer is accomplished by introducing a gas flow into a first cell culture chamber and transferring a supernatant containing the first cell product through the fluidic connector into a second cell culture chamber.

[0010] In certain aspects, the cell culture chambers of the exemplary embodiments use antigen-presenting cells from the same patient to provide for the expansion and stimulation of T cells, providing a therapeutic T cell product that can mobilize the patient's own immune system to selectively target the patient's tumor. These cell culture systems and methods significantly reduce the number of manual steps compared to conventional protocols. In this manner, the risk of contamination is greatly reduced and the robustness and reproducibility of the manufacturing technique are greatly increased, both of which are important considerations for the safe and reliable manufacture of therapeutic products such as precisely targeted, personalized T cell therapies.

[0011] In addition to simplifying and potentially shortening the process of generating immunotherapy products, the cell culture chamber of the exemplary embodiment significantly improves the utilization of patient cellular material for cell-based therapies and the reliability and robustness of the manufacturing process, while also reducing costs (e.g., labor costs). Furthermore, the method is easily scalable from processing just a few patient samples to processing hundreds of patient samples. The cell culture chamber configuration of the present embodiment allows automated fluid flow control to contact antigen-presenting cells with T cell-containing cells and refresh the antigen-presenting cells as needed to stimulate and expand antigen-presenting T cells to a number sufficient to produce a therapeutic response in the patient. This design is also easily scalable. For example, by designing a system with a series of cell culture chambers arranged in parallel, a single system can process samples ranging from one to tens to hundreds of samples. Each chamber can be independently controlled, and the number of chambers utilized at any given time can be scaled up or down depending on the number of samples. In other embodiments, a single central controller, such as a PLC logic controller, controls all of the chambers in the system.

[0012] An exemplary configuration is then described in which the systems and methods of exemplary embodiments of the present invention utilize one or more bioreactors, each including a cell culture chamber, configured to be fluidly connected to one another, for processing a patient's cellular material and generating an immunotherapy product. Those skilled in the art will recognize that this is an exemplary configuration described herein, and that other configurations are within the scope of the present invention.

[0013] In this exemplary embodiment, a cell culture chamber is provided, the cell culture chamber including a bottom surface constructed of a first material to which cells adhere, at least one additional surface, the at least one additional surface including a gas-permeable second material, and one or more inlets and one or more outlets, the one or more inlets and one or more outlets arranged to move fluid through the cell culture chamber at least partially along a vertical flow path. In certain aspects, the movement of fluid along the vertical flow path is such that the fluid flow rate is insufficient to overcome the settling rate of cells within the cell culture chamber.

[0014] The provision of at least one additional gas-permeable surface allows for a higher level of gas exchange compared to conventional protocols, and therefore allows for a larger number of cells to be processed in the chamber. In certain embodiments, the bottom surface and the at least one additional surface are bonded to each other without the use of adhesive. In certain embodiments, the at least one additional surface also comprises the first material.

[0015] The first material can include, for example, polystyrene. The second material can include one or more materials having a permeability to oxygen of a permeability coefficient of 350 or greater and a permeability to carbon dioxide of a permeability coefficient of 2000 or greater, where the permeability coefficient is in units of [cm 3 ][cm] / [cm 2

[0013] [s][cmHg]. In one embodiment, the second material is selected from one or both of silicone and polymethylpentene.

[0016] In certain embodiments, the cell culture chamber includes at least one fluid connector configured to fluidly couple the cell culture chamber to a second vessel, which can be a second cell culture chamber. To facilitate fluid flow through and between each chamber, the cell culture chamber can include one or more pumps. Each chamber can include its own pump, or one or more pumps can serve multiple chambers. In other embodiments, the cell culture chamber can further include one or more fluid reservoirs operably coupled to the one or more pumps. The fluid reservoirs are configured to supply the chambers with a medium containing nutrients and cytokines.

[0017] The cell culture chamber can also include one or more sensors operably coupled to the cell culture chamber in a manner that enables the sensors to measure one or more parameters within the cell culture chamber, such as pH, dissolved oxygen, total biomass, cell diameter, glucose concentration, lactate concentration, and cellular metabolite concentration. The cell culture chamber can further include a central processing unit communicatively coupled to the one or more sensors and configured to adjust the operating state of the one or more pumps as a function of the measured one or more parameters. In embodiments in which a flow-generating mechanism is used rather than a pump, such as an electrohydrodynamic mechanism, the central processing unit can vary the operating state of the flow-generating mechanism to adjust the flow rate of the first cell product as a function of the one or more parameters.

[0018] In certain embodiments, to help maintain a desired environment in and around the cell culture chamber, the chamber is sized and configured to fit within an incubator. In some embodiments, one or more pumps are located within the incubator. In other embodiments, one or more pumps are located outside the incubator and operably coupled to the cell culture chamber within the incubator.

[0019] In certain aspects, at least a portion of the system includes disposable components, and some or all of the disposable components may be housed within a non-disposable frame. In other aspects, all components of the system are disposable. Additionally, in some embodiments, the system includes a sample tracking component for tracking and recording patient material.

[0020] The systems and methods are designed so that any number of additional reactor or cell culture chambers can be provided. In some embodiments, the system includes two or more bioreactor chambers for developing T cells, as shown in Figures 6A and 6B. While the systems in Figures 6A and 6B are shown as having two cell culture chambers in fluid communication with each other, it should be understood that any number of additional chambers can be used with the system.

[0021] In certain embodiments, the systems of the present invention are capable of automatically calculating and setting a desired perfusion rate of a perfusion fluid given various inputs, such as, for example, the size of a cell culture chamber and the concentrations of two or more cell types, including dendritic cells and peripheral blood mononuclear cells. In an exemplary arrangement, a cell culture system is provided, comprising one or more cell culture chambers and a central processing unit including a memory having instructions, the instructions executable by the central processing unit, for receiving as a first input data including the size of the cell culture chamber, receiving as a second input data including 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 calculating, based on the first and second inputs, a perfusion rate of the perfusion fluid to be introduced into the cell culture chamber that maximizes the probability that the first cell type and the second cell type are in contact with each other within the cell culture chamber. In certain aspects, the first cell type is peripheral blood mononuclear cells, and the second cell type is dendritic cells.

[0022] The central processing unit can control the perfusion rate of the perfusion fluid by controlling one or more pumps (or valves) operably coupled to the one or more perfusion fluid reservoirs and the central processing unit. In certain embodiments, one or more sensors are operably coupled to the cell culture chamber in a manner that allows the sensors to measure one or more parameters within the cell culture chamber. These parameters include pH, dissolved oxygen, total biomass, cell diameter, glucose concentration, lactate concentration, and cellular metabolite concentration.

[0023] Another aspect of the invention provides a method for transferring cells from a first cell culture chamber into a second cell culture chamber. The method generally includes culturing cells in the first cell culture chamber in a manner to generate a supernatant containing a first cell product, and introducing a gas flow into the first cell culture chamber to transfer the supernatant containing the first cell product through a fluid connector into the second cell culture chamber. As the fluid enters the second cell culture chamber, the first cell product is further cultured.

[0024] Similar to the transfer of fluid from the first cell culture chamber to the second cell culture chamber, the method can also include introducing a gas flow into the second cell culture chamber and transferring a supernatant containing the cultured first cell product through the fluid connector into a third cell culture chamber. In one embodiment, the transfer of supernatant occurs at least three different times, such that at least four culture chambers are used to generate the desired amount of antigen-presenting T cells.

[0025] Additionally, in certain embodiments, as described above, one or more of the cell culture chambers are configured to move a fluid, such as a perfusion fluid, through the chamber along a vertical flow path (FIG. 8C), such that the fluid flow rate is insufficient to overcome the settling rate of cells within the cell culture chamber.

[0026] In yet another embodiment of the present invention, a method for generating an immunotherapeutic product includes culturing peripheral blood mononuclear cells and dendritic cells in a first cell culture chamber to produce a supernatant containing T cells, and introducing a gas flow into the first cell culture chamber to transfer the supernatant containing T cells through a fluidic connector into a second cell culture chamber. In a specific embodiment, fresh dendritic cells are contained in the second cell culture chamber, and the T cells are further cultured in the second cell culture chamber.

[0027] Culturing in the first culture chamber can occur in the presence of a first set of one or more stimulating antigens, and culturing in the second culture chamber can occur in the presence of a second set of stimulating antigens. In certain embodiments, the first set of stimulating antigens and the second set of stimulating antigens are the same, while in other embodiments, the first set of stimulating antigens and the second set of stimulating antigens are different.

[0028] The foregoing will become apparent from the following more particular description of exemplary embodiments, which are illustrated in the accompanying drawings, in which like reference characters represent the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the embodiments. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 illustrates a prior art manual technique for creating an immunotherapy product. [Figure 2] FIG. 1 illustrates an exemplary method for producing an immunotherapy product, according to an embodiment of the present invention. [Figure 3] FIG. 1 is a schematic diagram showing the system of the present invention in one embodiment, with one cell culture chamber shown. [Figure 4A] 1A-1C illustrate exemplary cell culture chamber configurations according to embodiments of the present invention. [Figure 4B] 1A-1C illustrate exemplary cell culture chamber configurations according to embodiments of the present invention. [Figure 4C] 1A-1C illustrate exemplary cell culture chamber configurations according to embodiments of the present invention. [Figure 4D] 1A-1C illustrate exemplary cell culture chamber configurations according to embodiments of the present invention. [Figure 4E]1A-1C illustrate exemplary cell culture chamber configurations according to embodiments of the present invention. [Figure 5] FIG. 1 shows a cell culture chamber surface having a configuration in which a permeable polymer is embedded within a frame of another polymer. [Figure 6A] 3 is a schematic representation of a process for connecting cell culture chambers and transferring fluids between them.FIG. 4 is a schematic diagram of a first cell culture chamber as shown in FIG. [Figure 6B] 1 is a schematic representation of a process for connecting cell culture chambers and transferring fluids between them, illustrating partially expanded T cells in a first cell culture chamber and moving a second cell culture chamber into an appropriate position for connection to the first cell culture chamber. [Figure 6C] 1 is a schematic representation of a process for connecting cell culture chambers and transferring fluids between them, illustrating the injection of sterile air into a first cell culture chamber and the transfer of supernatant containing expanded T cells into a second cell culture chamber. [Figure 7] FIG. 1 illustrates the flow of fluid from the outlet of one cell culture chamber to the inlets of two cell culture chambers, according to one embodiment. [Figure 8A] 1A-1C illustrate fluid flow through various chamber configurations, and FIG. 1D illustrates planar fluid flow into and out of the cell culture chamber. [Figure 8B] 1A-1C illustrate fluid flow through various chamber configurations, and FIG. 1D illustrates planar fluid flow into and out of the cell culture chamber. [Figure 8C] 1A-1C illustrate fluid flow through various chamber configurations, including a configuration with symmetric inflow and vertical outflow. [Figure 8D]1A-1C illustrate fluid flow through various chamber configurations, including a configuration with symmetric inflow and vertical outflow. [Figure 9] FIG. 1 shows kinetic parameter-based modeling of interactions between T cells and antigen-presenting cells. [Figure 10] FIG. 1 illustrates a system of the present invention, according to certain embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0030] The devices, systems, and methods of the present invention automate and remotely monitor and control the generation of sufficient quantities of antigen-specific T cells for personalized, targeted cancer or infectious disease therapy involving the cultivation of autologous cells. The systems and methods of the present invention include various technical features that enable the automation of the manual processes described above. These technical features include, but are not limited to, 1) constructing a cell chamber to include a bottom surface made of a material to which cells adhere and at least one additional surface made of a gas-permeable material to achieve a greater level of gas exchange; 2) arranging one or more inlet and outlet ports of the cell culture chamber to move fluid within the cell culture chamber along a vertical flow path as it leaves the chamber, ensuring that antigen-specific T cells and other cells involved in the culture process remain within the chamber during perfusion of the culture medium; and 3) automatically transferring antigen-specific T cells from one chamber to another by introducing a gas flow into the first cell culture chamber and transferring the cells through a fluidic connector into the second cell culture chamber.

[0031] The methods, devices, and systems of the present invention can be scaled up to provide multiple cell-based immunotherapy products and can operate on a single subject or on several subjects in parallel (whereby the cells and their progeny remain separate). Compared to prior art methods and devices, the methods and systems of the present invention are robust in operation, can provide high product yields, are simple and efficient, have less risk of contamination, and minimize labor costs.

[0032] FIG. 2 shows an overview of a method for generating a cell-based immunotherapy product using the system described herein. Briefly, steps in generating a cell therapy product according to certain embodiments of the present invention include co-culturing cells containing T cells with stimulated antigen-presenting 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 certain aspects, to generate a predetermined number of antigen-specific T cells sufficient to elicit a therapeutic response in a patient, the T cells must undergo additional culture in one or more additional cell culture chambers. To achieve 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 can 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. Furthermore, during each culture step, a perfusion fluid containing, for example, medium and cytokines, can be perfused through the chamber. In certain embodiments, the perfusion fluid flows through the chamber along a vertical flow path, ensuring that the cells remain within the chamber during culture. The only manual step required using the system of the present invention is providing the system with one or more subsequent biological reactors, each containing a cell culture chamber and each containing a new batch of antigenic peptide-pulsed autologous antigen-presenting cells.

[0033] Methods for generating cell-based immunotherapy products according to embodiments of the present invention are much simpler and more efficient than prior art methods. Figure 1 illustrates a conventional prior art protocol that requires at least 4,650 manual steps, as previously described. In contrast, as shown and described in Figures 2-4 and as described herein, the systems and methods of the present invention involve a 25-fold reduction in the number of manual steps that must be taken to generate the same cell dose, as shown in Table 1 below.

[0034] [Table 1]

[0035] The cell culture chamber of the present invention significantly improves immunotherapy manufacturing, providing a flow-based immunotherapy production technology with unparalleled consistency, quality, safety, economy, scalability, flexibility, and portability.

[0036] An exemplary configuration is then described, in which the systems and methods of the present invention utilize one or more bioreactors, each containing a cell culture chamber configured to be fluidly connected to one another to process a patient's cellular material and generate an immunotherapy product. It should be understood that the bioreactors, in certain embodiments, are provided in closed environments. It would be within the knowledge of one of ordinary skill in the art to scale up this exemplary embodiment by adding modules (e.g., biological reactors) to enable serial and / or parallel processing. Additionally, one of ordinary skill in the art will recognize that different or alternative configurations may be desired based on the product to be produced.

[0037] In an exemplary embodiment, as shown in FIG. 3 , a biological reactor 110 is provided that includes a cell culture chamber 120, which includes a bottom surface 122 and at least one additional surface 124. The bottom surface 122 is composed of a first material to which cells adhere, and the at least one additional surface 124 is composed of a second, gas-permeable material. The cell culture chamber also includes one or more inlet ports 126, 136 and one or more outlet ports 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 inlet ports 136 and outlet ports 138 for transporting fluids to and from the reservoirs 132, 134 and through the chamber 120.

[0038] With respect to cell culture chamber 120, the first material can be any material that is biocompatible and to which antigen-presenting cells (APCs), such as dendritic cells (DCs), 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 retrieve the expanded T cells.

[0039] In one exemplary embodiment, the first material comprises polystyrene. One benefit of using polystyrene for the bottom surface on which culture occurs 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 by differentiation of monocytes, for example, via culture in medium containing IL4 and GM-CSF. Using the same polystyrene surface for dendritic cell production throughout a single cycle of T cell stimulation is extremely valuable 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.

[0040] The bottom surface is 9.5 cm for 6-well and 24-well plates, respectively. 2 and 3.8 cm 2 The surface area can be smaller than that of a conventional well plate (e.g., having a surface area comparable to standard cell culture dishes and flasks) or much larger, e.g., about 2.0 cm. 2 Approximately 200cm from 2 surface area between, 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 also be understood that the surface area may be any surface area therebetween.

[0041] The at least one additional surface 124 can include any configuration, such as one or more sidewalls and a top wall. In one embodiment, the sidewalls can be disposed at a 90-degree angle relative to one another, such that a box shape is formed in conjunction with the bottom surface 122, as shown in FIG. 3. In another embodiment, the at least one additional surface 124 forms a curved sidewall, such that a cylinder, elliptical cylinder, or cone is formed, as shown in FIGS. 4A-4C. In another exemplary arrangement, the at least one additional surface can form a dome-like shape above the bottom surface, as shown in FIG. 4D. In yet another embodiment, the sidewall can form a triangular shape, as shown in FIG. 4E. 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 above exemplary configurations.

[0042] In another embodiment, at least one additional surface 124 comprises a second material, which is gas permeable to achieve gas exchange within the cell culture chamber. By fabricating the cell culture chamber so that the bottom surface is made of a material to which cells adhere, such as polystyrene, and so that at least one additional surface, such as a side wall and / or top wall, is made at least partially from a gas permeable material, high surface area gas exchange is achieved within the system of embodiments of the present invention. Having a larger surface with high permeability beyond the bottom surface offers the ability to achieve higher levels of gas exchange without having to sacrifice the adherent properties of the bottom surface compared to prior art culture systems, which were limited in terms of the amount of culture medium that could be contained and / or absent from a culture-friendly surface to which cells could adhere.

[0043] In certain embodiments, the second material comprises one or more materials having a permeability to oxygen of a permeability coefficient of 350 or greater and a permeability to carbon dioxide of a permeability coefficient of 2000 or greater, where the permeability coefficient is in units of [cm 3 ][cm] / [cm 2

[0013] [s][cmHg]. Exemplary materials include silicone-containing materials such as poly(dimethylsiloxane) (PDMS), which is well known for its high oxygen and carbon dioxide permeability (up to three orders of magnitude higher than materials such as polystyrene and PMMA) and polymethylpentene. In one exemplary embodiment, the cell culture chamber comprises a polystyrene floor and silicone side and top walls.

[0044] In certain aspects, in addition to the second material, at least one additional surface 124 can also include the first material. For example, and without limitation, the additional surface 124, e.g., one or more sidewalls and / or top wall, can incorporate a second material (e.g., a highly permeable polymer such as silicone) within a frame made of a first material (e.g., polystyrene), as shown in FIG. 5. It is also contemplated that the bottom surface can also include the second material. However, in some embodiments, the second material is only intermittently dispersed throughout the bottom surface, thereby ensuring that the first material covers a sufficient surface area to allow cells to attach to the surface.

[0045] The first and second materials can be joined to each other using any method known in the art, such as mechanical fastening, adhesives, solvent bonding, and welding. However, considering that the cellular immunotherapy products produced using the systems and methods of the present invention will subsequently be administered to human patients, regulatory issues may prevent the use of certain (or all) adhesives in assembling the cell culture chamber. Thus, in certain embodiments, the first and second materials are joined without the use of adhesives. In one embodiment, all surfaces of the cell culture chamber, such as the bottom wall, side wall, and top wall, comprise a first material (e.g., polystyrene), and at least a portion of the first material in the side wall and / or top wall is joined together using ultrasonic welding, as shown in FIG. 5, with cutouts to allow insertion of a second material (e.g., silicone material). It should be understood that the holes shown in FIG. 5 can be any desired shape and may not be simply circular. In an exemplary arrangement, the second material may be separately inserted into the hole in the same manner as a stopper inserted into the top opening of a vessel to seal the vessel. In another example, as shown in Figure 5, the second material may be fabricated to completely encompass and surround the outer surface of the first material frame, such that the second material is accessible through the hole in the frame. It should be understood that the above-described configurations are merely examples, and that other configurations for joining the first and second materials are also contemplated embodiments of the present invention.

[0046] The height of one or more cell culture chambers can vary. For example, and without limitation, exemplary ranges of cell culture chamber heights include any height from 0.5 mm to 100 mm, such as, for example, 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 more, or any height therebetween. In certain embodiments, the chamber height can be comparable to the liquid heights in cultures typically performed in 6-well and 24-well plates, e.g., between 2 mm and 6 mm, with a volume capacity of about 0.8 mL to 6 mL. In other embodiments, the cell culture chamber can be larger, e.g., between 10 mm and 50 mm, and can be approximately 50 cm. 2 It has a culture surface.

[0047] As briefly described above with respect to FIG. 3 , in certain embodiments, the bioreactor 110 also includes one or more pumps 140 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 120 and can be operably coupled to the one or more pumps 140. One or more tubes are also provided for connecting the fluid reservoirs to the pumps and the cell culture chamber. In certain aspects, the one or more pumps are configured for pumping fluid from the fluid reservoirs, through the cell culture chamber, and into a waste collection reservoir. In the exemplary embodiment shown in FIG. 3, fluid travels from fluid reservoir 132, travels through tubing 152 to pump 140, travels into cell culture chamber 120 via inlet port 136, travels out of cell culture chamber 120 via outlet port 138, travels through tubing 154, and travels into waste collection reservoir 134.

[0048] In certain embodiments, the fluid reservoirs and / or waste collection reservoirs can each be provided as one or more capped bottles, which are either contained within or fluidly connected to the cell culture chambers. Each reservoir contains an inlet port and an outlet port, or an outlet port and a vent fluidly connected to the inlet port or inlets of one or more cell culture chambers. In certain aspects, for example, luer connectors and silicone gaskets cut to fit around the luer connectors can be used to prevent leakage through either the inlet port or the outlet port, or through both the inlet and outlet ports.

[0049] In certain embodiments, one or more biological reactors are sized and configured to fit into an incubator, allowing the process to be carried out within the incubator. Conditions within the incubator include a sustained temperature of 37°C and 95-100% humidity. Therefore, materials selected must have the integrity to withstand these conditions, given that materials (including fluids and biologicals) tend to grow under such conditions. Furthermore, in some cases, conditions within the incubator remain stable, allowing automated temperature recording, with knowledge of temperature fluctuations and correlation with any aberrations in the reactions carried out within the incubator. Therefore, any power supply should not alter the environment within the incubator. For example, certain pumps generate heat. Thus, in one embodiment, the pump is housed separately from the biological reactor, but is still in fluid and operative communication with the reactor. In another embodiment, the pump is attached directly to the biological reactor and located within the incubator, but does not receive heat, or is operably connected to a heat sink and / or fan to dissipate heat. Regardless of the configuration, the pump is operably coupled to the biological reactor and to the cell culture chamber. Additional details regarding perfusion-based automated cell culture systems, such as small-scale culture systems for endothelial cell culture with on-board reagent storage and perfusion enabled by on-board disposable peristaltic pumps, and larger-scale culture systems for generating dendritic cells from monocytes using chambers with polystyrene bottom surfaces, can be found in U.S. Patent Nos. 5,929,995 and 5,929,995, both of which are incorporated by reference in their entireties.

[0050] The system can also include a heater to control the temperature of the cell culture reservoir and, optionally, the fluid reservoir. In such a configuration, an incubator is not required and the system can operate autonomously, relying only on a source of electrical power. If the system lacks a heater, the system can be operated inside a cell culture incubator.

[0051] In yet another aspect, the cell culture chamber includes one or more sensors (not shown) operably coupled to the cell culture chamber. The sensors can measure 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. In embodiments in which the system includes multiple cell culture chambers, the one or more sensors can be coupled to one or more of the cell culture chambers. In certain embodiments, the one or more sensors are coupled to one or more cell culture chambers, but not all of the chambers in the system. In other embodiments, the one or more sensors are coupled to all of the cell culture chambers in the system. In systems having multiple chambers operably coupled to one or more sensors, the sensors can be the same in each of the chambers to which they are coupled, the sensors can all be different, or some sensors can be the same and some sensors can be different. In certain aspects, the one or more sensors are operably coupled to a computer system (not shown in FIG. 3 ) having a central processing unit for executing instructions, allowing for automatic monitoring and adjustment of parameters. Additional details regarding computer systems for implementing the methods of the present invention using cell culture chambers are provided below.

[0052] Also, as shown in FIG. 3 , the cell culture chamber has an inlet port 126 and an outlet port 128, both of which can be used to fluidly connect the chamber to one or more additional vessels via fluid connectors. In certain embodiments, the additional vessels include one or more additional cell culture chambers, as described in more detail below. The systems of the present invention can include, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or any number therebetween, or even more than 100 cell culture chambers, configured to fluidly connect to each other in series to produce an immunotherapy product. Alternatively or additionally, one or more cell culture chambers can be arranged in parallel to each other to enable production of immunotherapy products for two or more individuals simultaneously. In a preferred embodiment, the cell culture chambers of the bioreactor are connected via sterile connections.

[0053] An exemplary configuration of a multi-bioreactor system can be seen in Figures 6B and 6C, and additional details regarding processes performed using this configuration are provided below. As shown in Figure 6B, when a second bioreactor 210 is involved, the second cell culture chamber 220 is moved into position and connected to the first cell culture chamber 120 via the outlet of the first chamber and the inlet of the second chamber. The connection is preferably a sterile connection, allowing for the injection of sterile air into the first cell culture chamber 120 and the transfer of the supernatant containing the expanded T cells into the second cell culture chamber 220. Alternative techniques known in the art of fluid flow can be used 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 can be shared between bioreactors.

[0054] In certain embodiments, as shown in Figures 6B and 6C, there is a 1:1 ratio of cell culture inlets to cell culture outlets, such as when one or more biological reactors are arranged in series with one another. In other embodiments, the ratio of outlets to inlets is 1:2 for at least a portion of the biological reactors. For example, the outlet of one cell culture chamber 120 is fluidly connectable to the inlets of two cell culture chambers 220a and 220b, such that the fluid flowing from the first cell culture chamber 120 is split into two streams, one into the second cell culture chamber 220a and the second into the third cell culture chamber 220b, as shown in Figure 7. In this configuration, both the second cell culture chamber 220a and the third cell culture chamber 220b can be used to further stimulate and expand T cells. Additionally or alternatively, one of the second cell culture chamber 220a or the third cell culture chamber 220b may be configured to allow monitoring of reaction and flow parameters using one or more sensors operably coupled to the chamber. In this manner, one of the chambers may be left without additional sensors, some of which may require penetration of the cell culture chamber walls, which may increase the risk of leakage and / or contamination.

[0055] In certain embodiments, one or more biological reactors may be provided in a system containing modules for accomplishing various other processes prior to, simultaneously with, or subsequent to the processes occurring in the cell culture chamber of the biological reactor.

[0056] The system and some or all of its components can be designed using CAD software and then transferred to a laser cutter, which allows plastic to be cut to specific sizes and shapes. Various connections, such as inlets and outlets, can be made by laser cutting through holes, which can then be manually tapped and provided with threads to accept male Luer fittings. Fluid can later be introduced into the system by connecting a Luer adapter to a blunt dispensing needle, with tubing pushed over the blunt needle portion. Additional details regarding the construction of fluid system components can be found in U.S. Patent Nos. 5,629,999 and 5,729,999, both of which are incorporated herein by reference in their entireties.

[0057] The above description focuses on system components and various possible configurations. The following description focuses on a process implemented using an exemplary embodiment system of the present invention. To stimulate and expand antigen-specific T cells, the process begins with co-culturing T cell-containing cells with 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 are co-cultured at a ratio (T cell-containing cells:APCs) of about 1000:1 to 1:1000, for example, and without limitation, 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, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 4 The cell culture chamber may be provided with a ratio of 1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 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 therebetween. In one embodiment, a 10:1 ratio is preferred.

[0058] To initiate T cell stimulation and proliferation from the interaction of the T cell-containing cells with the APC, the APC must be stimulated. This can be done through the use of one or more stimulatory molecules. In certain embodiments, the stimulatory molecules are not 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 antigenic peptides. In some embodiments, the stimulatory molecules are preferably added only at the beginning of the culture cycle. The stimulatory molecules can be added for a period of about a few minutes, an hour, several hours, or longer. In one preferred embodiment, the stimulatory molecules are added for a period of about an hour.

[0059] During the culture of the two cellular materials, a supernatant containing the lighter, non-adherent T cells is formed, while the heavier, mature APCs (e.g., dendritic cells) adhere to the bottom surface. In those embodiments in which DCs are used as APCs, the expanded T cells must be extracted from the cell culture chamber by the end of the seven-day period because primary DCs cannot be maintained in culture for more than seven days. Therefore, if additional expansion of T cells is desired, a supply of fresh dendritic cells is required. It should also be understood that culturing cells using a single batch of dendritic cells can be performed for any period of time less than seven days. For example, cells can be cultured for a period of anywhere from less than one minute to seven days, with the duration of culture depending on the degree of stimulation desired.

[0060] In an exemplary embodiment, after up to seven days in culture, the expanded T cells are extracted and transferred to a new cell culture chamber containing fresh DCs pulsed with, for example, the same antigenic peptide used in the first cell culture chamber. The stimulation process can be repeated as many times as necessary to generate a sufficiently large number of cells for a therapeutic dose of T cells. When using a culture surface area comparable to that of a typical well plate, the stimulation process is typically repeated four times to generate a sufficient supply of T cells.

[0061] Co-culture of APCs and T cells occurs in a culture medium. Exemplary culture media include, but are not limited to, RPMI medium and Cellgenix® medium. Any other suitable culture medium known in the art can be used according to embodiments of the present invention. Cytokines such as IL-4 and GM-CSF can also be added to the culture medium.

[0062] In one embodiment, perfusion of medium and cytokines is provided to the cell mixture within the cell culture chamber, supporting the formation of cell-based immunotherapy products. In plate-based protocols for T cell stimulation with DCs, an approximate 2 mL culture volume is maintained from the start, and cytokine infusion occurs twice within each 7-day stimulation period. The primary advantages of perfusion are the ability to maintain consistent local concentration profiles of medium and cytokines (ensuring greater yields) and the potential ability to speed 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 expansion of antigen-specific T cells, particularly with regard to fluid flow through the cell culture chamber. Therefore, in those embodiments in which medium and cytokines are provided via perfusion, the inventive system must be able to supply cells with nutrients and cytokines without removing them from the bioreactor, while also taking into account the shear sensitivity of certain antigen-presenting cells, such as DCs. Essentially, the systems and methods of some embodiments of the present invention aim to optimize the retention of autocrine / paracrine signals that favor T cell proliferation while refreshing growth factors and 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 taken into account.

[0063] In certain aspects, the fluid flow rate is maintained below the sedimentation rate of the antigen-presenting cells, so that the antigen-presenting cells remain within the culture chamber due to their mass. In other words, the antigen-presenting cells sink toward the bottom of the cell culture chamber and therefore remain within the cell culture chamber.

[0064] The flow rate below the settling rate can be calculated according to Equation 1.

number

[0065] In other embodiments, one or more inlet ports 136 and one or more outlet ports 138 of a cell culture chamber are arranged to move fluid, such as perfusion fluid, within the cell culture chamber along a vertical flow path. This configuration helps prevent cells (e.g., both DCs and T cells) from leaving the chamber, particularly when the flow rate through the chamber is in the range of 2-10 L / min. For example, as shown in Figures 8A and 8B, a planar in / out flow provides a region of high wall shear uniformity but requires a means to prevent cell removal from the chamber. In contrast, as shown in Figures 8C and 8D, a configuration with a symmetric inflow and a vertical outflow can prevent cells from leaving the chamber.

[0066] Although shown in FIG. 8C as having four inlets and one vertical outlet, any number of inlets and outlets may be provided, so long as fluid exiting the chamber flows vertically from the top of the chamber. For example, the chamber can have any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more perfusion fluid inlets, while also having any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more perfusion fluid outlets. Additionally, while the inlets are shown as symmetrical in FIG. 8C, configurations containing more than one inlet are contemplated that are not symmetrically arranged. The inlets can direct fluid to enter the chamber from any direction. In preferred embodiments, the inlets direct fluid to enter the chamber in a direction parallel to the bottom surface.

[0067] In certain embodiments, medium perfusion occurs at specific times over the period of time that cells are cultured in any one cell culture chamber, such as, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times per day or week. In other embodiments, medium is perfused continuously during culture. Continuous perfusion helps maintain a nearly constant culture volume throughout the process.

[0068] In certain aspects, cytokines are infused at one or more points during culture, such as, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. Alternatively, cytokines can be continuously perfused with culture medium. In these embodiments, continuous perfusion helps maintain a consistent local concentration profile of cytokines, which can help ensure greater yields and has the potential to increase the rate at which T cells are stimulated and expanded compared to static cell culture methods.

[0069] 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 have an impact on the efficacy of T stimulation. For example, in recent studies designing a culture chamber for monocyte proliferation to DCs, as described in U.S. Patent Nos. 5,629,999 and 5,729,999, we determined that a medium perfusion rate corresponding to a wall shear stress level of 0.1 dyn / cm2 can produce DCs phenotypically identical to those generated using conventional 6-well or 24-well plate-based protocols. Thus, by measuring one or more of the phenotypic and functional measures described above during the culture cycle, the effect of one or more perfusion parameters on efficacy can be monitored, allowing for appropriate adjustments.

[0070] According to certain embodiments, stimulatory efficacy can be assessed at any point during culture, preferably after 7 days. Both phenotypic and functional measures can be used to evaluate efficacy. For example, cell number (fold expansion) can be calculated using direct cell counting methods. Cell phenotype can be characterized by flow cytometry, including assessment of antigen specificity by tetramer staining. Functional assays can also be used to evaluate the ability of expanded T cells to recognize antigen-loaded target cells and autologous tumor cells. Results can be benchmarked against DC-based T cell stimulation performed in both 24-well plates and G-Rex® formats.

[0071] As explained above, because certain APCs, such as dendritic cells, cannot survive in culture for more than seven days, certain embodiments of the present invention require multiple cycles of T cell stimulation using two or more bioreactors in a semi-batch configuration. Each cycle is performed with freshly generated autologous antigen-presenting cells. In certain embodiments, the antigen-presenting cells are pulsed with the same set of antigens for each stimulation cycle. In other embodiments, different sets of antigens are used for one or more of the stimulation cycles.

[0072] Generally, as shown in FIG. 2, multiple cycle T cell stimulation involves culturing cells in a first cell culture chamber in a manner to generate a supernatant containing a first cell product, providing a second cell culture chamber, and then transferring the supernatant from the first cell culture chamber to the second cell culture chamber by introducing a gas flow into the first cell culture chamber.

[0073] An exemplary configuration of a multi-reactor system can be found in Figures 6A-6B. As shown, the process begins with one reactor containing mature, adherent DCs, which are loaded with PBMCs and subjected to a 7-day initial stimulation cycle by perfusion of medium and cytokines. Following completion of the first stimulation cycle, an optionally larger second reactor containing fresh DCs is connected to the first reactor, as illustrated in Figures 6B and 6C. An injection of sterile air then transfers the supernatant from the first reactor to the second reactor. This second bioreactor will contain its own supply of medium and cytokines in an on-board container, along with a disposable peristaltic pump. Following supernatant transfer, the first reactor can be disconnected and discarded. This process can be repeated as many times as desired with increasingly larger bioreactors to achieve the desired level of stimulation and T cell expansion. For example, in one embodiment, four stimulation cycles are performed, with the transfer of supernatant to a new cell culture chamber at three different times. Although the second chamber 220 is shown as being larger than the first cell culture chamber 120, it should be understood that the second and any subsequent cell culture chambers can be any size, for example, larger than, the same size as, or smaller than the first cell culture chamber. In certain embodiments, each subsequent cell culture chamber is larger in size than the preceding cell culture chamber from which the supernatant is transferred.

[0074] The modular design provides flexibility in terms of both the number of cycles and the type of antigen-presenting cells, which can be tailored by using either the same set of antigens for each cycle, a different set of antigens for each cycle, or a combination of the former and latter. In certain embodiments, the ability to generate T cells specific for multiple different antigens associated with a disease in one automated process is advantageous in treating the disease, as it allows for a multi-pronged attack.

[0075] In certain embodiments, a computational modeling approach is used to optimize the interaction of T cells with antigen-presenting cells, such as DCs. The computational model according to the present invention takes into account the effects of perfusion and the optimal time required for stimulation, incorporating both a particle-interaction-based approach and a kinetic-parameter-based approach. 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 used the following equation to describe the time required for a T cell to find an APC on the surface of a lymph node: where D is the T cell's diffusion rate and b is the radius of the APC centrally located within a spherical lymph node of radius R. See Non-Patent Document 1.

number

[0076] By incorporating both particle interaction-based and 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-infused medium) can be achieved to maximize the probability that two cell types are in contact with each other within the cell culture chamber.

[0077] For example, in certain embodiments, a cell culture system is provided that includes a cell culture chamber and a central processing unit that includes a memory having instructions executable by the central processing unit. In certain aspects, the instructions cause the system to receive as a first input data including the size of the cell culture chamber, receive as a second input data including 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 perfusion fluid introduced into the cell culture chamber that maximizes the probability that the first cell type and the second cell type are in contact with each other within the cell culture chamber. Additional details regarding computer systems for implementing the methods of the present invention in cell culture systems are provided below.

[0078] In some embodiments, the system also includes one or more pumps operably coupled to the one or more perfusion fluid reservoirs and to a central processing unit such that the central processing unit controls the perfusion rate of the perfusion fluid by controlling the one or more pumps.

[0079] As described above, the systems and methods of the present invention utilize modules (e.g., bioreactors containing cell culture chambers) that are fluidly connected to one another for processing an individual's cellular material to produce an immunotherapy product.

[0080] The systems or devices of the present invention are modular and can be fluidly connected to other similar devices in series (i.e., fluid flows from one device into another) and / or in parallel, and can be configured to be physically stacked on top of each other or physically located within related devices such as incubators, etc. The modular design of the system allows modules to be flexibly switched in and out depending, in particular, on the desired processes to be included within the system.

[0081] The fluidic devices of the present invention include biological reactors containing cell culture chambers, and may be provided in either microfluidic embodiments (i.e., one or more channels or chambers therein having dimensions ranging from about 1 μm to about 999 μm) or macrofluidic embodiments (all of the channels or chambers therein having dimensions of about 1 mm or greater), or both.

[0082] Fluidic devices can further include additional fluid channels or compartments, gaskets or seals, mixing zones, valves, pumps, vents, channels for pressurized gas, electrical conductors, reagents, ports, and tubing as required by a particular design. Fluidic devices can 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 device preferably contains only materials that are non-toxic to mammalian cells and are compatible with sterilization by the use of alcohol and / or heat or other means, such as exposure to gamma radiation or ethylene oxide gas.

[0083] The materials of the equipment are selected for their suitable chemical compatibility under different temperature and pressure ratings specific to each process. Additionally, the selection of pumps implemented in the device, such as syringe pumps, peristaltic pumps, pressure pumps, and rotary pumps, ranges from nL to mL in flow rates and from 10 psi to 10,000 psi in pressures depending on the flow and pressure requirements for different functions.

[0084] The systems of the invention can also include one or more sample solution reservoirs or wells or other devices for introducing samples into the device that are in fluid communication with the inlet channels at the various inlets of the modules. Reservoirs and wells used to load one or more samples onto the fluidic devices of the invention include, but are not limited to, syringes, cartridges, vials, Eppendorf tubes, and cell culture materials (e.g., 96-well plates).

[0085] Where useful, the surface of the device can be made more hydrophilic, for example 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 present invention are preferably leak-proof under operating conditions and capable of sterile operation over periods of days to weeks. Fluidic devices of the present invention also include a sampling mechanism that allows fluids to be removed from the system for testing without introducing new materials or contaminants into the system.

[0086] In certain aspects, at least a portion of the cell culture system includes disposable components, and some or all of the disposable components may 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 recording patient material.

[0087] At least one step, and sometimes multiple or all steps, during the manufacturing process are monitored for product attributes (e.g., purity and polymorphism) using various in-line process analytical tools (PAT) or miniaturized micro total analytical systems (microTAS).

[0088] As described above, the cell culture systems of the present invention can control the direction and flow of fluids and entities within the system. The systems of the present invention can utilize, for example, valves and pumps, using pressure-driven flow control to manipulate the flow of cells, reagents, etc. in one or more directions and / or into one or more channels of a fluidic device. However, other methods such as electroosmotic flow control, electrophoresis, and dielectrophoresis (Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5; Patent Document 3) can also be used alone or in combination with pumps and valves.

[0089] The systems of the invention may also include or be operably linked to one or more control systems for controlling the movement of fluids through the system, for monitoring and controlling various parameters within the system, such as temperature, and for detecting the presence of cell-based immunotherapy product, the amount of product (directly or indirectly), the 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 enable feedback control, and processes that allow integration and scale-up given the reaction and purification results obtained using the system.

[0090] In certain embodiments, the system includes a combination of microfluidic, millifluidic, or macrofluidic modules and tubing, which are interchangeable in terms of channel dimensions, flow geometry, and interconnections between different modules of the device. Each module and tubing can be designed for a specific function. In one embodiment, all of the modules in the system are designed for cell culture and T cell stimulation. In other embodiments, the modules comprising the system are designed for different functions, such as tissue processing, dendritic cell generation, cell culture, concentration, and / or purification, all integrated for the continuous production of immunotherapeutic products. Both homogeneous and heterogeneous processes are contemplated and suitable for flow applications. These processes are designed and optimized with respect to starting materials and operating conditions, such as temperature, pressure, and flow rate, to prevent the system from easily stalling during the flow process.

[0091] Device scale-up is achieved by adding modular reactors in parallel or expanding modular channels while maintaining a set of dimensionless parameters characteristic of each process constant and dimensional parameter within upper and lower boundary limits. During process integration and optimization, process decision variables, including temperature, pressure, flow rate, and channel dimensions, are varied to achieve the desired trade-off between yield, purity, and throughput. Throughout the optimization process, the aforementioned set of dimensionless parameters undergoes algebraic optimization with operating constraints. The operating constraints are the lower and upper bounds of the decision variables. The objective function considers the combination of purity, yield, and throughput operating variables. While dimensionless parameters determine the steady-state quality of the device, the device's start-up quality is also useful because it determines the time required to reach steady state and, in turn, the device's productivity in the form of lag time and waste. Start-up dynamics are analyzed using both simulations and experiments, and these results are used to implement real-time feedback control to perform start-up optimization.

[0092] Aspects of the disclosure described herein, such as the control of fluid movement through the system and the monitoring and control of various parameters as described above, can be implemented using any type of computing device, including a processor, e.g., a central processing unit, such as a computer or programmable logic controller (PLC), or any combination of computing devices, each performing at least a portion of a process or method. In some embodiments, the systems and methods described herein can be implemented by a handheld device, e.g., a smart tablet, a smart phone, or a specialized device created for the system.

[0093] The methods of the present disclosure may be implemented using software, hardware, firmware, hardwiring, or any combination thereof, and the features implementing the functionality may be physically located in various locations, including being distributed such that portions of the functionality are implemented in different physical locations (e.g., having imaging equipment in one room and a host workstation in another room or separate building, with either a wireless or wired connection).

[0094] Processors suitable for the execution of 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. Typically, a processor receives instructions and data from read-only memory or random-access memory, or both. Elements of a computer are a processor for executing instructions and one or more memory devices for storing instructions and data. Typically, a computer also includes one or more non-transitory mass storage devices, such as magnetic disks, magneto-optical disks, or optical disks, for storing data, or are operatively coupled thereto for receiving data from or transferring 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., EPROMs, EEPROMs, solid-state drives (SSDs), 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 the memory can be supplemented by, or incorporated in, special purpose logic circuitry.

[0095] To provide for user interaction, the subject matter described herein may be implemented on a computer having I / O devices, such as CRT, LCD, LED, or projection devices for displaying information to a user, as well as input or output devices such as keyboards and pointing devices (e.g., a mouse or trackball) by which a user can provide input to the computer. Other types of devices may be used to provide for user interaction as well. For example, feedback provided to a user can be any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback), and input from a user may be received in any form, including acoustic, speech, or tactile input.

[0096] The subject matter described herein may 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 a web browser through which a user can 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 may be interconnected through a network, such as by any form or medium of digital data communication, e.g., a communications network. 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.

[0097] The subject matter described herein may 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 control the operation of a data processing device (e.g., a programmable processor, computer, or multiple computers). Computer programs (also known as programs, software, software applications, apps, macros, or code) may be written in any form of programming language, including compiled or interpreted languages ​​(e.g., C, C++, Perl), and may be developed in any form, including as stand-alone programs or as modules, components, subroutines, or other units suitable for use in a computing environment. The systems and methods of the present invention may 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.

[0098] A computer program does not necessarily correspond to a file. A program can be stored in a file, or a portion of a file, that holds other programs or data, either in a single file dedicated to the program in question or in multiple cooperating files (e.g., files storing one or more modules, subprograms, or portions of code). A computer program can be deployed to run on one computer, or deployed to run on multiple computers at one site, or distributed across multiple sites and interconnected by a communications network.

[0099] A file can be a digital file stored, for example, on a hard drive, SSD, CD, or other tangible, non-transitory medium. A file can be sent from one device to another over a network (e.g., as packets sent from a server to a client, for example, through a network interface card, modem, wireless card, or the like).

[0100] 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., by a read / write head into a pattern of net charge or dipole moment magnetization), which then represents a new collocation of information about an objective physical phenomenon desired and useful by the user. In some embodiments, writing involves a physical transformation of material in the tangible, non-transitory, computer-readable medium (e.g., burning a CD-ROM with specific optical properties so that an optical read / write device can then read the new, useful collocation of information). In some embodiments, writing a file involves transforming a physical flash memory device, such as a NAND flash memory device, and includes storing 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 initiated, for example, manually or automatically, programmatically, or by a save command from software or a write command from a programming language.

[0101] Suitable computing devices typically include mass memory, at least one graphical user interface, and 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 can 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, 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.

[0102] As will be recognized by those skilled in the art as necessary or best suited for practicing the methods of the present invention, a computer system or machine used 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, which communicate with each other via a bus.

[0103] In the exemplary embodiment shown in FIG. 10 , system 600 can include a computer 649 (e.g., a laptop, desktop, or tablet). Computer 649 can be configured to communicate across network 609. Computer 649 includes one or more processors 659 and memory 663, as well as input / output mechanism 654. When the methods of the present invention employ a client / server architecture, operations of the methods of the present invention can be performed using a server 613, which can include one or more of processor 621 and memory 629, and can obtain data, instructions, etc., or provide results via interface module 625 or as file 617. Server 613 can be engaged via network 609 through computer 649 or terminal 667, or server 613 can be directly connected to terminal 667, which can include one or more processors 675 and memory 679, as well as input / output mechanism 671.

[0104] 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)) in connection with any of I / O 649, 637, or 671. Computer systems or machines 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.

[0105] Memory 663, 679, or 629 according to exemplary embodiments of the invention may include a machine-readable medium on which one or more sets of instructions (e.g., software) embodying any one or more of the methodologies or functions described herein are stored. The software may also reside, completely or at least partially, in main memory and / or in the processor during its execution by the computer system, with the main memory and processor also constituting machine-readable media. The software may also be transmitted or received over a network via a network interface device.

[0106] Incorporation by Reference References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web content, etc., have been made throughout this disclosure. All such documents are incorporated herein by reference in their entirety for all purposes.

[0107] equivalent While the present invention has been described in connection with specific embodiments, those skilled in the art, after reading the foregoing specification, will be able to realize various modifications, substitutions of equivalents, and other alternatives to the compositions and methods described herein.

Claims

1. 1. A method for generating an immunotherapeutic product, the method comprising: culturing peripheral blood mononuclear cells and dendritic cells in a first cell culture chamber to produce a supernatant containing T cells; transferring the supernatant containing the T cells from the first cell culture chamber through a fluidic connector into a second cell culture chamber; Including, The first cell culture chamber comprises: a bottom surface composed of a first material to which cells adhere; at least one additional surface constructed from a gas-permeable second material; at least two inlets arranged to direct one or more fluids into the first cell culture chamber in a substantially parallel and symmetric inflow relative to the bottom surface; one or more outlets positioned to direct one or more fluids from the first cell culture chamber as an outflow substantially perpendicular to the bottom surface; A method comprising:

2. 10. The method of claim 1, wherein the second cell culture chamber contains fresh dendritic cells, and the method further comprises the step of further culturing the T cells.

3. 10. The method of claim 1, wherein culturing in the first cell culture chamber occurs in the presence of a first set of stimulatory antigens and culturing in the second cell culture chamber occurs in the presence of a second set of stimulatory antigens.

4. 4. The method of claim 3, wherein the first set of stimulating antigens and the second set of stimulating antigens are the same.

5. 4. The method of claim 3, wherein the first set of stimulating antigens and the second set of stimulating antigens are different.

6. 10. The method of claim 1, further comprising measuring one or more parameters within at least the first or second cell culture chamber via one or more sensors.

7. 7. The method of claim 6, wherein the one or more parameters are selected from the group consisting of pH, dissolved oxygen, total biomass, cell diameter, glucose concentration, lactate concentration, and cellular metabolite concentration.

8. 10. The method of claim 1, wherein the movement of the vertical outflow along the vertical flow path is such that the fluid flow rate is insufficient to overcome the settling rate of cells in the first cell culture chamber.

9. 10. The method of claim 1, further comprising the step of further culturing and expanding the T cells in the second cell culture chamber.

10. 10. The method of claim 9, further comprising transferring the cultured and expanded T cells through the fluidic connector to a third cell culture chamber.

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