Cell culture system

Abstract

This invention relates to a cell culture system. In some embodiments, the cell culture system uses autologous antigen-presenting cells to provide T cell expansion and stimulation to deliver therapeutic T cell products that can mobilize the patient's own immune system in a manner that selectively targets the patient's tumor.

Description

[0001] This application is a divisional application. The international application number of the original application is PCT / US2017 / 039538, the international application date is June 27, 2017, the Chinese national application number is 201780038219.2, the entry date into the Chinese national phase is December 19, 2018, and the invention title is "Cell Culture Chamber and Method of Using the Same".

[0002] Cross-references to related applications

[0003] This application claims the interests and priorities of 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 entirety.

[0004] Government funding

[0005] This invention was made with government funding under grant number 1645205 from the National Science Foundation, and the government owns certain rights to this invention. Technical Field

[0006] This invention generally relates to cell culture chambers and their methods of use. Background Technology

[0007] Cell-based cancer immunotherapy has garnered significant attention due to the promising therapeutic potential of chimeric antigen receptor T-cell (CAR-T) therapy, T-cell receptor (TCR) therapy, dendritic cell vaccines, and neoantigen-based T-cell therapy for certain cancers. In neoantigen-based T-cell therapy, the ability to predict candidate neoantigens from tumor sequencing data and monitor neoantigen-specific T-cell responses in patients provides a foundation for designing highly personalized immunotherapeutic agents.

[0008] While T-cell therapy for cancer holds great promise, existing methods for isolating, preparing, and expanding cancer antigen-specific T cells are limited. Currently, the preparation of T-cell therapies using labor-intensive, manual, multi-step methods poses significant challenges to the large-scale production of such therapies. Automation of these methods has been unsuccessful due to the complex biological processes associated with T-cell therapy production, as well as the biological processes and regulatory requirements related to autologous cell handling. Additional challenges exist, such as cell preparation time, maintenance of optimal phenotype, expansion to sufficient cell numbers, and the quality and safety of the cell products.

[0009] like Figure 1As shown, a conventional existing protocol for stimulating human T cells with autoantigen-presenting dendritic cells (DCs) involves several manual steps, including the transfer of supernatant between culture plates, replacement of culture medium, and the addition of cytokines and cell culture medium. T cells are exposed to dendritic cells (DCs) for 7 days, during which time they are stimulated and expand. This process is typically repeated four times, with each stimulation cycle requiring the extraction of T cells from the supernatant and plated with fresh DCs, while the culture medium and growth factors (cytokines) are manually replaced, typically twice every seven days. The number of manual steps required to perform this protocol is very high. For example, using this conventional four-week protocol, approximately 4,650 manual steps are involved in total, all of which increase the risk of contamination and may compromise the quality and safety of the cell product. Summary of the Invention

[0010] This invention recognizes the need to develop new technologies for the automated production of antigen-specific T cells. The cell culture chamber of an exemplary embodiment of this invention includes various technical features that allow for the automation of the aforementioned manual process, significantly reducing user intervention and thus significantly reducing the risk of contamination. For example, the cell culture chamber of this embodiment is manufactured to include a bottom surface and at least one additional surface, the bottom surface being made of a material for cell attachment, and the additional surface, such as sidewalls and / or top walls, being made of a breathable material. In this way, a higher level of gas exchange is achieved without sacrificing the attachment properties of the bottom surface compared to prior art culture systems. Additionally, the cell culture chamber of this embodiment is configured to allow the perfusion of culture medium and cytokines into the chamber, thereby enabling a more consistent level of perfusion. To ensure that antigen-specific T cells and other cells involved in the culture process remain in the chamber during perfusion, one or more inlets and outlets of the cell culture chamber are arranged such that fluid within the cell culture chamber moves at least partially along a vertical flow path upon exiting the chamber. The cell culture chambers are also configured to be fluidly connected to each other, allowing for the automatic transfer of antigen-specific T cells between chambers to allow for further culture and expansion of T cells in new cell culture chambers. In some embodiments, the transfer is achieved by introducing an airflow into the first cell culture chamber, so that the supernatant containing the first cell product is transferred through a fluid connector into the second cell culture chamber.

[0011] In some aspects, the cell culture chambers of exemplary embodiments use antigen-presenting cells from the same patient to provide T-cell expansion and stimulation, thereby providing therapeutic T-cell products that can mobilize the patient's own immune system in a manner that selectively targets the patient's tumor. These cell culture systems and methods significantly reduce the number of manual steps compared to conventional methods. In this way, the risk of contamination is greatly reduced, and the robustness and reproducibility of the manufacturing technology are greatly increased, both of which are key considerations for the safe and reliable manufacture of therapeutic products, such as precisely targeted, personalized T-cell therapies.

[0012] In addition to simplifying and potentially shortening the process for producing immunotherapy products, the cell culture chamber of the exemplary embodiment significantly improves the utilization of patient cellular material in cell-based therapies and the reliability and robustness of the manufacturing process, while also leading to reduced costs (e.g., labor costs). Furthermore, the method can be easily scaled from processing only a few patient samples to 100 patient samples. The construction of the cell culture chamber in this embodiment allows for automated fluid flow control to contact antigen-presenting cells with cells containing T cells, and to refresh the antigen-presenting cells as needed to stimulate and expand the antigen-presenting T cells to a number capable of generating a therapeutic response in patients. 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 1-10 to 100 or hundreds of samples. Each chamber can be controlled independently, and the number of chambers in use at any given time can be increased or decreased proportionally according to the number of samples. In other embodiments, a single central controller (e.g., a PLC logic controller) controls all chambers in the system.

[0013] Exemplary arrangements will now be described, in which systems and methods of exemplary embodiments of the present invention utilize one or more bioreactors, each bioreactor including a cell culture chamber, the bioreactors being configured to be fluidly connected to each other to perform processing of patient cellular material, thereby producing an immunotherapy product. Those skilled in the art will understand that these are exemplary arrangements described herein, and other arrangements are within the scope of the invention.

[0014] In this exemplary embodiment, a cell culture chamber is provided, comprising a bottom surface containing a first material for attaching cells; at least one additional surface containing a breathable second material; one or more inlets; and one or more outlets, wherein the inlets and outlets are arranged to allow fluid to move at least partially along a vertical flow path within the cell culture chamber. In some respects, the fluid movement along the vertical flow path results in a fluid velocity insufficient to overcome the settling rate of cells within the cell culture chamber.

[0015] Compared to conventional methods, providing at least one additional breathable surface allows for a higher level of gas exchange, thereby allowing for the processing of a larger number of cells in the chamber. In some aspects, the bottom surface and at least one additional surface are bonded together without the use of adhesives. In some embodiments, at least one additional surface also comprises the first material.

[0016] The first material may include, for example, polystyrene. The second material may include one or more materials having an oxygen permeability coefficient equal to or greater than 350 and a carbon dioxide permeability coefficient greater than or equal to 2000, wherein the unit of permeability coefficient is [cm²]. 3 ][cm] / [cm 2 [s][cm Hg]. In one aspect, the second material is selected from one or both of polysiloxane and polymethylpentene.

[0017] In some aspects, the cell culture chamber includes at least one fluid connector configured to fluidly connect the cell culture chamber to a second container, which may be a second cell culture chamber. To facilitate fluid flow through each chamber and between chambers, the cell culture chamber may include one or more pumps. Each chamber may include its own pump, and the one or more pumps may serve multiple chambers. In other aspects, the cell culture chamber also includes one or more fluid reservoirs operatively connected to the one or more pumps. The fluid reservoirs are configured to supply the chambers with a culture medium comprising nutrients and cytokines.

[0018] The cell culture chamber may also include one or more sensors operatively connected to the cell culture chamber in a manner that allows 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 concentration of cell metabolites within the cell culture chamber. The cell culture chamber may also include a central processing unit communicatively connected to the one or more sensors and configured to adjust the operating state of the one or more pumps based on the measured one or more parameters; in embodiments employing a flow-generating mechanism instead of pumps, such as an electrohydrodynamic mechanism, the central processing unit may modify the operating state of the flow-generating mechanism to adjust the flow rate of the first cell product based on one or more parameters.

[0019] In some embodiments, the chamber is sized and constructed to help maintain the desired environment inside and around the cell culture chamber. In some embodiments, one or more pumps are located inside the incubator. In other embodiments, one or more pumps are located outside the incubator and operatively connected to the cell culture chamber inside the incubator.

[0020] In some aspects, at least a portion of the system includes disposable components, and some or all of these components may be housed within a non-disposable frame. In other aspects, all components of the system are disposable. Furthermore, in some embodiments, the system includes a sample tracking component for tracking and recording patient materials.

[0021] The system and method are designed to allow for the provision of any number of additional reactors or cell culture chambers. In some embodiments, the system includes two or more bioreactor chambers for generating T cells, such as... Figure 6 As shown in A and 6B. Although Figure 6 The systems in A and 6B are shown as having two cell culture chambers in fluid communication with each other. It should be understood that the systems can use any number of additional chambers.

[0022] In some embodiments, the system of the present invention has the ability to automatically calculate and set a desired perfusion rate of the perfusion fluid given various inputs, such as the size of the 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, the central processing unit including a memory containing instructions executable by the central processing unit, such that the system receives data including the size of the cell culture chamber as a first input, receives 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 as a second input, and calculates, based on the first and second inputs, a perfusion rate of the perfusion fluid to be introduced into the cell culture chamber, the perfusion rate maximizing the probability that the first cell type and the second cell type come into contact with each other within the cell culture chamber. In some aspects, the first cell type is peripheral blood mononuclear cells, and the second cell type is dendritic cells.

[0023] The central processing unit can control the perfusion rate of the perfusion fluid by controlling one or more pumps (or valves) operably connected to one or more perfusion fluid reservoirs and the central processing unit. In some respects, one or more sensors are operably connected 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 cell metabolite concentration.

[0024] In another aspect of the invention, a method for transferring cells from a first cell culture chamber to a second cell culture chamber is provided. The method typically includes culturing cells in the first cell culture chamber to produce a supernatant containing a first cell product, and introducing an airflow 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. Once the fluid enters the second cell culture chamber, the first cell product is further cultured.

[0025] Similar to transferring fluid from a first cell culture chamber to a second cell culture chamber, the method may further include introducing an airflow into the second cell culture chamber to transfer a supernatant containing the first cell product for further culture through a fluid connector and into a third cell culture chamber. In one embodiment, the transfer of the supernatant occurs at at least three different times, thus using at least four culture chambers to produce the desired amount of antigen-presenting T cells.

[0026] Furthermore, in some respects, as described above, one or more cell culture chambers are configured to allow fluid (e.g., perfusion fluid) to move along a vertical flow path within the chamber. Figure 8 C). The fluid movement makes the fluid velocity insufficient to overcome the sedimentation rate of cells in the cell culture chamber.

[0027] In another aspect of the invention, a method for producing an immunotherapy 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 an airflow into the first cell culture chamber to transfer the supernatant containing T cells through a fluid connector into a second cell culture chamber. In some aspects, fresh dendritic cells are contained in the second cell culture chamber, and T cells are further cultured in the second cell culture chamber.

[0028] Culture in the first culture chamber can be carried out in the presence of one or more stimulating antigens from a first group, while culture in the second culture chamber can be carried out in the presence of a second group of stimulating antigens. In some embodiments, the first and second groups of stimulating antigens are the same. In other embodiments, the first and second groups of stimulating antigens are different. Attached Figure Description

[0029] The foregoing will become apparent from the following more detailed description of exemplary embodiments as illustrated in the accompanying drawings, in which the same reference numerals refer to the same parts in different views. The drawings are not necessarily drawn to scale, but rather focus on illustrating the embodiments. The patent or application document contains at least one color-drawn drawing. A copy of the disclosure of this patent or patent application with color drawings will be provided by the competent authority upon request and after payment of the necessary fees.

[0030] Figure 1 Existing manual techniques for producing immunotherapy products are shown.

[0031] Figure 2 An exemplary method for producing an immunotherapy product according to one embodiment of the present invention is shown.

[0032] Figure 3 This is a schematic diagram depicting the system of the present invention in one embodiment, showing a cell culture chamber.

[0033] Figures 4A-4E An exemplary cell culture chamber structure according to an embodiment of the present invention is depicted.

[0034] Figure 5 The surface of a cell culture chamber is depicted, which has a structure that allows a permeable polymer to be introduced into another polymer.

[0035] Figure 6 AC depicts a schematic representation of the process used to connect cell culture chambers and transfer fluids between them. Figure 6 A is as follows Figure 3 The diagram shows the first cell culture chamber. Figure 6 B shows partially expanded T cells in the first cell culture chamber, and the second cell culture chamber moved to a position connected to the first cell culture chamber. Figure 6 C illustrates the injection of sterile air into the first cell culture chamber to transfer the supernatant containing expanded T cells to the second cell culture chamber.

[0036] Figure 7 The flow of fluid from the outlet of one cell culture chamber to the inlet of two cell culture chambers is depicted according to one embodiment.

[0037] Figure 8 AD depicts fluid flow through various chamber configurations. Figure 8 A and B depict the planar flow of fluid entering and exiting the cell culture chamber. Figure 8 C and D depict structures with symmetrical inflow and vertical outflow.

[0038] Figure 9 A kinetic parameter-based modeling of the interaction between T cells and antigen-presenting cells was developed.

[0039] Figure 10 A system according to certain embodiments of the present invention is described. Detailed Implementation

[0040] The apparatus, systems, and methods of the present invention enable the automation and remote monitoring and control of methods for generating sufficient quantities of antigen-specific T cells for use in personalized targeted therapies for cancer or infectious diseases involving cultured autologous cells. The systems and methods of the present invention include various technical features that allow for the automation of the aforementioned manual processes. These technical features include, but are not limited to: 1) configuring the cell chamber to include a bottom surface made of a material for attaching cells and at least one additional surface made of a breathable material, thereby enabling a higher level of gas exchange; 2) arranging one or more inlets and outlets of the cell culture chamber such that fluid flows along a vertical flow path within the cell culture chamber as it exits the chamber, ensuring that antigen-specific T cells and other cells involved in the cell culture process remain in the chamber during perfusion of the culture medium; and 3) automatically transferring antigen-specific T cells from one chamber to another by introducing airflow into a first cell culture chamber to transfer cells via a fluid connector and into a second cell culture chamber.

[0041] The methods, apparatus, and systems of the present invention can be scaled up to provide large quantities of cell-based immunotherapy products and can be operated on in parallel for a single subject or for several subjects (thereby keeping their cells and progeny isolated). Compared to prior art methods and apparatus, the methods and systems of the present invention are robust in operation, capable of providing high product yields, simple and effective, involve less risk of contamination, and minimize labor costs.

[0042] Figure 2 This document provides an overview of a method for producing cell-based immunotherapy products using the system described herein. In summary, the steps for producing cell therapy products according to certain embodiments of the invention include co-culturing stimulated antigen-presenting cells and cells containing T cells in a bioreactor containing a cell culture chamber. During culture, a supernatant containing expanded therapeutic T cell products is produced. In some aspects, to produce an amount of antigen-specific T cells sufficient to elicit a therapeutic response in a patient, the T cells must be further cultured in one or more additional cell culture chambers. To achieve this additional culture, a transfer of supernatant from the culture chamber in which the supernatant was produced must occur to a subsequent cell culture chamber containing a fresh supply of antigen-presenting cells. The transfer of supernatant between cell culture chambers may include introducing an airflow into a first cell culture chamber, which transfers the supernatant containing the first cell product to the new cell culture chamber via a fluid connector. Furthermore, in each culture step, a perfusion fluid containing, for example, culture medium and cytokines may be perfused into the chamber. In some aspects, the perfusion fluid flows through the chamber along a vertical flow path to ensure that the cells remain within the chamber during culture. The only manual step involved in using the system of the present invention is to provide the system with one or more subsequent bioreactors, each containing a cell culture chamber, each containing a new batch of autoantigen-presenting cells stimulated by antigen peptide shock.

[0043] The method for generating cell-based immunotherapy products according to embodiments of the present invention is much simpler and more effective than existing methods. Figure 1 The conventional approach of the prior art is illustrated, which requires at least 4,650 manual steps, as previously described. In contrast, the system and method of the present invention, as... Figure 2 As shown in Figure 4 and described herein, the number of manual steps that must be taken to produce the same cell dose is reduced by 25-fold, as shown in the table below.

[0044] Table 1. Typical steps in antigen-specific T cell generation

[0045]

[0046] The cell culture chamber of this invention significantly improves the manufacturing of immunotherapy products, providing a flow-based immunotherapy agent production technology with unparalleled consistency, quality, safety, economy, scalability, flexibility, and portability.

[0047] Exemplary arrangements will now be 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 each other, for processing patient cellular material to produce an immunotherapeutic product. It should be understood that in some embodiments, the bioreactors are situated in a closed environment. Scale-up of this exemplary embodiment will be possible by adding modules (e.g., bioreactors) to allow for serial and / or parallel processing. Those skilled in the art will also recognize that different or alternative arrangements may be required depending on the product to be produced.

[0048] In an exemplary implementation, such as Figure 3 As shown, a bioreactor 110 is provided, which includes a cell culture chamber 120. The cell culture chamber 120 includes a bottom surface 122 and at least one additional surface 124. The bottom surface 122 is composed of a first material for attaching cells, and the at least one additional surface 124 is composed of a breathable second material. The cell culture chamber also includes one or more inlets 126, 136 and one or more outlets 128, 138. In some embodiments, the bioreactor 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 conveying fluid to and from the reservoirs 132, 134 and through the chamber 120.

[0049] Regarding cell culture chamber 120, the first material can be any biocompatible material to which antigen-presenting cells (APCs) such as dendritic cells (DCs) will attach. During the T cell stimulation and expansion process occurring in cell culture chamber 120, mature APCs will develop and preferably attach to the bottom surface 122, while the T cells remain in the supernatant located above the bottom surface, which makes it easier to obtain the expanded T cells separately.

[0050] In one exemplary embodiment, the first material comprises polystyrene. One benefit of using polystyrene as the substrate surface for culturing is the useful role this material plays in the process of generating dendritic cells from PBMCs. Specifically, the polystyrene surface can be used to enrich monocytes from a heterogeneous suspension of PBMCs. This is the first step in a culture process for generating DCs by differentiating monocytes through culture in a medium containing, for example, IL4 and GM-CSF. From a biological process perspective, the use of the same polystyrene surface is highly valuable for the production of dendritic cells entirely through a single cycle of T cell stimulation, as it eliminates numerous transfer steps that would otherwise be required, thereby allowing for the use of closed systems for the therapeutic T cell production of DC-stimulated cells.

[0051] The bottom surface can have a surface area comparable to that of conventional perforated plates, such as 6-well and 24-well plates (9.5 cm² each). 2 and 3.8cm 2 It should also be understood that the surface area can be much smaller or even much larger than that of a conventional well plate (e.g., having a surface area comparable to that of standard cell culture dishes and flasks), for example, a surface area of ​​approximately 2.0 cm². 2 Approximately 200cm 2 For example, approximately 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 , and any surface area in between.

[0052] At least one additional surface 124 may include any configuration, such as one or more sidewalls and a top wall. In one embodiment, such as Figure 3 As shown, the sidewalls can be arranged at 90-degree angles to each other, thereby allowing the box shape to be formed in conjunction with the bottom surface 122. In another embodiment, at least one additional surface 124 forms curved sidewalls, thereby forming a cylinder, elliptical cylinder, or cone, such as Figure 4AAs shown in -C. In another exemplary arrangement, at least one additional surface may be formed into a dome shape on the bottom surface, as shown in Figure 1. Figure 4D As shown. In other embodiments, the sidewalls can be formed in a triangular shape, such as... Figure 4E As shown. It should be understood that the above exemplary construction is non-limiting, and at least one additional surface may have other constructions not provided in the foregoing exemplary construction.

[0053] In another embodiment, at least one additional surface 124 comprises a breathable second material to facilitate gas exchange occurring within the cell culture chamber. High surface area gas exchange is achieved in the system of this embodiment by fabricating the cell culture chamber such that the bottom surface is made of a material for cell attachment (e.g., polystyrene), and at least one additional surface (e.g., sidewalls and / or top walls) is at least partially made of a breathable material. In addition to the bottom surface, the large, highly permeable surface provides the ability to achieve a higher level of gas exchange without sacrificing the adhesion properties of the bottom surface, compared to prior art culture systems (which are limited in the amount of culture medium they can contain and / or lack culture-friendly surfaces to which cells can attach).

[0054] In some embodiments, the second material comprises one or more materials having a permeability coefficient for oxygen equal to or greater than 350 and a permeability coefficient for carbon dioxide greater than or equal to 2000, wherein the unit of permeability coefficient is [cm]. 3 ][cm] / [cm 2 [s][cm Hg]. Exemplary materials include polysiloxane-containing materials and polymethylpentene, such as poly(dimethylsiloxane) (PDMS), which is known for its high oxygen and carbon dioxide permeability (three orders of magnitude higher than materials such as polystyrene and PMMA). In one exemplary embodiment, the cell culture chamber comprises a polystyrene base plate and polysiloxane sidewalls and top walls.

[0055] In some respects, in addition to the second material, at least one additional surface 124 may also comprise the first material. For example, but not limitingly, the additional surface 124, such as one or more sidewalls and / or top walls, may incorporate a second material (e.g., a highly permeable polymer, such as polysiloxane) within a framework made of the first material (e.g., polystyrene). Figure 5 As shown. It is also envisioned that the bottom surface may also include a second material. However, in some embodiments, the second material is only intermittently dispersed throughout the bottom surface to ensure that the first material covers a sufficient surface area so that cells can attach to the surface.

[0056] The first and second materials can be joined together using any method known in the art (e.g., mechanical fastening, adhesive and solvent bonding, and welding). However, given that cell immunotherapy products produced using the systems and methods of embodiments of the present invention will be administered to human patients, regulatory issues may prevent the use of some or all adhesives in assembling the cell culture chamber. Therefore, in some 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, sidewalls, and top wall, comprise the first material (e.g., polystyrene) and are joined together using ultrasonic welding, with at least a portion of the first material cut out in the sidewalls and / or top wall to allow insertion of the second material (e.g., a polysiloxane material), such as... Figure 5 As shown. It should be understood that the hole can be any desired shape, not just as shown. Figure 5 The circle shown. In an exemplary arrangement, the second material can be inserted into the hole individually in the same way as a plug that seals the container by being inserted into the top opening. In another example, the second material can be manufactured to completely cover and surround the outer surface of the first material frame, such that the second material is accessible through holes within the frame, as... Figure 5 As shown. It should be understood that the foregoing construction is merely an example, and other constructions for connecting the first material and the second material are also contemplated embodiments of the present invention.

[0057] The height of one or more cell culture chambers can vary. For example, but not limited to, exemplary ranges of cell culture chamber height include any height 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 higher, or any height between these values. In some embodiments, the chamber height may be comparable to the liquid height typically found in cultures performed in 6-well and 24-well plates, for example, from 2 mm to 6 mm, with a volumetric capacity of about 0.8 mL to 6 mL. In other embodiments, the cell culture chamber will have a large size, for example, 10 mm to 50 mm, with a culture surface of approximately 50 cm². 2 .

[0058] As mentioned above Figure 3As briefly mentioned, in some embodiments, the bioreactor 110 may also include one or more pumps 140 operatively connected to the cell culture chamber 120 for perfusing perfusion media into the cell culture chamber. The bioreactor 110 may also include one or more fluid reservoirs 132. The fluid reservoirs 132 are in fluid communication with the cell culture chamber 110 and may be operatively connected to 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 some aspects, the one or more pumps are configured to pump fluid from the fluid reservoir through the cell culture chamber and into a waste collection reservoir. Figure 3 In the example embodiment shown, fluid moves from fluid reservoir 132 through pipe 152 to pump 140 and enters cell culture chamber 120 via inlet 136, exits cell culture chamber 120 via outlet 138, passes through pipe 154, and enters waste collection reservoir 134.

[0059] In some embodiments, the fluid reservoir and / or waste collection reservoir may each be provided as one or more capped bottles contained within or in fluid connection to a cell culture chamber. Each reservoir includes an inlet and an outlet, or an outlet and a vent in fluid connection to the inlet of one or more cell culture chambers. In some aspects, for example, Luer connectors and polysiloxane liners cut to mate with the Luer connectors may be used to prevent leakage through the inlet and / or outlet.

[0060] In some embodiments, one or more bioreactors are sized and constructed to fit within an incubator, such that the process takes place within the incubator. Conditions within the incubator include a sustained temperature of 37°C and 95%–100% humidity. Therefore, the selected materials must be robust enough to withstand these conditions, as materials (including fluids and biological agents) tend to expand under such conditions. Furthermore, in some cases, conditions within the incubator remain stable, and automated temperature recording can provide knowledge of temperature fluctuations to correlate with any distortions in the reaction occurring within the incubator. Therefore, any power supply 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 remains fluidly connected and operatively communicated with it. In another embodiment, the pump is directly connected to the bioreactor and located within the incubator, but without thermal or operative connection to a radiator and / or fan for heat dissipation. Regardless of the construction, the pump is operatively connected to the bioreactor and, consequently, to the cell culture chamber. Further details regarding perfusion-based automated cell culture systems, such as small-scale culture systems for endothelial cell culture with onboard reagent storage and perfusion achieved by an onboard disposable peristaltic pump, and larger-scale culture systems for the generation of dendritic cells from monocytes using chambers with polystyrene bottom surfaces, can be found in International Patent Applications PCT / US2016 / 040042 and PCT / US2016 / 60701, both of which are incorporated herein by reference in their entirety.

[0061] The system may also include heaters for controlling the temperature of the cell culture reservoir and, optionally, the fluid reservoir. In this configuration, an incubator is not required, and the system can operate automatically using only electricity. If the system does not have a heater, it can operate inside an incubator for cell culture.

[0062] In other aspects, the cell culture chamber includes one or more sensors (not shown) operatively connected to the cell culture chamber. The sensors may be able 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 cell metabolite concentration. In embodiments where the system includes multiple cell culture chambers, one or more sensors may be connected to one or more cell culture chambers. In some embodiments, one or more sensors are connected to one or more cell culture chambers, but not to all chambers in the system. In other embodiments, one or more sensors are connected to all cell culture chambers in the system. In a system with multiple chambers operatively connected to one or more sensors, the sensors may be the same in each chamber to which they are connected, they may all be different, or some sensors may be the same while some may be different. In some aspects, one or more sensors are operatively connected to a computer system (…). Figure 3 (Not shown in the image), this computer system has a central processing unit for executing instructions, enabling automated monitoring and adjustment of parameters. Further details regarding a computer system for implementing the methods of the present invention using a cell culture chamber are provided below.

[0063] For example Figure 3 As shown, the cell culture chamber has an inlet 126 and an outlet 128, both of which can be used to fluidly connect the chamber to one or more additional containers via fluid connectors. In some embodiments, the additional containers include one or more additional cell culture chambers, as will be described in more detail below. The system of the present invention may 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 theream or more than 100 cell culture chambers configured to be fluidly connected in series to produce an immunotherapy product. Alternatively or additionally, one or more cell culture chambers may be arranged in parallel to each other to allow for the production of immunotherapy products for more than one individual at a time. In a preferred embodiment, the cell culture chambers of the bioreactor are connected via aseptic connections.

[0064] An exemplary construction of a multi-bioreactor system is shown in Figure 6 For B and C, the following provides further details regarding the methods used to execute this construct. Figure 6 As shown in Figure B, in the case of the second bioreactor 210, the second cell culture chamber 220 is moved into position to connect with the first cell culture chamber 120 via the outlet of the first chamber and the inlet of the second chamber. This connection is preferably a sterile connection. This connection allows sterile air to be injected into the first cell culture chamber 120 to transfer supernatant containing expanded T cells into the second cell culture chamber 220. Alternative techniques known in the field 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 reservoir, pump, and associated piping. However, it should be understood that the reservoir and pump can be shared between bioreactors.

[0065] In some embodiments, the ratio of cell culture inlet to cell culture outlet is 1:1, for example, when one or more bioreactors are arranged in series with each other. Figure 6 As shown in B and C. In other embodiments, the outlet-to-inlet ratio of at least a portion of the bioreactor is 1:2. For example, the outlet of a cell culture chamber 120 can be fluidly connected to the inlets of two cell culture chambers 220a and 220b, such that the fluid flowing out of the first cell culture chamber 120 is split into two streams, one stream being sent to the second cell culture chamber 220a and the second stream being sent to the third cell culture chamber 220b, as shown. Figure 7 As shown. In this configuration, both the second cell culture chamber 220a and the third cell culture chamber 220b can be used for further stimulation and expansion of T cells. Alternatively or complementaryly, one of the second cell culture chamber 220a and the third cell culture chamber 220b can be configured to allow monitoring of reaction and flow parameters using one or more sensors operatively connected to the chamber. In this way, one of the chambers remains without additional sensors, some of which may need to penetrate the walls of the cell culture chamber, potentially increasing the risk of leakage and / or contamination.

[0066] In some implementations, one or more bioreactors may be arranged in a system containing modules for performing various other processes before, simultaneously with, or after the processes occurring within the cell culture chamber of the bioreactor.

[0067] The system and some or all of its components can be designed using CAD software and then transferred to a laser cutting machine, which allows the plastic to be cut to specified sizes and shapes. Various connections (e.g., inlets and outlets) can be fabricated by laser-cutting through holes, which can then be manually tapped to provide threads for receiving male Luer connectors. Fluid can then be introduced into the system by connecting a Luer adapter to a blunt dispensing needle and pushing the tube onto the blunt needle portion. Further details regarding the construction of the fluid system components can be found in International Patent Applications PCT / US2016 / 040042 and PCT / US2016 / 60701, the entire contents of which are incorporated herein by reference.

[0068] The above description focuses on system components and various possible configurations. The following description focuses on the process performed using a system according to an exemplary embodiment of the present invention. To stimulate and amplify antigen-specific T cells, the process begins with the co-culture of cells containing T cells with APCs obtained from the same individual in a cell culture chamber. In a specific embodiment, the cells containing T cells include peripheral blood mononuclear cells (PBMCs), and the APCs include dendritic cells (DCs). Cells containing T cells and APCs can be supplied to the cell culture chamber in a ratio (cells containing T cells:APCs) of approximately 1000:1 to 1:1000, such as, but not limited to, approximately 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, 1... 0:1, 5:1, 4:1, 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 between them. In one aspect, a ratio of 10:1 is preferred.

[0069] To stimulate and expand T cells from the interaction between APCs and cells containing T cells, APCs need to be stimulated. This can be accomplished by using one or more stimulating molecules. In some embodiments, the stimulating molecules are non-tumor-specific. In other embodiments, the stimulating molecules are tumor-specific. For example, the stimulating molecules may be selected from one or more characteristics of an individual tumor, such as different antigenic peptides. In some embodiments, the stimulating molecules are preferably added only at the beginning of the culture cycle. The stimulating molecules may be added over a period of only a few minutes, an hour, several hours, or longer. In a preferred embodiment, the stimulating molecules are added over a period of about one hour.

[0070] During the culturing of both cell materials, a supernatant is formed containing lighter, non-attached T cells, while heavier, mature APCs (e.g., dendritic cells) attach to the substrate surface. In embodiments where DCs are used as APCs, the expanded T cells must be extracted from the cell culture chamber at the end of seven days because primary DCs cannot be sustained in culture for more than seven days. Therefore, if additional T cell expansion is required, a fresh supply of dendritic cells is necessary. It should also be understood that cells can be cultured using a batch of dendritic cells for any duration less than seven days. For example, cells can be cultured for any time, from less than one minute to seven days, depending on the desired level of stimulation.

[0071] In one exemplary embodiment, after culturing for up to seven days, the expanded T cells are extracted and transferred to a new cell culture chamber containing fresh dendritic cells (DCs), which are pulse-stimulated with the same antigenic peptide used, for example, in the first cell culture chamber. The stimulation process can be repeated multiple times as needed to generate a sufficient 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.

[0072] The co-culture of APCs and T cells occurs in a culture medium. Exemplary culture media include, but are not limited to, RPMI medium and... Culture medium. Any other suitable culture medium known in the art can be used according to embodiments of the invention. Cytokines such as IL-4 and GM-CSF can also be added to the culture medium.

[0073] In one embodiment, culture medium and cytokines can be perfused into a cell culture chamber to aid in the formation of cell-based immunotherapeutic products. In plate-based protocols for stimulating T cells via dendritic cells (DCs), a culture volume of approximately 2 mL is maintained from the outset, with cytokine infusions performed twice within each 7-day stimulation cycle. The main advantage of perfusion is the ability to maintain a consistent local concentration distribution of culture medium and cytokines, which ensures higher yields and the potential to accelerate the differentiation of monocytes into DCs compared to prior art plate-based protocols. However, the combination of attached (DC) and non-attached (T cell) types, and the high sensitivity of DCs to mechanical forces, pose challenges to the stimulation and expansion of antigen-specific T cells, particularly with fluid flow through the cell culture chamber. Therefore, in those embodiments where culture medium and cytokines are provided via perfusion, the systems of the present invention must be able to supply nutrients and cytokines to cells without removing cells from the bioreactor, while also taking into account the shear sensitivity of certain antigen-presenting cells (e.g., DCs). Essentially, some embodiments of the systems and methods of the present invention are designed to optimize the retention of autocrine / paracrine signaling to promote T cell proliferation, while renewing growth factors and maintaining minimal physical stimulation of DCs. To take this into account, the direction and rate of perfusion flow through the cell culture chamber must be considered.

[0074] In some respects, the fluid flow rate is maintained below the sedimentation rate of the antigen-presenting cells. Therefore, the antigen-presenting cells will remain in the culture chamber due to their mass. In other words, the antigen-presenting cells will settle to the bottom of the cell culture chamber and thus remain there.

[0075] The flow velocity below the settling rate can be calculated using Equation 1:

[0076] v_max=〖(ψd_p)〗^2 / 150μg(ρ_cell - ρ_fluid)∈^3 / (1-∈)

[0077] Where v_max is the liquid velocity beyond which the cell will rise, ψ is the cell shape factor (the ratio of the cell surface area to the surface area of ​​a sphere of equal volume; note that the cell is not a perfect sphere, and this factor is expected to be less than 1), d_p is the diameter of the spherical particle with a volume equal to that of the cell, μ is the viscosity of the liquid containing the cell, g is the gravitational constant, ρ_cell is the density of the cell, ρ_liquid is the density of the liquid containing the cell, and ε is the fraction of the volume of interest not occupied by the cell.

[0078] In other respects, one or more inlets 136 and one or more outlets 138 of the cell culture chamber are arranged to allow liquid (e.g., perfusion fluid) to move along a vertical flow path within the cell culture chamber. This configuration helps prevent cells (e.g., DCs and T cells) from leaving the chamber, especially when the flow rate through the chamber is 2–10 L / min. For example, planar inflow / outflow, such as… Figure 8 As shown in A and B, regions with high wall shear uniformity are provided, but measures are needed to prevent cells from being removed from the chamber. In contrast, configurations with symmetrical inflow and vertical outflow, such as... Figure 8 As shown in C and D, this prevents cells from leaving the chamber.

[0079] Despite Figure 8 C is shown as having four inlets and one vertical outlet, but any number of inlets and outlets can be provided, as long as the fluid flowing out of the chamber exits the top of the chamber in a vertical direction. For example, the chamber can have any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more inlet inlets, and also any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more inlet outlets. Additionally, although the inlets are... Figure 8 Figure C shows a symmetrical arrangement, but constructions with more than two inlets are also contemplated as asymmetrical arrangements. The inlets can also guide fluid into the chamber from any direction. In a preferred embodiment, the inlets guide fluid into the chamber in a direction parallel to the bottom surface.

[0080] In some respects, culture medium is perfused at specific time points within the time frame during cell culture in any cell culture chamber, such as daily or weekly (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times). In other respects, culture medium is perfused continuously during the culture period. Continuous perfusion helps maintain a nearly constant culture volume throughout the process.

[0081] In some aspects, cytokines are infused at one or more points during culture, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. Alternatively, cytokines can be continuously perfused with the culture medium. In those embodiments, continuous perfusion helps maintain a consistent local concentration distribution of cytokines, which helps ensure higher yields compared to static cell culture methods and has the ability to increase the rate of T cell stimulation and expansion.

[0082] Perfusion parameters can vary at any time during the culture cycle. Exemplary parameters include, but are not limited to, median flow rate, cytokine concentration, and culture cycle duration. Each of these parameters can potentially affect the efficacy of T stimulation. For example, in recent work on the design of culture chambers for the diffusion of monocytes to DCs, as described in International Patent Applications PCT / US2016 / 040042 and PCT / US2016 / 60701, we have determined that 0.1 dyn / cm 2 A moderate perfusion rate at a wall shear stress level can produce DCs with the same phenotype as those produced using conventional 6-well or 24-well plate-based protocols. Therefore, by measuring one or more of the aforementioned phenotypes and functional metrics during the culture cycle, the effect of one or more perfusion parameters on efficacy can be monitored, allowing for appropriate adjustments.

[0083] Depending on certain aspects, the efficacy of stimulation can be assessed at any point during culture, preferably after 7 days. Both phenotypic and functional measurements can be used to assess efficacy. For example, cell numbers (fold expansion) can be counted using directed cell counting methods. Cell phenotypes (including assessment of antigen specificity via tetramer staining) can be characterized by flow cytometry. Functional assays can also be used to assess the ability of expanded T cells to recognize antigen-carrying target cells as well as autologous tumor cells. Results can be evaluated for values ​​in 24-well plates. The format was used to benchmark DC-based T cell stimulation.

[0084] As described above, because some APCs (e.g., dendritic cells) cannot survive in cultures exceeding 7 days, certain embodiments of the present invention involve multiple cycles of T cell stimulation in a semi-batch configuration using more than one bioreactor. Each cycle utilizes newly generated autoantigen-presenting cells. In some embodiments, the antigen-presenting cells are subjected to a shock stimulation with the same set of antigens for each stimulation cycle. In other embodiments, different sets of antigens are used for one or more stimulation cycles.

[0085] Typically, multi-cycle T cell stimulation involves culturing cells in a first cell culture chamber to produce a supernatant containing first cell products, providing a second cell culture chamber, and subsequently transferring the supernatant from the first cell culture chamber to the second cell culture chamber by introducing an airflow into the first cell culture chamber, such as... Figure 2 As shown.

[0086] Exemplary constructions of multi-reactor systems can be found in Figure 6 A-6B. As shown, the process begins in a reactor containing mature, attached DCs, which are loaded together with PBMCs and subjected to an initial stimulation cycle of 7 days with perfusion culture medium and cytokines. After the first stimulation cycle, a second (optionally larger) reactor containing fresh DCs is connected to the first reactor, as shown. Figure 6 As shown in B and 6C. The injection of sterile air then transfers the supernatant from the first reactor to the second reactor. This second bioreactor will contain its own culture medium and cytokine supply, as well as a disposable peristaltic pump. After the supernatant transfer, the first reactor can be detached and discarded. For progressively larger bioreactors, this process can be repeated multiple times as needed to achieve the desired stimulation level and T cell proliferation. For example, in one embodiment, four stimulation cycles are performed, which include transferring the supernatant to three new cell culture chambers at three different times. It should be understood that although the second chamber 220 is shown as larger than the first cell culture chamber 110, the second cell culture chamber and any subsequent cell culture chambers can be of any size, such as larger than, equal to, or smaller than the first cell culture chamber. In some embodiments, the size of each subsequent cell culture chamber is larger than the cell culture chamber from which the supernatant was transferred.

[0087] Modular design offers flexibility in terms of cycle number and antigen-presenting cell type, allowing for the use of antigens from the same group for each cycle, antigens from different groups for each cycle, or a combination of both. In some respects, the ability to generate T cells specific to multiple different disease-related antigens in an automated process is advantageous in disease treatment because it allows for multi-directional attack.

[0088] In some respects, computational modeling methods are used to optimize the interaction between T cells and antigen-presenting cells (e.g., dendritic cells). The computational model according to the invention considers the effects of perfusion and the optimal time required for stimulation, and combines particle interaction-based and kinetic parameter-based methods. Exemplary particle interaction-based and kinetic parameter-based methods are known in the art, some of which are described herein. For example, regarding particle interaction-based methods, Day and Lythe describe using the following equation to determine the time required for T cells to find an APC on the surface of a lymph node, where D is the diffusivity of the T cell and b is the radius of the APC located at the center of a spherical lymph node of radius R. See Day et al., Mathematical Models and Immune Cell Biology; 2011.

[0089]

[0090] Regarding methods based on kinetic parameters, Valitutti has developed models of the interaction between T cells and antigen-presenting cells, such as... Figure 9 As shown in Valitutti et al., FEBS Lett. 2010. However, this interaction has not yet been modeled on the scale of culture chambers or bioreactors.

[0091] By incorporating both particle interaction-based and kinetic parameter-based approaches into the computational model of this invention, it is possible to automatically determine and monitor the optimal perfusion rate of the perfusion fluid (e.g., the culture medium for infusing cytokines) to maximize the probability of two cell types coming into contact with each other within the cell culture chamber.

[0092] For example, in some embodiments, a cell culture system is provided, comprising a cell culture chamber and a central processing unit (CPU), the CPU including a memory containing instructions executable by the CPU. In some aspects, the instructions cause the system to receive data including the size of the cell culture chamber as a first 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 as a second input, and to calculate, based on the first and second inputs, a perfusion rate of the perfusion fluid to be introduced into the cell culture chamber, the perfusion rate maximizing the probability that the first and second cell types will come into contact with each other within the cell culture chamber. Further details regarding a computer system for implementing the methods of the present invention within a cell culture system are provided below.

[0093] In some aspects, the system also includes one or more pumps operably connected to one or more infusion fluid reservoirs and operably connected to a central processing unit, such that the central processing unit also controls the infusion rate of the infusion fluid by controlling one or more pumps.

[0094] As described above, the systems and methods of the present invention utilize modules (e.g., bioreactors containing cell culture chambers, etc.) that are fluidly connected to each other for processing individual cellular materials to produce immunotherapeutic products.

[0095] The systems or apparatus of the present invention are modular and can be fluidly connected in series (i.e., fluid flows from one device to another) and / or in parallel with other similar devices, and can also be configured to be physically stacked on top of each other, or physically arranged within related devices such as incubators. The modular design of the system in particular allows for the flexible switching on and off of modules according to the desired processes to be included in the system.

[0096] The fluid device of the present invention (including a bioreactor containing a cell culture chamber) can be provided in a microfluidic embodiment (i.e., wherein one or more channels or chambers have a size of about 1 μm to about 999 μm) and / or a macrofluidic embodiment (where all channels or chambers have a size of about 1 mm or more).

[0097] Fluid devices may also include additional fluid channels or compartments, gaskets or seals, mixing zones, valves, pumps, vents, channels for pressurized gases, electrical conductors, reagents, ports, and tubing required for specific designs. They may also include one or more control modules, transmitters, receivers, processors, memory chips, batteries, displays, buttons, controllers, motors, pneumatic actuators, antennas, and electrical connectors. Preferably, the devices contain only materials that are non-toxic to mammalian cells and compatible with sterilization through the use of alcohol and / or heat or other methods (e.g., exposure to gamma radiation or ethylene oxide gas).

[0098] For each method, at different temperature and pressure levels, equipment materials with appropriate chemical compatibility are selected. Furthermore, depending on the flow and pressure requirements of different functions, the pumps implemented in the equipment are selected, such as syringe pumps, peristaltic pumps, pressure pumps, and rotary pumps, with flow rates ranging from nL to mL and pressures ranging from 10 psi to 10,000 psi.

[0099] The system of the present invention may also include one or more sample solution reservoirs or orifices or other devices for introducing samples into the device at each inlet of the module, the inlets being in fluid communication with the inlet channel. Reservoirs and orifices for loading one or more samples into the fluid device of the present invention include, but are not limited to, syringes, cartridges, vials, eppendorf tubes, and cell culture materials (e.g., 96-well plates).

[0100] Where useful, the surface of the device can be made more hydrophilic, for example, by exposure to plasma, or by coating with one or more gels, chemically functionalized coatings, proteins, antibodies, proteoglycans, glycosaminoglycans, cytokines, or cells. The fluid device of the present invention preferably operates without fluid leakage under conditions and is capable of aseptic operation for days to weeks. The fluid device of the present invention also includes a sampling mechanism that allows fluid to be removed from the system for testing without introducing new materials or contaminants into the system.

[0101] In some aspects, at least a portion of the cell culture system includes disposable components, some or all of which may be housed within a non-disposable frame. In other aspects, all components of the system are disposable. Furthermore, in some embodiments, the cell culture system includes a sample tracking component for tracking and recording patient materials.

[0102] Various online process analysis tools (PAT) or miniaturized micro-total analysis systems (micro-TAS) are used to monitor product characteristics (e.g., purity and polymorphism) at at least one step in the manufacturing process, and sometimes multiple or all steps.

[0103] As described above, the cell culture system of the present invention is capable of controlling the direction and flow of fluids and entities within the system. The system of the present invention can use pressure-driven fluid control (e.g., using valves and pumps) to manipulate the flow of cells, reagents, etc., in one or more directions and / or manipulate the flow of cells, reagents, etc., into one or more channels of a fluid device. However, other methods, such as electroosmotic flow control, electrophoresis, and dielectrophoresis, can also be used alone or in combination with pumps and valves (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).

[0104] The system of the present invention may also include one or more control systems or be operatively connected to one or more control systems for controlling the movement of fluid through the system; monitoring and controlling various parameters within the system, such as temperature; and detecting the presence of cell-based immunotherapy products, product quality (direct or indirect), conversion rates, etc. The system may also be equipped with various types of software, such as advanced real-time process monitoring and control processes that allow feedback control, and processes that allow the integration and scaling of reaction and purification results obtained using the system.

[0105] In some embodiments, the system comprises a combination of microfluidic, nanofluidic, or macrofluidic modules and conduits that are interchangeable in terms of channel size, flow geometry, and interconnections between different modules of the device. Each module and conduit can be designed for a specific function. In one embodiment, all modules within the system are designed for cell culture and T-cell stimulation. In other embodiments, modules of 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 manufacture of immunotherapeutic products. Homogeneous and heterogeneous methods suitable for flow applications are considered. These processes are designed and optimized for starting materials and operating conditions (e.g., temperature, pressure, and flow rate) to minimize system clogging during flow.

[0106] A method for scaling up the plant is implemented by adding modular reactors in parallel or expanding modular channels, while maintaining a set of dimensionless parameters, characterized by each process constant and dimensional parameter being within upper and lower limits. During process integration and optimization, process decision variables (including temperature, pressure, flow rate, and channel size) are varied to achieve desired trade-offs between yield, purity, and flux. Throughout the optimization process, the aforementioned set of dimensionless parameters undergoes algebraic optimization with operational constraints. Operational constraints are the lower and upper limits of the decision variables. The objective function considers combinations of purity, yield, and flux operational variables. While the dimensionless parameters determine the steady-state quality of the plant, the start-up quality is also useful because it determines the time required to reach steady state, which in turn determines the plant's productivity and waste in hysteresis form. Start-up dynamics are analyzed using simulation and experiments, and the results are used to perform start-up optimization by implementing real-time feedback control.

[0107] As described above, various aspects of this disclosure, such as controlling fluid movement through the system and monitoring and controlling various parameters, can be performed using any type of computing device (e.g., a computer or programmable logic controller (PLC) including a processor, such as a central processing unit) or any combination of computing devices (where each device performs at least a portion of the process or method). In some embodiments, the systems and methods described herein can be performed using handheld devices, such as smart tablets, smartphones, or dedicated devices manufactured for the system.

[0108] The methods of this disclosure can be performed using software, hardware, firmware, hardwired, or any combination thereof. Features implementing the functionality can also be physically located in various locations, including being distributed such that different parts of the functionality are 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, for example, utilizing wireless or wired connections).

[0109] For example, processors suitable for executing computer programs include general-purpose microprocessors and special-purpose microprocessors, as well as any one or more processors in any type of digital computer. Typically, the processor receives instructions and data from read-only memory and / or random access memory. The components of a computer are a processor for executing instructions and one or more memory devices for storing instructions and data. Typically, a computer will also include, or be operatively coupled to, receiving data from and / or transferring data to one or more non-transitory mass storage devices for storing data, such as magnetic disks, magneto-optical disks, or optical disks. Suitable information carriers for embodying computer program instructions and data include all forms of non-volatile memory, including, for example, semiconductor memory devices (e.g., EPROM, EEPROM, 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., CDs and DVDs). The processor and memory may be supplemented by or incorporated into special-purpose logic circuitry.

[0110] To provide interaction with the user, the subjects described herein can be implemented on a computer with I / O devices, such as CRT, LCD, LED, or projection devices for displaying information to the user, as well as input or output devices. For example, keyboards and pointing devices (e.g., mice or trackballs) can be used by the user to provide input to the computer. Other types of devices can also be used to provide interaction with the user. 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.

[0111] The subject matter described herein may include backend components (e.g., data servers), middleware components (e.g., application servers), or frontend components (e.g., client computers with graphical user interfaces or web browsers through which users can interact with implementations of the subject matter described herein), or any combination of such backend, middleware, and frontend components. Components of the system may be interconnected via a network through digital data communication (e.g., communication networks) of any form or medium. Examples of communication networks include cellular networks (e.g., 3G or 4G), local area networks (LANs), and wide area networks (WANs), such as the Internet.

[0112] 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 for controlling the operation of a data processing device (e.g., a programmable processor, computer, or multiple computers). Computer programs (also referred to as programs, software, software applications, applications, 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 standalone programs or as modules, components, subroutines, or other units suitable for 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.

[0113] 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, in a single file dedicated to the program in question, or in multiple coordinating files (e.g., a file that stores portions of one or more modules, subroutines, or code). Computer programs can be deployed to execute on one computer or multiple computers at one site, or distributed across multiple sites and interconnected via a communication network.

[0114] Files can be digital files, such as those stored on hard drives, solid-state drives, CDs, or other tangible, non-transitory media. Files can be sent from one device to another over a network (e.g., as data packets sent from a server to a client via a network interface card, modem, wireless card, etc.).

[0115] Writing a document according to embodiments of the invention involves, for example, transforming a tangible, non-transitory computer-readable medium by adding, removing, or rearranging particles (e.g., where electrostatic charge or dipole moment enters a magnetization mode performed by a read / write head), and these modes then represent a new configuration of information about objective physical phenomena that are desired and useful to the user. In some embodiments, writing involves a physical transformation of the material in the tangible, non-transitory computer-readable medium (e.g., where certain optical properties allow an optical read / write device to then read the new, useful information configuration, e.g., recording a CD-ROM). In some embodiments, writing a document includes transforming a physical flash memory device, such as a NAND flash memory device, and storing information by transforming the physical elements in a memory cell array made of floating gate transistors. Methods of writing documents are well known in the art and can be invoked manually or automatically, for example, by a program or by a save command in software or a write command from a programming language.

[0116] Suitable computing devices typically include mass storage, at least one graphical user interface, at least one display device, and typically include communication between devices. Mass storage describes a computer-readable medium, i.e., a computer storage medium. Computer storage media can include volatile, non-volatile, removable, and non-removable media implemented in any method or technology for storing 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 technologies, CD-ROM, Digital Universal Optical Disc (DVD) or other optical storage, cassette tape, magnetic tape, 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 can be accessed by a computing device.

[0117] As will be recognized by those skilled in the art when it is necessary or most suitable to carry out the methods of the present invention, the computer system or machine used in the embodiments of the present invention may include one or more processors (e.g., a central processing unit (CPU) and / or a graphics processing unit (GPU)), main memory and static memory, which communicate with each other via a bus.

[0118] exist Figure 10In the exemplary embodiment shown, system 600 may include computer 649 (e.g., laptop computer, desktop computer, or tablet computer). Computer 649 may be configured to communicate via network 609. Computer 649 includes one or more processors 659 and memory 663, as well as input / output mechanism 654. When the method of the present invention adopts a client / server architecture, the operation of the method of the present invention may be performed using server 613, which includes one or more processors 621 and memory 629, capable of obtaining data, instructions, etc., or providing results through interface module 625 or providing results as file 617. Server 613 may be connected to network 609 via computer 649 or terminal 667, or server 613 may be directly connected to terminal 667, including one or more processors 675 and memory 679, as well as input / output mechanism 671.

[0119] The system 600 or machine according to an exemplary embodiment of the present invention may further include a video display unit (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)) for any one of I / O 649, 637, or 671. The 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 touchscreen, 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 wireless network card, or a cellular modem.

[0120] 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 methods or functions described herein are stored. During execution by a computer system, the software may also reside wholly or at least partially in main memory and / or a processor, which also constitute the machine-readable medium. The software may also be transmitted or received over a network via a network interface device.

[0121] References

[0122] Throughout this disclosure, references and citations have been made to other literature (e.g., patents, patent applications, patent publications, journals, books, papers, web content). All of these references are incorporated herein by reference in their entirety for all purposes.

[0123] Equivalent scheme

[0124] Although the invention has been described in conjunction with certain embodiments, those skilled in the art, upon reading the foregoing specification, will be able to make various changes, equivalent substitutions and other modifications to the compositions and methods presented herein.

Claims

1. A cell culture system comprising: A cell culture chamber, comprising: At least two inlets, arranged to guide one or more liquids into the cell culture chamber in a symmetrical flow substantially parallel to the bottom surface; and At least one outlet is arranged to guide one or more liquids to exit the cell culture chamber in a substantially perpendicular flow relative to the bottom surface, wherein at least one of the one or more outlets is located on the top surface of the cell culture chamber; and Central processing unit, including memory containing instructions executable by the central processing unit to cause the system to: Receive data including the size of the cell culture chamber as the first input; Receive data containing 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 as a second input; and Based on the first input data and the second input data, the perfusion rate of the perfusion fluid to be introduced into the cell culture chamber is calculated, the perfusion rate maximizing the probability that the first cell type and the second cell type come into contact with each other in the cell culture chamber, wherein the calculation includes a particle interaction-based model of the interaction between the first cell type and the second cell type and a model based on kinetic parameters.

2. The cell culture system of claim 1, wherein, The first cell type is peripheral blood mononuclear cells, and the second cell type is dendritic cells.

3. The cell culture system of claim 1, further comprising one or more pumps operably connected to one or more perfusion fluid reservoirs and operably connected to the central processing unit, wherein the central processing unit controls the perfusion rate of the perfusion fluid by controlling the one or more pumps.

4. The cell culture system as described in claim 1, wherein, The cell culture chamber includes one or more sensors operatively connected to the cell culture chamber in a manner that enables the sensors to measure one or more parameters within the cell culture chamber.

5. The cell culture system of claim 1, wherein... The bottom surface contains a first material for attaching cells; and The top surface and at least one or more sidewalls comprise a breathable second material.

6. The system of claim 5, wherein, The movement of the fluid along the vertical flow path makes the fluid velocity insufficient to overcome the sedimentation rate of the cells in the cell culture chamber.

7. The system of claim 4, wherein, The one or more parameters are selected from the group consisting of the following parameters: pH, dissolved oxygen, total biomass, cell diameter, glucose concentration, lactate concentration, and cell metabolite concentration.

8. The system of claim 7, wherein, The central processing unit is communicatively connected to the one or more sensors and is configured to adjust the operating state of the one or more pumps based on the measured one or more parameters.

9. The system as claimed in claim 1, wherein, Calculating the perfusion rate involves applying one or more computational models to one or more interactions of one or more cell types within a cell culture chamber.

10. The system of claim 9, wherein, The one or more computational models include one or more of particle interaction-based models and kinetic parameter-based models of the interaction between T cells and antigen-presenting cells.

11. The system of claim 10, wherein, The system is configured to automatically calculate and monitor the optimal perfusion rate to maximize the probability of T cells and antigen-presenting cells coming into contact with each other in the cell culture chamber.

Images