Dendritic cell generation apparatus and method

Abstract

A cell culture cartridge is provided that includes a plurality of regions that are geometrically configured to provide fluid flow symmetrical to each of the plurality of regions to avoid flow dead zones within each of the plurality of regions. In some embodiments, at least eight inlets are provided, where the inlets are located at each corner of the cell culture cassette. In some embodiments, the shared outlet is located on the top surface of the cell culture cartridge.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201980089180.6, application date November 11, 2019, and invention name: Dendritic Cell Generation Device and Method.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. application serial number 16 / 192,062, filed November 15, 2018, the contents of which are incorporated by reference in their entirety.

[0004] Government support

[0005] This invention was made with government support under Grant No. 1819306 awarded by the National Science Foundation. The government has certain rights in this invention. Technical Field

[0006] The present invention generally relates to cell culture chambers and methods of using the same. Background Art

[0007] Cell-based cancer immunotherapy is a method for treating cancer using immune-active cells comprising dendritic cells (DCs). Since DCs cannot be harvested in sufficient quantities by other means, they are typically generated by differentiation of monocytes extracted from peripheral blood. However, generating clinically relevant amounts of monocyte-derived dendritic cells for therapeutic purposes may be challenging. Conventional generation techniques (e.g., standard well plates and T-flask cultures) involve cumbersome processes with multiple manual steps that expose cell cultures to the external environment and require trained technicians.

[0008] Conventional generation techniques have numerous safety and contamination issues, such as mix-up and misidentification of patient samples, exposure to unknown contaminants inside laminar flow hoods (e.g., particles and bacteria / fungi resistant to standard sterilization techniques (e.g., 70% ethanol), and incidental contact of cultures with a sterile environment. Furthermore, scaling up artificial DC generation techniques is generally not feasible without adding more culture vessels to the workflow. Automated systems that continuously perfuse fresh culture medium into culture vessels and simultaneously remove spent medium are an alternative to conventional manual generation techniques.

[0009] Although automated systems generally have fewer safety and contamination issues than conventional techniques, there are scale-up and other issues with automated systems. For example, many commercially available automated systems cannot be expanded for research or clinical production of DCs. Moreover, automated systems suffer from uneven flow or flow dead spots within the cell culture vessel. Flow dead spots (dead zones) are areas in the cell culture vessel where uniform flow cannot be maintained when providing fresh culture medium and removing depleted culture medium, thereby affecting the generation of DCs. Summary of the Invention

[0010] The present invention provides an automated cell culture cassette and system for generating dendritic cells with uniform, symmetrical flow within the cell culture cassette. Various aspects of the present invention are achieved by designing the cell cassette chamber to have multiple zones, each geometrically configured to provide symmetrical fluid flow and avoid dead zones within the cell culture chamber. The geometric design provides uniform flow and avoids dead zones or dead spots in the flow. In this way, the present invention provides an optimal and more efficient method for automating the generation of dendritic cells (DCs).

[0011] In certain embodiments, the cell culture chamber comprises a plurality of corners. An inlet is located at each of the plurality of corners, while an outlet is located on the top surface of the cell culture chamber. The placement of the inlet of the cell culture chamber allows for symmetrical fluid flow paths within the cell culture chamber. In some instances, the cell culture chamber comprises an octagon having eight corners, each corner comprising an inlet. The outlet is located at the center of the top surface of the cell culture chamber.

[0012] The cell culture chamber also includes various technical features that allow automation of manual processes, thereby greatly reducing user intervention in the process, thereby significantly reducing the risk of contamination. The cell culture chamber allows culture medium and cytokines to be perfused into the chamber, thereby allowing more consistent levels to be maintained. The realization of consistent nutrient and cytokine levels is crucial for ensuring effective cell culture and processing, and is therefore crucial for predictable and effective scale expansion. In addition, a vertical flow path is provided when the fluid leaves the chamber, which ensures that DCs, antigen-specific T cells and other cells involved in the culture process remain in the chamber during perfusion.

[0013] In addition, the present invention includes additional features designed to achieve uniform flow. As an example, the cell culture cassette further includes one or more posts extending between the bottom surface and the top surface. As another example, the bottom surface includes one or more notches at the periphery of the bottom surface. Some embodiments of the present invention further include one or more stopcocks operably connected to the cell culture chamber.

[0014] The cell culture chamber of the present invention can be manufactured to include a bottom surface made of a material to which cells adhere. In some embodiments, cells do not adhere to the bottom surface material. In some embodiments, the material of the bottom surface is treated with air or oxygen plasma in a glow discharge or corona discharge. In some embodiments, the material of the bottom surface is modified with proteins or polyamino acids, such as fibronectin, laminin, and collagen. In some embodiments, the material of the bottom surface is modified with proteins or polyamino acids, such as fibronectin, laminin, and collagen. The cell culture chamber is made of any suitable material. In certain instances, one or more materials are selected from the group consisting of polystyrene and acrylate. In some embodiments of the present invention, the cell culture box is transparent. In some embodiments, the height of the cell culture box is 10 times or more smaller than the largest of the length or width dimensions.

[0015] The cell culture cassette and system may further include one or more stopcocks. One or more stopcocks may be operably coupled to the cell culture chamber. When attached to the filter, the stopcock on the cassette allows for air exchange when inoculating the cassette with a cell solution or harvesting the cassette. When attached to a Luer-activated transfer valve, the stopcock allows for the aseptic transfer of differentiation medium to fill the inlet bottle and remove waste from the outlet bottle. This arrangement allows the tubing and cassette system to remain sterile from setup to harvest without compromising the system's sterile seal.

[0016] In addition, the present invention provides a completely enclosed sterile iDC generation system for producing immature DC (iDC) at a clinical scale, thereby effectively eliminating the need for a large number of well plates (or T bottles / bags), ensuring a sterile and particle-free culture system, and reducing the technician time required to maintain cell culture. The present invention is an automated cell culture system for aseptically generating a therapeutically relevant number of iDCs in a single cell culture box. The system is also capable of further processing iDCs to mature them by adding maturation reagents and stimulated by adding one or more antigens to the cell culture chamber. The cell culture system includes a cell culture box, which includes multiple areas that are geometrically configured to provide symmetrical fluid flow channels in the cell culture chamber and avoid flow dead zones in the cell culture chamber. The cell culture system further includes one or more pumps operably associated with the cell culture chamber. In some embodiments, the peristaltic pump continuously perfuses fresh culture medium into the culture container at a specified flow rate of, for example, 8 μL / min per inlet, while removing the exhausted culture medium into a waste reservoir. Transfer of fresh medium, removal of spent medium, cell plating, and iDC harvesting were performed aseptically.

[0017] In some embodiments, the cell culture system further includes at least one fluid connector configured to couple the cell culture chamber fluid to a second container, which may be a second cell culture chamber. For example, the cell culture chambers are configured to be fluidically connected to each other so that the cells are concentrated into a smaller volume (if such concentration is desired for maturation and antigen stimulation (also referred to as pulse) steps). When using this system to stimulate DC with T cells, these T cells can be automatically transferred between each chamber to allow further culture and amplification of T cells in a new cell culture chamber. In some embodiments, transfer is achieved by introducing a gas flow into the first cell culture chamber to transfer the supernatant containing the first cell product to the second cell culture chamber via a fluid connector.

[0018] In certain aspects, the cell culture chamber of the exemplary embodiment provides expansion and stimulation of T cells using antigen-presenting cells from the same patient to provide therapeutic T cell products that can mobilize the patient's own immune system in a manner that selectively targets the patient's tumor. Compared to conventional protocols, these cell culture systems and methods greatly reduce the number of manual steps. In this way, the risk of contamination is greatly reduced, and the robustness and repeatability of the manufacturing technology are greatly increased, which are two key considerations for safely and reliably manufacturing therapeutic products (e.g., personalized T cell therapies capable of precise targeting).

[0019] In other aspects, the cell culture chamber further comprises one or more fluid reservoirs operably coupled to the one or more pumps. The fluid reservoirs are configured to supply culture medium comprising nutrients and cytokines to the chamber.

[0020] In some embodiments, the present invention further comprises one or more sensors operably coupled to the cell culture cassette. The one or more sensors can measure any suitable parameter. In one example, the one or more sensors measure one or more parameters selected from the group consisting of: pH, dissolved oxygen, total biomass, cell diameter, glucose concentration, lactate concentration, and cellular metabolite concentration.

[0021] The cell culture chamber can further include a central processing unit (CPU). The CPU can be communicatively coupled 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 one embodiment employing a flow generating mechanism rather than a pump (e.g., an electrohydrodynamic mechanism), the central processing unit can change the operating state of the flow generating mechanism to adjust the flow rate of the first cell product based on the one or more parameters.

[0022] In one embodiment, a central processing unit executes instructions to cause the system to receive first input data including the size of a cell culture chamber. Then, second input data is received, the 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. Based on the first and second inputs, a perfusion rate of the perfusion fluid to be introduced into the cell culture chamber is calculated. The calculated perfusion rate maximizes a probability that the first cell type and the second cell type come into contact with each other within the cell culture chamber. The first cell type is peripheral blood mononuclear cells, and the second cell type is dendritic cells.

[0023] In one embodiment, the system further comprises one or more pumps operably coupled to the one or more perfusion fluid reservoirs and operably coupled 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.

[0024] In certain embodiments, the present invention provides a method for culturing dendritic cells. The method includes providing a cell culture cassette. The cell culture cassette includes multiple regions that are geometrically configured to provide symmetrical fluid flow with respect to each of the multiple regions to avoid dead zones within each of the multiple regions. In some embodiments, the cell culture cassette includes a cell culture chamber that includes multiple corners, an inlet located at each of the multiple corners, and an outlet located on the top surface of the cell culture chamber. The fluid flows symmetrically through the cell culture chamber.

[0025] Monocytes are seeded in a cell culture chamber to generate dendritic cells by continuously perfusing culture medium into the cell culture chamber via an inlet and removing spent culture medium into a waste reservoir via an outlet. In some embodiments, the method further comprises harvesting the dendritic cells, and the harvesting of the cells comprises a cooling chamber.

[0026] In one embodiment, the method further comprises transferring the immature dendritic cells to a second cartridge, wherein the second cartridge is smaller than the cell culture cartridge. The immature dendritic cells undergo maturation and antigen pulse in the second cartridge. In one embodiment, maturation and antigen pulse can be performed in the cell cartridge without using a second cartridge.

[0027] In some embodiments, the methods of the invention further comprise maturing the dendritic cells and pulsing the cells with an antigen.

[0028] 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 external to the incubator and are operably coupled to the cell culture chamber within the incubator.

[0029] In some aspects, at least a portion of the system includes disposable components, some or all of which can be housed within a non-disposable frame. In other aspects, all components of the system are disposable. Additionally, in some embodiments, the system includes a sample tracking component for tracking and recording patient materials.

[0030] The systems and methods are designed so that any number of additional cassettes or cell culture chambers can be provided. In some embodiments, the system comprises two or more cell culture cassettes for generating T cells.

[0031] In certain embodiments, the systems of the present invention have the ability to automatically calculate and set a desired perfusion rate for a perfusion fluid given various inputs, such as 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 one exemplary arrangement, a cell culture system is provided that includes 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 as a first input data including the size of the cell culture chamber, receives 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 calculates, based on the first and second inputs, a perfusion rate for 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 come into 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 An embodiment of a cell culture chamber of a cell culture cassette according to the present invention is shown.

[0033] Figure 2 Shown is a front view of the cell culture cassette and system.

[0034] Figure 3 Shown is a top view of the cell culture cassette and system.

[0035] Figure 4 Shown is a left side view of the cell culture cassette and system.

[0036] Figure 5 Shown is a right side view of the cell culture cassette and system.

[0037] Figure 6 One embodiment of the system 100 of the present invention is shown.

[0038] Figure 7 An embodiment of the invention is shown having two cassettes.

[0039] Figure 8 One embodiment of the present invention is shown showing the transfer from a smaller cassette to an infusion bag.

[0040] Figure 9 Disposable and non-disposable components of the present invention are shown.

[0041] Figure 10 One embodiment of an EDEN automated fluid system is shown.

[0042] Figure 11 A cell culture cassette design showing the cell culture cassette flow channels.

[0043] Figure 12 Cell culture cassette design showing the polystyrene surface (shaded) at the base of the cassette where cells reside.

[0044] Figure 13 Cell culture cassette design showing streamlines resulting from perfusion within the cassette.

[0045] Figure 14 Cell culture cassette design showing gauge pressure due to perfusion within the cassette.

[0046] Figure 15 Perfusion of cytokines into the cell culture cassette is shown.

[0047] Figure 16 Shown are the phenotypes of iDCs generated from cell culture cassettes and 6-well plates differentiated from MO for 6 days.

[0048] Figure 17 The phenotypes of IDCs and mDCs from cell culture cassettes are shown. IDCs were generated in a cell culture cassette and then seeded into the cell culture system of the present invention for 1 or 3 days of maturation. The labels above the figures indicate the gate from which the figures are derived.

[0049] Figure 18 An exemplary method for producing an immunotherapeutic product according to one embodiment of the present invention is shown.

[0050] Figure 19 A system of the present invention according to certain embodiments is described.

[0051] Figure 20 The MicroDEN N3iDC phenotype is shown. Data from experiments N1-N2 are Figure 28 and 30 Shown in.

[0052] Figure 21 The phenotype of N3 iDCs in a 6-well plate is shown. Data from experiments N1-N2 are shown in Figure 29 and 31 Shown in.

[0053] Figure 22 The differentiation data of iDCs generated in the cell culture system and 6-well plates of the present invention are shown, particularly with respect to the cassette of the present invention (39.7 cm 2 ) or 6-well plate (9.5cm 2 iDCs were harvested and normalized for surface area (μg / well).

[0054] Figure 23 Differentiation data for iDCs generated in the cell culture system of the present invention and in 6-well plates are shown, particularly the average harvested iDCs normalized to the surface area of ​​the cassette or 6-well plate of the present invention. Data are presented as mean ± standard deviation of the indicated experiments. Data are listed in Tables 1-3.

[0055] Figure 24 Differentiation data of iDCs generated in the cell culture system of the present invention and in 6-well plates are shown, particularly at 200k-600k MO / cm for each experiment. 2 IDC yield at different inoculation densities.

[0056] Figure 25 Differentiation data of iDCs generated in the cell culture system of the present invention and in 6-well plates are shown, particularly at 200k-600k MO / cm for each experiment. 2 Average iDC yields at seeding densities. Data are presented as mean ± standard deviation for the indicated experiments. Data are listed in Tables 1-3.

[0057] Figure 26 Shown in 200k-600k MO / cm 2 Allogeneic functional assay proliferation statistics of iDC generated in the cell culture system of the present invention or 6-well plates at differentiation seeding density. The legend indicates the iDC source (cell culture system of the present invention or 6-well plates) and the number of iDCs co-cultured with 1 million allogeneic T cells from a single donor. The data are listed and shown in Tables 4-6.

[0058] Figure 27The allogeneic functional assay proliferation histogram of experiment N1 is shown. The columns indicate the MO seeding density of iDC generation in the cell culture system of the present invention or 6-well plates. The rows indicate the iDC source (cell culture system of the present invention or 6-well plates) and the number of iDCs co-cultured with 1 million allogeneic T cells for 5 days. The green vertical line indicates the position of the unstimulated control peak of the staining, which is also the position of the undivided cells. The thicker curve indicates the overall fit, while the thinner curve indicates the single T cell generation. The histograms of experiments N2-N3 are shown in Figure 32 and 33 The unstimulated T cell control was shown in Figures 34-36 Shown in.

[0059] Figure 28 The phenotype of N1iDC, a cell culture system of the present invention, is shown.

[0060] Figure 29 The phenotype of N1iDCs in a 6-well plate is shown.

[0061] Figure 30 The phenotype of N2iDC in the cell culture system of the present invention is shown.

[0062] Figure 31 N2iDC phenotypes in 6-well plates are shown.

[0063] Figure 32 Experiment N2: Allogeneic functional assay histogram is shown. T cell control Figures 34-36 Shown in.

[0064] Figure 33 Experiment N3: Allogeneic functional assay histogram is shown. T cell control Figures 34-36 Shown in.

[0065] Figure 34 Shown is an allogeneic functional assay T cell control for N1. One million T cells (same donor for N1-N3) were cultured for 5 days in the absence of iDCs.

[0066] Figure 35 An allogeneic functional assay T cell control for N2 is shown. One million T cells (same donor as N1-N3) were cultured for 5 days in the absence of iDCs.

[0067] Figure 36 Allogeneic functional assay T cell control for N3 is shown. One million T cells (same donor for N1-N3) were cultured for 5 days in the absence of iDCs. DETAILED DESCRIPTION

[0068] Dendritic cells (DCs) are antigen-presenting cells that reside in the circulating blood and other parts of the body. DCs are a key component of the immune system. The presentation of antigens by these cells drives the mobilization of the immune system against various infections and the development and maintenance of immunological memory. Vaccines specifically designed to target DCs have recently been developed for various diseases, including cancer, and personalized DC vaccines are currently being developed for infectious diseases, cancer, and transplant rejection. Within these disease categories, cell-based therapies using in vitro expanded T cells represent another frontier that has recently made significant progress. DCs are the most potent antigen-presenting cells (APCs) and are the only APCs capable of inducing naive T cells. DCs play a key role in the in vivo expansion of T cells and can be used to expand T cells in vitro. From a mechanistic perspective, DCs are an essential part of studying human responses that are important for protective immunity against cancer and infectious diseases, as well as for preventing autoimmunity and transplant rejection.

[0069] Despite the crucial role of DCs in both clinical and basic research settings, methods for obtaining these cells from individuals remain an underdeveloped and inefficient process. Because DCs are present in very low concentrations in blood (<1%), these cells must be generated from monocytes, involving laborious static culture and stimulation with cytokines (IL-4 and GM-CSF) contained in culture medium. In particular, multiple manual steps are required to obtain sufficient numbers of DCs for vaccine development, T cell therapy, or mechanistic studies from patient-derived whole blood samples, leukapheresis products, or peripheral blood mononuclear cells (PBMCs). For studies involving one or two conditions or a single blood draw, this is cumbersome even when scaling to dozens of samples, requiring extensive human resources and manual steps. Given the larger-scale current and projected uses of these cells (e.g., in autologous DC-based cell therapies and vaccines), conventional DC generation methods impose an exceptionally large burden, primarily in terms of the efficiency and reliability of the manufacturing process, as well as supply and labor costs.

[0070] The present invention provides an automated cell culture box and system for generating dendritic cells, which have uniform, symmetrical flow in the cell culture box. A cell culture box is provided, which includes a cell culture chamber formed between the top surface of the cell culture box and the bottom surface of the cell culture box. The cell culture chamber includes multiple regions, which are geometrically configured to provide symmetrical fluid flow channels in the cell culture chamber and avoid dead spots or dead zones in the cell culture chamber. Flow dead spots (dead zones) are regions in the cell culture container that do not maintain uniform flow when providing fresh culture medium and removing depleted culture medium, thereby affecting the generation of DCs. By providing multiple regions in the cell culture box, the present invention provides symmetrical flow channels without fluid flow dead spots or dead zones. In addition, the cell culture chamber of an exemplary embodiment provides the feature of allowing uniform flow of fresh culture medium and removal of depleted culture medium.

[0071] Figure 1 A top view of a cell culture chamber 1000 is shown. The cell culture chamber 1000 is formed between the top and bottom surfaces of a cell culture cassette. A plurality of fluid flow inlets 1130 are provided in the chamber. Figure 1 1135. The embodiment shown in FIG1000 includes eight inlets, one of which is shown by dotted line 1135. Inlet 1130 is arranged at each corner 1120 of the cell culture box. Inlet 1130 can be located on the top surface of the cell culture box. An outlet 1150 is located at the center of the cell culture chamber on the top surface of the cell culture box. The chamber 1000 includes multiple areas 1160, which are geometrically configured to provide symmetrical fluid flow channels in the cell culture chamber 1000. Recesses 1110 are arranged on the outer periphery of the cell culture chamber and help avoid dead zones or dead spots (wherein the fluid flow in the cell culture chamber is uneven). Post 1140 extends from the bottom surface to the top surface so that the top surface does not sag or bend and does not generate increased pressure in the chamber. Figure 1 The embodiment shown in is an exemplary non-limiting embodiment of the present invention. Other non-limiting embodiments can include different numbers of inlets. In some examples of non-limiting embodiments, box according to the present invention can include 2 inlets, 5 inlets, 10 inlets, 13 inlets, 14 inlets, 20 inlets, 30 inlets and 100 inlets. Non-limiting embodiments can further include different numbers of angles. In some examples of non-limiting embodiments, box according to the present invention can include 5 angles, 10 angles, 17 angles, 25 angles, 50 angles and 100 angles.

[0072] In the present invention, the symmetry of fluid flow is achieved in the cell culture box. For example, the box contains separate areas, and each separate area is the space between two fluid inlets. Figure 1As shown in , each region has a triangular base that tapers in the middle, wherein each region is symmetrical with other regions. In some instances, the number of fluid inlets in this box can be greater than or less than 8 fluid inlets. In a preferred embodiment, this box is divided into 8 separate regions (region / zone) with an inlet and a shared outlet (center). This ensures that the entire box is filled with fresh differentiation culture medium and does not form a flow dead zone or dead spot. In addition, 4 triangular recesses 1110 are located around the periphery to avoid the flow dead zone or dead spot that may occur in these regions. This box includes 8 posts to support the top surface of the cell culture box, which can be constructed of poly (methyl methacrylate) (PMMA or acrylate). In the absence of a post, the PMMA top surface would sag, and because culture medium would support the top of the box, pressure in the box would accumulate.

[0073] The box can be constructed from any suitable material. In some examples, the box is constructed from polystyrene, acrylic, or a combination thereof. As an example, the base or bottom surface comprises polystyrene, while the top and side surfaces are acrylic. As another example, for high-volume manufacturing, the box can be made entirely of polystyrene.

[0074] In an exemplary embodiment, the bottom surface includes polystyrene and / or acrylate. One benefit of using polystyrene for the bottom surface to be cultivated is that this material has played a useful role in the process of generating dendritic cells by PBMC. Specifically, the polystyrene surface can be used to enrich monocytes from the heterogeneous suspension of PBMC. This is the first step in the culture process for generating DC by differentiating monocytes via cultivating in a culture medium containing, for example, IL4 and GM-CSF. From the perspective of biological processes, it is very valuable to use the same polystyrene surface to produce dendritic cells throughout a T cell stimulation cycle because it eliminates a large number of transfer steps that would otherwise be necessary, thereby allowing a closed system to be used for DC stimulation therapeutic T cell manufacturing.

[0075] In addition, any suitable material process can be carried out on this box.In certain embodiments, the bottom polystyrene surface can be modified to promote cell adhesion.For example, the bottom polystyrene surface can be processed with air or oxygen plasma, which is also referred to as glow discharge or corona discharge.For example, the bottom polystyrene surface can be modified with known proteins or polyamino acids that promote cell adhesion, and these proteins or polyamino acids include but are not limited to fibronectin, laminin and collagen.

[0076] The surface area of ​​the bottom surface can be comparable to that of conventional well plates, such as 6-well plates and 24-well plates (9.5 cm 2 and 1.9cm 2 ) or T bottle (25cm2 to 225cm 2 It will also be appreciated that the surface area can be smaller or even much larger than conventional well plates (e.g., comparable in surface area to standard cell culture dishes and flasks), e.g., with a surface area of ​​approximately 2.0 cm 2 and about 500cm 2 , 100.0, 125.0, 150.0, 175.0, 200.0, 400.0, 500.0 cm 2 and any surface area in between.

[0077] The surfaces of the cell culture box can be joined together using any method known in the art, such as mechanical fastening, adhesive and solvent bonding and welding. However, given that the 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 cell culture chambers. Therefore, in certain embodiments, the surfaces are joined without the use of adhesives. In one embodiment, all surfaces of the cell culture chamber (e.g., bottom wall, side walls, and top wall) comprise a first material (e.g., polystyrene) and are joined together using ultrasonic welding. It should be understood that the above configurations are merely examples, and other configurations for joining surfaces are also contemplated embodiments of the present invention.

[0078] The height of one or more cell culture chambers can be different. For example, but not limitation, the exemplary range of the cell culture chamber height comprises any height from 0.5mm to 100mm, 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, 100.0mm or larger height or any height therebetween. In certain embodiments, the height of the chamber can be suitable with the liquid height in the culture carried out conventionally in 6 orifice plates and 24 orifice plates, for example, between 2 and 6mm, and the volume capacity is about 0.8mL to 6mL. In other embodiments, the cell culture chamber will be larger in size, for example between 10 mm and 50 mm, with a culture surface of approximately 50 cm 2 .

[0079] In some embodiments of the present invention, the cartridge is optically clear (clear / transparent). This optical transparency, combined with appropriately isolated fluid ports, allows the user to view cells in any vertical plane within the cartridge. Figure 2-5 As shown in , embodiments of the present invention include an optically clear cell culture cassette. Figure 2 Shown is a front view of the cell culture cassette and system. Figure 3 Shown is a top view of the cell culture cassette and system. Figure 4 Shown is a left side view of the cell culture cassette and system. Figure 5 Shown is a right side view of the cell culture cassette and system.

[0080] In addition, if Figure 2-5 As shown in , stopcock can be placed on box or on reservoir bottle. Especially, stopcock is placed on the specific port on box, and each stopcock has specific function. Placement is specific to each function, and work has been carried out to determine the best position, to ensure that process is successful and workflow is simple. For example, the stopcock in front is used for inoculation and results, and the Luer activated valve (LAV) at the stopcock top allows syringe to be connected aseptically. The stopcock on the right front side is used for inoculation and results (adding cold buffer to wash), and when cell solution is inoculated into box, the air in box will flow out by the filter at this stopcock. As another example, the stopcock on the left rear side is used for results, and when removing cell solution, the air in box inside will flow into box. When adding liquid or removing liquid from box, the filter attached to the stopcock avoids pressure or vacuum accumulation in box.

[0081] In the present invention, LAV can be used on the bottle to add and / or remove culture medium. Traditionally, LAV is sold and marketed for anesthesia and IV lines. Therefore, using LAV to add or remove culture medium is different from traditional use.

[0082] Computational fluid dynamics (CFD) aided in the design of the current EDEN box. In particular, CFD aided in the design of the box size, the placement of the posts, and the placement and size of the triangular recesses.

[0083] In some embodiments, a perfusion flow rate of 8 μL / min can be maintained. Because this is the same perfusion rate as a cell culture system (e.g., MicroDEN), linear scale-up of MicroDENs can be achieved using a system according to the present invention (EDEN). Each of the eight subsections of an EDEN cartridge is slightly larger than a single MicroDEN cartridge, so the effect of perfusion on cells in an EDEN should be similar to that in a MicroDEN. Thus, the present invention allows for easy scaling of MicroDEN experiments to EDEN without unknown factors (e.g., different fluid flow rates).

[0084] Figure 6 An embodiment of a system 100 of the present invention is shown. A peristaltic pump 110 is provided. The pump 110 is used to pump fluid into or out of a cell culture cassette 120. The cell culture cassette 120 has a bottom surface 125 to which cells adhere. In other embodiments, the cells do not adhere to the bottom surface. The cell culture cassette 120 has eight fluid inlets 145 arranged at the corners of the cell culture cassette 120. A fluid outlet 135 is arranged at the center of the cell culture cassette 120. Connecting tubing 140 connects the fluid inlets to a differentiation medium reservoir (perfusion source) 180 containing differentiation medium 182. The differentiation medium reservoir 180 contains differentiation medium 182 to be pumped into the cell culture cassette 120. Connecting tubing 140 also connects the fluid outlet 135 to a waste reservoir 184. Spent medium is pumped out of the cell culture cassette 120 through the outlet 135 and into the waste reservoir 184. The covers 170 and 175 on the differentiation medium reservoir 180 and the waste reservoir 184 are non-removable, thereby maintaining a sterile system. In other embodiments, the covers 170 and 175 are removable.

[0085] The stopcocks on the reservoir bottles 180 and 184 and / or the LAVs 160 and 165 allow for the aseptic transfer of differentiation medium to fill the inlet bottle and remove waste from the outlet bottle. A console 190 provides a designated space for arranging the aforementioned components and also provides a display / user interface 192, connections 194, and an on / off switch 196.

[0086] Figure 7 An embodiment of the present invention is shown with two cassettes. A cell culture cassette 200 is provided for monocyte to dendritic cell differentiation. A smaller cassette 220 is provided for maturation and antigen pulsing. In other embodiments, maturation and antigen pulsing can be performed in the main cell culture cassette without the use of a second cassette.

[0087] Figure 8 One embodiment of the present invention is shown with a smaller cassette 320 for maturation and antigen pulsing. The smaller cassette 320 is fluidly connected to an infusion bag 330 containing the final product transferred from the smaller cassette 320.

[0088] Figure 9 Disposable and non-disposable components of the present invention are shown. EDEN console 410 is non-disposable and has a length L. In this embodiment, length L is 14 inches. Smaller cartridge 420 is used for maturation and antigen pulsing. Connecting tubing 430 connects the inlet and outlet to the reservoir and cartridge. Smaller cartridge 420 and connecting tubing 430 are single-use and disposable.

[0089] Figure 10An embodiment of the EDEN automated fluidics system is shown. The EDEN system generates monocyte-derived iDCs while continuously perfusing fresh differentiation medium into the cell culture chamber.

[0090] Figure 11-14 A cell culture cassette design according to an embodiment of the present invention is shown. Figure 11 A cell culture cassette design showing the cell culture cassette flow channels. Figure 12 Cell culture cassette design showing the polystyrene surface (shaded) at the base of the cassette where cells reside. Figure 13 Cell culture cassette design showing streamlines resulting from perfusion within the cassette. Figure 14 Cell culture cassette design showing gauge pressure due to perfusion within the cassette.

[0091] Figure 15 The perfusion of cytokines into a cell culture cassette is shown. In this example, the cassette is initially filled with water (culture medium) without cytokines. Cytokines are perfused into the cassette at 1.16 mol / m3 (IL-4) at eight inlet ports, flow through the cassette driven by the perfusion, and exit through the outlet port at the center. In practice, the cell culture cassette is filled with culture medium containing cytokines. This data is acquired at the lower or bottom surface of the flow channel, as shown in FIG. Figure 12 As shown in .

[0092] Figure 16 The phenotypes of iDCs generated from cell culture cassettes and 6-well plates differentiated from MO for 6 days are shown. The labels above the figures indicate the gate from which the graphs are derived.

[0093] Figure 17 The phenotypes of IDCs and mDCs from cell culture cassettes are shown. IDCs were generated in a cell culture cassette and then seeded into the cell culture system of the present invention for 1 or 3 days of maturation. The labels above the figures indicate the gate from which the figures are derived.

[0094] Figure 18 An exemplary method for producing an immunotherapeutic product according to one embodiment of the present invention is shown. Figure 18An overview of a method for generating a cell-based immunotherapy product using the system described herein is shown. In short, the steps for generating a cell therapy product according to certain embodiments of the present invention include co-culturing stimulated antigen-presenting cells with cells containing T cells in a bioreactor containing a cell culture chamber. During the culture period, a supernatant containing an amplified therapeutic T cell product is generated. In some aspects, in order to produce an amount of antigen-specific T cells sufficient to elicit a therapeutic response in a patient, the T cells must be cultured additionally in one or more additional cell culture chambers. To achieve this additional culture, the supernatant must be transferred from the culture chamber in which the supernatant is generated to a subsequent cell culture chamber containing a fresh supply of antigen-presenting cells. The transfer of supernatant between cell culture chambers can involve introducing a gas flow into a first cell culture chamber that transfers the supernatant containing the first cell product through a fluid connector and into a new cell culture chamber. In addition, during each culture step, a perfusion fluid containing, for example, culture medium and cytokines can be perfused into these chambers. In some aspects, the perfusion fluid flows through these chambers along a vertical flow path to ensure that the cells remain within the chamber during the culture period. The only manual step involved in using the system of the present invention is providing the system with one or more subsequent cell culture cassettes, each containing a cell culture chamber, wherein each chamber contains a new batch of antigen peptide-pulsed autologous antigen-presenting cells. The use of gas-facilitated transfer may also involve manual steps to manipulate the system settings, but does not compromise the sterility of the system.

[0095] In certain embodiments of the present invention, cells are harvested. This is typically accomplished by injecting a cold buffer into the cartridge. In some embodiments of the present invention, a Peltier device can be integrated beneath the cartridge to cool the cartridge to a temperature between about 20°C and about 30°C, allowing release without diluting the cells in a larger volume of fluid.

[0096] In certain embodiments, the dendritic cells generated in the octagonal box can be moved into a smaller box. When manufacturing an immunotherapy based on dendritic cells, the immature dendritic cells generated by the differentiation (first step) of the mononuclear cell will typically undergo additional steps (maturation and antigen pulse). Routinely, this is accomplished by carrying out the first step in multiple bottles or wells and then merging the immature dendritic cells into a single bottle or well. This type of concentration / consolidation allows for less use and subsequent waste of reagents for maturation and antigen pulse, and these reagents are expensive. In the present invention, the immature dendritic cells from the octagonal box are transferred to a smaller box to carry out maturation and antigen pulse when the first step is carried out. In some embodiments of the present invention, maturation and antigen pulse are carried out in the main cell culture box, and there is no need to use a second box.

[0097] Some embodiments of the present invention can use a Luer-activated valve (LAV) for inoculating and harvesting mononuclear cells (MO) and immature dendritic cells (iDC), respectively. This improves the workflow so that cell solution is not lost during inoculation / harvesting. A syringe can be connected to the LAV, for example in a MicroDEN system. The syringe can be used as a funnel to add MO solution (for inoculation) and cold buffer (for harvesting). Due to the turbulence caused by the "pipetting" action, the syringe can be used to "pipet" up and down to de-adhere and resuspend the iDC. This up and down "pipetting" is actually pushing and pulling the syringe plunger.

[0098] An exemplary arrangement is now described in which the systems and methods of the present invention utilize one or more cell culture cassettes, each containing a cell culture chamber that is configured to be fluidically coupled to one another for processing of the patient's cell material, thereby producing an immunotherapy product. It will be appreciated that in certain embodiments, the cell culture cassette is provided in a closed environment. By adding modules (e.g., cell culture cassettes) to allow serial and / or parallel processing, the scale-up of this exemplary embodiment will be within the knowledge of the skilled person. The skilled person will also appreciate that, based on the product to be produced, a different or alternative arrangement may be desired.

[0099] In certain embodiments, one or more pumps are operably connected to the cell culture chamber to perfuse the perfusion medium into the cell culture chamber. The perfusion medium includes any suitable culture medium. In some embodiments, the perfusion medium is a differentiation medium. The cell culture box may also include one or more fluid reservoirs. The fluid reservoir is in fluid communication with the cell culture chamber and can be operably connected to one or more pumps. One or more tubes for connecting the fluid reservoir to the pump and the cell culture chamber are also provided. In some aspects, one or more pumps are configured to pump the fluid from the fluid reservoir through the cell culture chamber and into a waste collection reservoir. In one embodiment, the fluid moves from the fluid reservoir to the pump via a pipeline and moves into the cell culture chamber via an inlet, then moves out of the cell culture chamber via an outlet, then moves through a pipeline and into a waste collection reservoir.

[0100] In certain embodiments, the fluid reservoir and / or waste collection reservoir can each be provided as one or more capped bottles contained within or fluidically coupled to the cell culture chamber. Each reservoir can include an inlet port and an outlet port, or an outlet and a vent that is fluidically coupled to the inlet of one or more cell culture chambers. In certain aspects, for example, a Luer connector and a silicone gasket cut to fit around the Luer connector can be used to prevent leakage through one or both of the inlet and outlet ports.

[0101] In certain embodiments, one or more cell culture cassettes are sized and configured to fit within an incubator so that the process will be performed within the incubator. The conditions within the incubator include a continuous temperature of 37°C and 95-100% relative humidity. Therefore, given that materials (including fluids and biologicals) tend to expand under such conditions, the selected materials must have the integrity to withstand these conditions. In addition, in some cases, the conditions within the incubator remain stable, and automated recording of the temperature can provide information on whether temperature fluctuations are related to any anomalies in the reactions being performed in the incubator. Therefore, any power supply should not alter the environment within the incubator. For example, certain pumps generate heat.

[0102] In one embodiment, the pump is separately housed with the cell culture box, but is still operably connected to the cell culture box fluid. In another embodiment, the pump is directly attached to the cell culture box and is located in the incubator, but is calorimetrically or operably connected to a radiator and / or fan to radiate heat. No matter how the configuration is, the pump is operably connected to the cell culture box, then connected to the cell culture chamber. For example, the small-scale culture system for endothelial cell cultivation with the on-board reagent storage and perfusion function realized by the disposable peristaltic pump on the plate, and the larger-scale culture system for generating dendritic cells by monocytes using the chamber with a polystyrene bottom surface) can be found in International Patent Application No. PCT / US2016 / 040042 and PCT / US2016 / 60701, and both patent applications are incorporated herein by reference in their entirety.

[0103] The present system can also include a heater for controlling the temperature of the cell culture reservoir and the optional fluid reservoir. In this configuration, an incubator is not required, and the system can operate autonomously using only a power source. If the system does not have a heater, it can operate inside the cell culture incubator. Some embodiments of the present invention include a carbon dioxide (CO2) environment for the culture medium buffer.

[0104] In other aspects, the cell culture chamber includes one or more sensors (not shown) that are operably coupled to the cell culture chamber. The sensor may be capable of measuring any suitable parameter. For example, the sensor may be capable of measuring 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, the one or more sensors may 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 to all chambers in the system. In other embodiments, the one or more sensors are coupled to all cell culture chambers in the system. In a system with multiple chambers that are operably coupled to one or more sensors, the sensors may be the same in each chamber to which they are coupled, they may all be different, or some sensors may be the same, while some may be different. In some aspects, the one or more sensors are operably coupled to a computer system having a central processing unit for performing instructions so that parameters can be automatically monitored and adjusted. Additional details regarding a computer system for implementing the method of the present invention using a cell culture chamber are provided below.

[0105] In certain embodiments, the cell culture chamber has an inlet and an outlet, both of which can be used to couple the chamber to one or more other container fluids via a fluid connector. In certain embodiments, other containers include one or more other cell culture chambers. System 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 more than one hundred cell culture chambers, which are configured to be connected to each other in series fluid to produce immunotherapy products. Alternatively or additionally, one or more cell culture chambers can be arranged in parallel to each other to allow one or more individual immunotherapy products to be produced at a time. In a preferred embodiment, the cell culture chambers of the cell culture box are connected via aseptic connection.

[0106] The system and some or all of its components can be designed using CAD software and then transferred to a laser cutter, allowing the plastic to be cut into specified sizes and shapes. Various connections (e.g., inlets and outlets) can be formed by laser cutting through-holes, which can then be manually tapped to provide threads for receiving male Luer fittings. Later, fluid can be introduced into the system by connecting a Luer adapter to a blunt dispensing needle and pushing the tubing onto the blunt needle portion. Additional details on the construction of the fluid system assembly can be found in International Patent Application Nos. PCT / US2016 / 040042 and PCT / US2016 / 60701, both of which are incorporated herein by reference in their entirety. The system and some or all of its components can also be produced using injection molding.

[0107] The above description focuses on system components and various possible configurations. The following description focuses on the process performed using an exemplary embodiment system of the present invention. In order to stimulate and amplify antigen-specific T cells, the process begins by co-culturing 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 comprise peripheral blood mononuclear cells (PBMCs), and the APCs comprise DCs. The cells containing T cells and APCs can be co-cultured at a dilution ratio of about 1000:1 to 1:1000 (such as, for example, but not limited to, 1000:1, 900:1, 800:1, 700:1, 600:1, 500:1, 400:1, 300:1, 200:1, 100:1, 75:1, 50:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1: In one embodiment, the T cells are provided to the cell culture chamber at a ratio (T cell-containing cells:APCs) of 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 aspect, a ratio of 10:1 is preferred.

[0108] In order to trigger the stimulation and expansion of T cells through the interaction of APCs with cells containing T cells, the APCs need to be stimulated. This can be accomplished by using one or more stimulatory molecules. In certain embodiments, the stimulatory molecules are non-tumor specific. In other embodiments, the stimulatory molecules are tumor specific. For example, the stimulatory molecules can be selected from one or more characteristics of an individual tumor, such as different antigenic peptides. In some embodiments, the stimulatory molecules are preferably added only at the beginning of the culture period. The stimulatory molecules can be added for only about a few minutes, an hour, several hours, or longer. In a preferred embodiment, the stimulatory molecules are added over a period of about an hour.

[0109] In the culture period of two kinds of cell materials, the supernatant containing lighter non-adhesive T cells is formed, while heavier mature APC (for example, dendritic cells) adheres to or resides on the bottom surface. In those embodiments in which DC is used as APC, the T cells amplified must be extracted from the cell culture chamber within seven days because the primary DC can not be kept in culture for more than seven days. Therefore, if the other amplification of the desired T cell is desired, dendritic cells need to be re-supplied. It should also be understood that the cell culture using a batch of dendritic cells can continue to be less than any time of seven days. For example, the cell culture can be less than any time of one minute to seven days, wherein the duration of the culture depends on the desired stimulation level.

[0110] In an 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 DC 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 needed 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 an adequate supply of T cells.

[0111] Co-culture of APCs and T cells is performed in a culture medium. Exemplary culture media include, but are not limited to, RPMI medium and DC culture medium sold under the trademark CELLGENIX by CellGenix Inc. (Portsmouth, New Hampshire). Any other suitable culture medium known in the art may be used according to embodiments of the present invention. Cytokines (e.g., IL-4 and GM-CSF) may also be added to the culture medium.

[0112] In one embodiment, the perfusion of culture medium and cytokine can be provided to the cell mixture in one or more cell culture chambers to assist in the formation of cell-based immunotherapy products. In the experimental scheme based on plate by DC stimulating T cells, the culture volume of about 2mL is maintained from the beginning, wherein cytokine is infused twice in each 7-day stimulation period. The main advantage of perfusion is that the consistent local concentration distribution of culture medium and cytokine can be maintained, and compared with the scheme based on plate of prior art, this ensures a larger yield and the potential ability of accelerating the process of monocyte differentiation to DC. However, the combination of adhesion (DC) and non-adhesion (T cell) type and DC's high sensitivity to mechanical force have challenged the stimulation and amplification of antigen-specific T cells, especially in terms of the flow of liquid by cell culture chamber. Therefore, in those embodiments in which culture medium and cytokine are provided via perfusion, the system of the present invention must be able to supply nutrition and cytokine to cells without removing cells from cell culture box, while also considering the shear sensitivity of some antigen presenting cells (e.g., DC). Basically, some embodiments of the present invention system and method are intended to optimize the retention of the autocrine / paracrine signal that is conducive to T cell proliferation, while refreshing growth factors and keeping the minimum physical stimulation of DC. To address this issue, both the direction and rate of perfusion flow through the cell culture chamber must be considered.For example, some embodiments of the present invention may include medium flow arrangements other than unidirectional flow, such as a countercurrent medium flow arrangement.

[0113] In certain aspects, the fluid flow rate is maintained below the sedimentation rate of the antigen-presenting cells. Thus, the antigen-presenting cells will remain within the cell culture chamber due to their mass. In other words, the antigen-presenting cells will sink toward the bottom of the cell culture chamber and thus remain in the cell culture chamber.

[0114] In other aspects, the multiple inlets and outlets of the cell culture chamber are arranged to move fluid (e.g., perfusion fluid) 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 in the range of 2-10 mL / min. A configuration with symmetrical inflow and vertical outflow prevents cells from leaving the chamber. Figure 1 As shown in , certain embodiments of the cell culture cassette of the present invention have eight inlets and one vertical outlet.

[0115] Despite Figure 1 The chamber is shown as having eight inlets and one vertical outlet, but any number of inlets and outlets may be provided, as long as the fluid flowing out of the chamber flows vertically out of the top of the chamber and into the symmetrical fluid channels within the chamber. For example, the chamber may have any of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more perfusion fluid inlets and / or outlets.

[0116] In certain aspects, culture medium is perfused at specific time points within the time period during which 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 per week. In other aspects, culture medium is perfused continuously during the culture period. Continuous perfusion helps maintain a nearly constant culture volume throughout the process.

[0117] In some aspects, cytokines are infused at one or more points during the culture period (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 those embodiments, continuous perfusion helps to maintain a consistent local concentration distribution of cytokines, which can help to ensure greater productivity and has the ability to increase the rate of stimulation and amplification of T cells compared to static cell culture methods.

[0118] 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 may affect the efficacy of T stimulation. For example, in recent work designed for monocyte to DC proliferation culture chambers, as described in International Patent Application Nos. PCT / US2016 / 040042 and PCT / US2016 / 60701, a flow rate corresponding to 0.1 dyn / cm 2 The perfusion rate of culture medium at a wall shear stress level was determined to produce DCs that are phenotypically equivalent to those generated using conventional 6-well or 24-well plate-based protocols. Therefore, by measuring one or more of the above-mentioned phenotypic and functional metrics during the culture period, the impact of one or more perfusion parameters on efficacy can be monitored, allowing for appropriate adjustments.

[0119] According to certain aspects, stimulation efficacy can be assessed at any point during the culture period (preferably after seven days). Phenotypic and functional metrics can be used to assess efficacy. For example, a directed cell counting method can be used to calculate the number of cells (expansion multiples). Cell phenotype (including antigen specificity assessed by tetramer staining) can be characterized by flow cytometry. Functional assays can also be used to assess the ability of amplified T cells to recognize antigen-loaded target cells and autologous tumor cells. The results can be compared with the results of 24-well plates and G- DC-based T cell stimulation was measured in a novel format.

[0120] As mentioned above, because some APC (for example, dendritic cells) can not survive more than seven days in culture, certain embodiments of the present invention relate to multiple cycles of T cell stimulation using more than one cell culture box of semi-batch configuration. Each cycle is carried out with freshly generated autologous antigen-presenting cells. In certain embodiments, for each stimulation cycle, antigen-presenting cells are pulsed with the same antigen group. In other embodiments, one or more stimulation cycles are carried out using different antigen groups.

[0121] Typically, multi-cycle T cell stimulation involves culturing cells in a first cell culture chamber in a manner to generate a supernatant including 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.

[0122] For example, in certain embodiments, a cell culture system is provided that includes a cell culture chamber and a central processing unit (CPU) including a memory containing instructions executable by the CPU. In certain aspects, the instructions cause the system to receive, as a first input, data including a size of the cell culture chamber, and, 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 to calculate, based on the first and second inputs, a perfusion rate for a perfusion fluid to be introduced into the cell culture chamber that maximizes a probability that the first cell type and the second cell type come into contact with each other within the cell culture chamber.

[0123] In some aspects, the system further comprises one or more pumps operably coupled to the one or more perfusion fluid reservoirs and operably coupled to the central processing unit, such that the central processing unit also controls the perfusion rate of the perfusion fluid by controlling the one or more pumps.

[0124] In certain embodiments, the systems and methods of the present invention utilize modules (e.g., cell culture boxes containing cell culture chambers, etc., and systems thereof) that are fluidically coupled to each other to process individual cellular materials to produce immunotherapy products. The systems or devices of the present invention are modular and can be fluidically coupled in series and / or in parallel (i.e., fluid flows from one device to another) to other similar devices, and can also be configured to be physically stacked on each other or physically arranged in related devices (e.g., incubators). The modular design of the system particularly allows modules to be flexibly switched on and off depending on the desired process to be included in the system.

[0125] The fluidic devices of the present invention (including cell culture cassettes comprising cell culture chambers) can be provided as microfluidic embodiments (i.e., wherein one or more channels or chambers have dimensions ranging from about 1 μm to about 999 μm) or macrofluidic embodiments (wherein all channels or chambers have dimensions of about 1 mm or greater), or both.

[0126] Fluidic devices may further comprise additional fluid channels or compartments, gaskets or seals, mixing regions, valves, pumps, vents, pressurized gas channels, electrical conductors, reagents, ports, and tubing as required for a particular design. They may also contain one or more control modules, transmitters, receivers, processors, memory chips, batteries, displays, buttons, controls, motors, pneumatic actuators, antennas, electrical connectors, etc. These devices preferably contain only materials that are non-toxic to mammalian cells and are compatible with sterilization by use of alcohol and / or heat or other means (e.g., exposure to gamma radiation or ethylene oxide gas).

[0127] The materials of the equipment are selected for their appropriate chemical compatibility at the various temperature and pressure levels specific to each process. Furthermore, the pumps implemented in the device (e.g., syringe, peristaltic, pressure, and rotary pumps) are selected with flow rates ranging from nL to mL and pressures ranging from 10 to 10,000 psi, depending on the flow and pressure requirements of the various functions.

[0128] The system of the present invention may also include one or more sample solution reservoirs or wells or other devices for introducing samples into the device at each inlet of the module, these inlets being in fluid communication with the inlet channel. The reservoirs and wells for loading one or more samples onto the fluidic device of the present invention include, but are not limited to, syringes, boxes, vials, microcentrifuge tubes, and cell culture materials (e.g., 96-well plates).

[0129] 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. In one embodiment, the cell culture cassette and system are centrally located. The device is single-use, meaning that patient material is processed in bags, tubing, and cell culture vessels used only for a single patient's cells.

[0130] The fluidic device of the present invention preferably has no fluid leakage under operating conditions and can be operated aseptically for days to weeks.The fluidic 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.

[0131] In some aspects, at least a portion of the cell culture system includes disposable components, some or all of which can be housed within a non-disposable frame or console. 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. In one embodiment, the cell culture cassette and system are centrally located. The device is single-use, meaning that patient material is processed in bags, tubing, and cell culture vessels that are used only for a single patient's cells.

[0132] Various in-line process analytical 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, and sometimes multiple or all steps, during the manufacturing process.

[0133] As described above, the cell culture system of the present invention can control the direction and flow of the fluid and entity in the system. The system of the present invention can use pressure-driven flow control, such as using valves and pumps to manipulate cells, reagents, etc. to flow in one or more directions and / or flow into one or more channels of a fluid device. However, other methods can also be used alone, or other methods can be used in combination with pumps and valves, such as electroosmotic flow control, electrophoresis and dielectrophoresis (Fulwyer, " Science (Science)", 156,910 (1974); Li and Harrison, " Analytical Chemistry (Analytical Chemistry)", 69,1564 (1997); Fiedler et al., " Analytical Chemistry (Analytical Chemistry)", 70,1909-1915 (1998); and U.S. Patent No. 5,656,155, each of which is incorporated herein by reference.

[0134] The systems of the present invention may also include or be operably coupled to one or more control systems for controlling the movement of fluids through the system; monitoring and controlling various parameters within the system (e.g., temperature); and detecting the presence of cell-based immunotherapy products, product quantity (directly or indirectly), conversion rates, etc. The systems may also be equipped with various software, such as advanced real-time process monitoring and control processes (thereby allowing feedback control) and processes that allow for integration and scale-up given the reaction and purification results obtained using the system.

[0135] In certain embodiments, the system comprises a combination of microfluid, millifluid or macrofluid module and pipeline, which is interchangeable in terms of the interconnection between the different modules of channel size, flow geometry and device. Each module and pipeline can be designed for specific functions. In one embodiment, all modules in the system are designed for cell culture and T cell stimulation. In other embodiments, the modules in the system are designed for different functions, such as tissue processing, dendritic cell generation, cell culture, concentration and / or purification, all of which are integrated for continuous manufacture of immunotherapy products. Homogeneous and heterogeneous processes are all considered to be suitable for flow applications. For starting material and operating conditions (such as, temperature, pressure and flow rate), these processes have been designed and optimized, so that during the flow process, they are not prone to blocking the system.

[0136] A method for scaling up a plant by adding modular reactors in parallel or expanding modular channels while holding a set of dimensionless parameters specific to each process constant and maintaining dimensional parameters within upper and lower 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 set of dimensionless parameters is algebraically optimized with operational constraints. The operational constraints are the upper and lower limits of the decision variables. The objective function considers the combination of purity, yield, and throughput operational variables. While the dimensionless parameters determine the steady-state quality of the plant, the startup quality of the plant is also useful because it determines the time required to reach steady state and, in turn, the productivity and waste of the plant in the form of lag time. Startup dynamics are analyzed using simulations and experiments, and the results are used to perform startup optimization by implementing real-time feedback control.

[0137] Any type of computing device (e.g., a computer or programmable logic controller (PLC) containing a processor, such as a central processing unit) or any combination of computing devices (where each device performs at least a portion of a process or method) can be used to perform the various aspects of the present disclosure described herein, such as controlling the movement of fluids through the system as described above and monitoring and controlling various parameters. In some embodiments, the systems and methods described herein can be performed using a handheld device, such as a smart tablet, a smart phone, or a dedicated device produced for the system.

[0138] The methods of the present disclosure may be performed using software, hardware, firmware, hardwiring, or any combination thereof. Features implementing the functionality may also be physically located at various locations, including being distributed such that portions of the functionality are implemented at different physical locations (e.g., the imaging device is located in one room, while the host workstation is located in another room, or in separate buildings, e.g., via wireless or wired connections).

[0139] For example, processors suitable for executing computer programs include general-purpose and special-purpose microprocessors, and any one or more processors of any type of digital computer. Typically, the processor will receive instructions and data from a read-only memory or a random access memory or both. The elements of a computer are a processor for executing instructions and one or more memory devices for storing instructions and data. Typically, a computer will also include one or more non-transitory mass storage devices or be operably connected to receive data from it or transmit data to it (or both), and the non-transitory mass storage device is used to store data (e.g., a magnetic disk, a magneto-optical disk, or an optical disk). In some embodiments, the sensors on the system send process data to a central data collection unit located outside the incubator via Bluetooth.

[0140] In some embodiments, data is sent directly to the cloud rather than to a physical storage device. Information carriers suitable for embodying computer program instructions and data include all forms of nonvolatile memory, including, for example, semiconductor memory devices (e.g., EPROM, EEPROM, solid-state drives (SSD), and flash memory devices); magnetic disks (e.g., internal hard disks or removable disks); magneto-optical disks; and optical disks (e.g., CDs and DVDs). The processor and memory can be supplemented by, or incorporated in, special purpose logic circuitry.

[0141] To provide for interaction with a user, the subject matter described herein can be implemented on a computer having I / O devices (e.g., a CRT, LCD, LED, or projection device for displaying information to the user) and input or output devices (e.g., a keyboard and pointing device (e.g., a mouse or trackball) through which the user can provide input to the computer). Other types of devices can also be used to provide for interaction with the user. For example, the 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, voice, or tactile input.

[0142] The subject matter described herein can be implemented in a computing system that includes a back-end component (e.g., a data server), a middleware component (e.g., an application server), or a front-end component (e.g., a client computer having a graphical user interface or a web browser through which a user can interact with an embodiment of the subject matter described herein), or any combination of such back-end, middleware, and front-end components. The components of the system can be interconnected through a network using any form or medium of digital data communication (e.g., a communication network). Examples of communication networks include cellular networks (e.g., 3G or 4G), local area networks (LANs), and wide area networks (WANs) (e.g., the Internet).

[0143] 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., a non-transitory computer-readable medium) to be executed by a data processing device (e.g., a programmable processor, a computer, or multiple computers) or to control the operation thereof. A computer program (also referred to as a program, software, software application, app, macro, or code) can be written in any form of programming language, including compiled or interpreted languages ​​(e.g., C, C++, Perl), and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. The systems and methods of the present invention can include instructions written in any suitable programming language known in the art, including but not limited to C, C++, Perl, Java, ActiveX, HTML5, Visual Basic, or JavaScript.

[0144] A computer program does not necessarily correspond to a file. A program may be stored in a file or portion of a file that stores other programs or data, in a single file dedicated to the program, or in multiple coordinated files (e.g., files storing one or more modules, subroutines, or code portions). A computer program may be deployed to execute on one computer or on multiple computers at one location, or distributed across multiple locations and interconnected by a communications network.

[0145] A file can be a digital file stored on, for example, 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., when a data packet is sent from a server to a client, such as via a network interface card, modem, wireless card, etc.).

[0146] Writing a file according to an embodiment of the present invention involves converting a tangible, non-transitory computer-readable medium, for example, by adding, removing, or rearranging particles (e.g., placing a net charge or dipole moment into a magnetization pattern of a read / write head), whereby these patterns then represent a new collocation of information about an objective physical phenomenon desired and useful to the user. In some embodiments, writing involves physical conversion of the material in a tangible, non-transitory computer-readable medium (e.g., having certain optical properties so that an optical read / write device can then read the new and useful collocation of information, such as burning a CD-ROM). In some embodiments, writing a file comprises converting a physical flash memory device (e.g., a NAND flash memory device) and storing the information by converting physical elements in a memory cell array made of floating-gate transistors. Methods for writing a file are well known in the art and can be invoked manually or automatically, for example, by a program, a save command from software, or a write command from a programming language.

[0147] Suitable computing devices typically include mass storage, at least one graphical user interface, at least one display device, and typically include communication between the devices. Mass storage represents a type of computer-readable medium, i.e., computer storage media. Computer storage media can include volatile, non-volatile, removable, and non-removable media implemented in any method or technology for storing information (e.g., 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 the desired information and can be accessed by the computing device.

[0148] As those skilled in the art will recognize as necessary or most suitable for carrying out the methods of the present invention, the computer system or machine employed in embodiments of the present invention may include one or more processors (e.g., a central processing unit (CPU), a graphics processing unit (GPU), or both), main memory, and static memory, which communicate with each other via a bus.

[0149] exist Figure 19 In an exemplary embodiment shown in FIG, system 600 may include a computer 649 (e.g., a laptop, desktop, or tablet). Computer 649 may be configured to communicate across a network 609. Computer 649 includes one or more processors 659, memory 663, and input / output mechanisms 654. Where the method of the present invention employs a client / server architecture, the operations of the method of the present invention may be performed using a server 613, which includes one or more processors 621 and memory 629 and is capable of obtaining data, instructions, etc. or providing results via an interface module 625 or as a file 617. Server 613 may be accessed via network 609 via computer 649 or terminal 667, or server 613 may be directly connected to terminal 667 and include one or more processors 675, memory 679, and input / output mechanisms 671.

[0150] For any of I / O 649, 637, or 671, 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)). 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 generating 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).

[0151] The memory 663, 679 or 629 according to an exemplary embodiment of the present invention may include a machine-readable medium on which is stored one or more sets of instructions (e.g., software) embodying any one or more of the methods or functions described herein. During execution of the software by the computer system, the software may also reside completely or at least partially in the main memory and / or in the processor, the main memory and the processor also constituting the machine-readable medium. The software may be further transmitted or received over a network via a network interface device.

[0152] Effect of monocyte seeding density on DC generation

[0153] Dendritic cells (DC) are becoming increasingly important in research and clinical use, but obtaining sufficient numbers of DCs is an increasing challenge. The effect of mononuclear cell (MO) seeding density on the generation of monocyte-derived immature DCs (iDCs) was studied in a perfusion-based culture system and 6-well plates of the present invention. Cell surface markers and the ability of iDCs to induce allogeneic T cell proliferation were investigated. The data showed a strong relationship between iDC phenotype (specifically CD80 / 83 / 86 expression) and T cell proliferation. In the 200k-600k MO / cm studied, the 100k MO / cm 2 iDCs generated by the cell culture system of the present invention were shown to be superior to those generated in well plates in inducing T cell proliferation across a range of seeding densities. This can be attributed to the perfusion in the cell culture system of the present invention, which continuously supplies fresh differentiation medium to the differentiation MO while removing depleted medium and toxic byproducts of cellular respiration. At lower MO seeding densities, the cell culture system of the present invention produced fewer iDCs than well plates on a standardized basis, but a comparable number of iDCs were generated at a 600k MO seeding density. The results demonstrate that the cell culture system of the present invention can generate larger numbers of iDCs with less manual effort than standard well plate cultures, and that iDCs generated by the cell culture system of the present invention have a greater ability to induce T cell proliferation.

[0154] Dendritic cells are antigen-presenting cells that reside primarily in solid tissues and play a crucial role in activating both adaptive and humoral immune responses. The main function of dendritic cells (DCs) is to identify and capture foreign antigens that pose a threat to the human body, process them into smaller peptides, and then present these peptides to naive T or B cells. After antigen presentation, DCs can activate CD4 + Helper T cells and CD8 + Cytotoxic T cells and naive and memory B cells. In addition, DC activates natural killer (NK) and natural killer T (NKT) cells. In view of its ability to elicit responses from a variety of immune cells, DC is an attractive target for therapeutic manipulation. Vaccines containing antigen-loaded DCs for in vivo activation and amplification of T and B cells have been used for infectious disease treatment, and they are being developed into specialized targeting cancer cells in multiple clinical and preclinical research trials. In addition, DC plays a vital role in the emerging field of T cell-based immunotherapy and has been used to amplify activated T cells in vitro.

[0155] Direct isolation of patient-specific DCs is a challenge because they reside in solid tissues and are present in very low concentrations (<1%) in human blood. Therefore, DCs are typically generated ex vivo from monocytes or stem cell precursors that can be easily isolated from circulating blood. To generate DCs for therapeutic manipulation, the standard approach is to isolate peripheral blood mononuclear cells (PBMCs) from peripheral blood leukapheresis products, enrich for CD14+ monocytes (MOs) by plastic adherence, elutriation, or by magnetic bead positive selection, and then culture with IL-4 and GM-CSF for 5-10 days. This method is traditionally performed in well plates and T-flasks, requiring extensive manual manipulation and involving replenishing differentiation medium throughout the culture duration. The counts of immature DCs (iDCs) generated range from approximately 9 to 15 million in 6-well plates and 6 to 20 million in T-175 flasks; a single dose of therapy may require approximately 150 million DCs. Scaling up current DC generation technologies to produce the relevant numbers of DCs needed for clinical immunotherapy is challenging due to the extensive manual manipulation required, the large number of well plates / T-flasks, and the significant labor costs. Furthermore, identifying the optimal monocyte seeding density is a significant challenge associated with the scale-up of the MO to iDC differentiation process, and this information is difficult to determine from the literature.

[0156] To overcome the above-mentioned shortcomings of artificial DC generation, a closed, automated cell culture system was designed to generate DCs from monocytes (which functions similarly to iDCs generated in well plates). The cell culture system of the present invention combines a closed tubing and a cell culture cassette system that continuously perfuses fresh differentiation medium into the cassette while simultaneously removing spent medium and waste products (CO2 and lactate). This setup also reduces the manual steps required for priming and replenishing the culture medium.

[0157] The effects of MO seeding density on iDC yield, phenotype, and function were investigated. Three seeding densities were studied in the cell culture system of the present invention and in a 6-well plate control: 200,000 MO / cm 2 、400,000MO / cm 2 and 600,000MO / cm 2 . As the iDC seeding density increases, the IDC yield increases in the cell culture system of the present invention and remains constant in the well plate. At 200k and 400k MO seeding densities, the IDC yield in the cell culture system of the present invention is lower than that in the well plate, and is comparable to the well plate at 600k MO seeding density. The IDC phenotype shows a strong dependence on MO seeding density in the cell culture system of the present invention, wherein the iDC generated from the low seeding density induces greater T cell proliferation. The iDC generated by the cell culture system of the present invention is phenotypically similar to the iDC generated in 6-well plates (thereby confirming previous studies), and this shows that the iDC generated by the cell culture system of the present invention induces greater T cell proliferation compared to the iDC generated by the well plate.

[0158] A total of three identical experiments (N1, N2, N3) were systematically carried out to evaluate the performance of the cell culture system of the present invention. Each experiment consists of three boxes of the present invention (one box for each inoculation density) and one or two 6-well plates (two to three holes for each inoculation density). For each individual experiment (N1, N2 or N3), MO from a single donor was used, thereby requiring a total of three donors to generate iDC. All allogeneic functional assays were performed using T cells from the fourth donor. CellGenix GMP DC culture medium was used as the basal culture medium for MO differentiation into iDC. The culture medium was supplemented with 1% penicillin-streptomycin (Gibco 15140122) and 350U / mL preclinical IL-4 and GM-CSF (CellGenix) to generate iDC.

[0159] Each MO-to-iDC differentiation experiment lasted 6 days. All experiments were performed in a standard cell culture incubator maintained at 37°C and 5% CO2 with near-saturated humidity. All work was performed under sterile conditions in a laminar flow hood. All cell counts were performed using a Countess II automated cell counter.

[0160] Cell culture system of the present invention

[0161] Experiments with the cell culture system of the present invention were performed using the automated cell culture system previously described by our group. The polystyrene surface of each cell culture box was treated with O plasma at 50W for 90 seconds. The polystyrene surface area of ​​each box was 39.7 cm and the volume was approximately 12.7 mL. Throughout the experiment, the perfusion rate of differentiation medium was 8.0 μL / min. For each experiment, one box of the present invention was used for each seeding density, for a total of three boxes. This required two pumping instruments because each instrument controlled two boxes. Previous experiments showed that during the experiment, no cells were removed from the box by perfusion of the culture medium at 8.0 μL / min, as verified by centrifugation of the effluent and subsequent cell counting.

[0162] At day 0, differentiation medium was added to the inlet bottle to allow the medium to perfuse for 3 days. On day 3, when the inlet bottle was almost empty, fresh differentiation medium was added to allow the medium to perfuse for another 3 days. On day 3, the outflow was removed from the outlet bottle. On day 6, cells were collected by aspirating the cell culture medium and washing the cassette twice with cold DPBS (4°C). Adherent cells remaining after the two DPBS washes were not collected.

[0163] 6-well plate

[0164] Corning Costar 6-well plates (3516) were used as controls for each experiment. A volume of 2.5 mL of differentiation medium was added to each well. 3.0 mL of DPBS was filled into the empty wells to minimize evaporation. On the 3rd day, 1 mL of fresh differentiation medium was added to each well. On the 6th day, cells were harvested by aspirating the culture medium and washing each well 2 times with cold DPBS (4°C). The adherent cells remaining after the two DPBS washes were not collected.

[0165] PBMC and MO / T cell isolation

[0166] Four units of whole blood (approximately 470 mL / unit) drawn from normal healthy donors were purchased from StemExpress. Blood was collected via venipuncture and processed on the same day. PBMCs were isolated using Ficoll density gradient medium and suspended in CryoStor CS10 cryopreservation medium (at a concentration of approximately 50 million PBMCs / mL). The cells were cooled at -80°C in Mr. Frosty containers for 12-24 hours and then transferred to a cryogenic LN2 storage facility for at least one week before being revived. MO or T cells were enriched from PBMCs using Miltenyi Biotec CD 14 or CD3 microbeads and passed through two LS columns.

[0167] Allogeneic T cell function assay

[0168] Allogeneic T cell function assay was performed in Corning Costar 24-well plates (3526) using 0, 200,000 (200k) or 500,000 (500k) iDC generated in MicroDEN or 6-well plates. Each well contained 1 million allogeneic T cells. CellGenix DCM was used as basal culture medium and supplemented with 1% penicillin-streptomycin (Gibco15140122) and 5% human AB serum (Sigma Aldrich H4522). T cells were stained with CellTrace far red to evaluate proliferation. The 24-well plates were covered with foil to avoid light and then placed in an incubator (37°C and 5% CO2) for 5 days. During harvest, the cell solution was aspirated and the wells were washed twice with cold DPBS (4°C) to collect residual cells. Data were analyzed using FCS Express6Flow software.

[0169] Immunophenotyping

[0170] Flow cytometry was performed on an ACEA Biosciences NovoCyte instrument with 488 nm and 640 nm laser lines and four fluorescence channels. After viability staining and before antibody staining, cells were stained 10 minutes with Fc Block (BD Biosciences 564220). Group A tested viability (live / dead fixed green; Invitrogen L34970), CD209 / DC-SIGN (R&D Systems FAB161P100), CD14 (Abeam abl573 12) and CD45 (R&D Systems FAB1430A). Group B tested CD80 (BD Biosciences 557226), CD83 (BD ​​Biosciences 556855), CD86 (BD Biosciences 561128) and CD45. Group C tested viability, HLA-DR (R&D Systems FAB4869P), CD11c (BD Biosciences 565227) and CD45 (R&D Systems FAB1430A). Group D tested viability, CD3 (BD ​​Biosciences 555333), CD45 (BD Biosciences 340953) and CellTrace far red. The CD209 isotype control (R&D Systems IC0041P) and fluorescence minus one (FMO) control of group A were used to set the gate. FlowJo software was used to analyze the data.

[0171] Flow cytometry gating strategies

[0172] Large cells were gated in the SSC-A / FSC-A plot, and single cells were gated in the FSC-A / FSC-H plot. + The cells were gated and CD14 / CD209 was plotted to identify the presence of CD147CD209 cells. + Panel B: Lymphocytes were gated on the CD45 histogram and then plotted for CD80 / 83 and CD80 / 86 to determine the iDC phenotype. Panel C: Live / CD45 + Cells were gated and then plotted for HLA-DR / CD11c to determine iDC phenotype. Panel D: Live cells were gated, followed by CD3 / CD45 plots to isolate T cells, and then CellTrace Far Red histograms were performed to deconvolute T cell proliferation.

[0173] Three experiments (N1, N2, N3) were run continuously (starting on different days), and each experiment included 1 box of the present invention and 2-3 wells of a 6-well plate at each MO seeding density. Viability and iDC immunophenotype were performed via flow cytometry. The generated iDC count and iDC yield were calculated by the following:

[0174] Live harvested iDCs = [harvested cells] x [live / CD45+ cells] x [CD209+ / CD14- cells]

[0175] iDC yield = live harvested iDC ÷ inoculated MO

[0176] iDC phenotype

[0177] The iDCs generated by the cell culture system of the present invention and the 6-well plate are similar in phenotype, with slight differences in the expression of CD209 (DC-SIGN) / 80 / 83 / 86 depending on the MO seeding density. + and may have lower CD14 expression depending on the differentiation conditions. For this study, only CD209 + CD14 - The cells were considered as iDCs. Figure 20 and 21 The phenotypic expression of iDCs generated by the cell culture system of the present invention and 6-well plates in experiment N3 is shown. Figure 28-32 Shown in.

[0178] For experiments N1 and N3, the viability of the harvested cells was >90% and comparable between the cell culture system of the present invention and the 6-well plate. Experiment N2 had significantly lower viability (about 70-90% for the cell culture system of the present invention and about 77% for the well plate). There was no correlation between MO seeding density and viability, and all harvested cells were CD45+ leukocytes. The CD209 expression of iDC did not show dependence on MO seeding density in either the cell culture system of the present invention or the 6-well plate, but for experiments N1 and N3, the CD209 expression of the cell culture system iDC of the present invention was indeed slightly less than that of the well plate iDC (indicated by the left shift of CD209 fluorescence). Experiment N2iDC had similar CD209 expression in both the cell culture system of the present invention and the well plate.

[0179] A large number of live CD45+ cells harvested from the cell culture system of the present invention are CD209 - This CD209 - The population comprised approximately 20-40% of the harvested cells in experiments N1 / N3 and 2-7% in experiment N2. 6-well plates generated approximately 2-4% CD209 in all three experiments. -Cells. There was no obvious trend between the CD209- population and the MO seeding density, but compared to the 600kMO seeding density, the 200k and 400k MO seeding densities produced fewer CD209- cells. The dichotomy of the CD209- cells harvested from the cell culture system of the present invention and the 6-well plates may be due to the perfusion in the cell culture system of the present invention during differentiation under the test conditions. Perfusion may play a role in slowing down the MO to iDC dynamics, thereby potentially requiring a longer differentiation duration or higher cytokine concentrations to further differentiate this population into CD209+ iDC. The cell culture system of the present invention generates iDCs that are less differentiated under certain conditions, and it is believed that further optimization of the differentiation conditions (i.e., differentiation duration and cytokine concentrations, particularly IL-4 concentrations) in the cell culture system of the present invention is necessary.

[0180] In the cell culture system of the present invention, CD80 / 83 / 86 iDC expression was significantly dependent on the MO seeding density. iDCs generated in the well plate showed relatively constant CD83 / 86 expression at a 200k MO seeding density, while CD80 expression was maximal and decreased with increasing seeding density. All iDCs in the cell culture system of the present invention and in the 6-well plate were HLA-DR + and CD11c + Collectively, the phenotypic expression of cells harvested from the cell culture system and well plates of the present invention indicated that they were MO-derived iDCs. Under similar conditions, iDCs generated by the cell culture system of the present invention were phenotypically similar to iDCs generated in 6-well plates, with slight differences at low MO seeding density.

[0181] Harvesting iDCs

[0182] A direct comparison of the total counts of harvested cells between the cell culture system of the present invention and 6-well plates is not instructive because the cell culture system of the present invention has a larger number of seeded MOs, so the number of harvested iDCs normalized to the surface area and iDC yield of the cell culture system of the present invention or the well plate is not instructive. Figure 22-25 For direct comparison, plotting was performed in the cell culture system and well plates of the present invention. There were differences between each experiment (N1-N3), which is to be expected when using different donor cells for each experiment. For MO-derived iDCs, CD209 (DC-SIGN) expression is generally high, and the relatively high percentage of CD209- cells generated in the cell culture system of the present invention negatively impacts the number of harvested iDCs. Table 1 shows experimental data for iDC generation experiments in the cell culture system of the present invention and in 6-well plates.

[0183] Standardized harvest of iDCs

[0184] Figure 22and 23 The number of harvested iDCs and the average data normalized to the cell culture surface area for each experiment are shown. The cell culture system and 6-well plates of the present invention exhibit a "per cm 2 There was a positive correlation between the MO seeding density and iDC harvest, indicating that more iDCs were generated when more MOs were seeded. At lower MO seeding densities, the well plate had more iDCs per cm compared to the cell culture system of the present invention. 2 Generate more iDCs. At a seeding density of 600k MO, the cell culture system and well plate of the present invention have a high seeding density per cm 2 Similar numbers of iDCs were generated.

[0185] iDC yield

[0186] Figure 24 and 25 The iDC yield and average data for each experiment are shown. When the data were averaged across the three experiments, the cell culture system of the present invention showed a slight positive correlation between MO seeding density and iDC yield; however, there was no clear trend within each individual experiment. At 600k MO seeding density, the average iDC yield of the cell culture system of the present invention and the 6-well plate was similar. In experiments N1 and N3, the iDC yield of the 6-well plate was relatively constant with increasing MO seeding density; however, for experiment N2, the iDC yield dropped sharply with MO seeding density.

[0187] Plate N2 exhibits a trend inconsistent with data generated in this study and other experiments performed in our laboratory. The experimental procedures for this plate were identical, and we are not aware of the specific issue that caused this unusual trend. Viability in experiment N2 was lower than expected, which may be related to the inconsistent iDC yields in this experiment. Interestingly, the iDCs generated in these plates were phenotypically normal. Average iDC data from 6-well plates with and without data from N2 are plotted.

[0188] The cell culture system of the present invention and the 6-well plate had similar iDC yields at the highest seeding density, and diverged as the seeding density decreased. This indicates that the MO seeding density affects the ability of MOs to differentiate into iDCs, and that the iDC yield of the cell culture system of the present invention is greatest at higher seeding densities, with yields similar to those of the 6-well plate. Further increasing the MO seeding density to over 600k can improve the iDC yield in the cell culture system of the present invention, but this needs to be determined experimentally, as increasing the number of MOs beyond a critical upper limit may negatively affect the differentiation and phenotype of the generated cells. The similar iDC yields between the cell culture system of the present invention and the well plate at a 600k MO seeding density indicate that the cell culture system of the present invention generates phenotypically similar iDCs at a yield similar to that of the well plate. Furthermore, more MOs can be seeded into a single cassette of the present invention, allowing more iDCs to be harvested from a single cassette of the present invention compared to using multiple wells / well plates. This ultimately reduces user time and minimizes potential errors and contamination.

[0189] Table 1: Differentiation data of the cell culture system and 6-well plate of the present invention

[0190]

[0191]

[0192] As shown in Table 1 above, for a 6-well plate, N1 uses 3 wells, while N2-N3 uses 2 wells. Phenotypic data are in Figure 20 (Cell culture system of the present invention) and Figure 21 (6-well plate) is shown.

[0193] Allograft functional assay

[0194] The ability of generated iDCs to induce T cell proliferation was investigated via an allogeneic functional assay.For each experiment (N1, N2, N3), 1 million T cells from a single donor were co-cultured with 200k or 500kiDCs from different MO donors. Figure 26 Proliferation statistics are shown, and Figure 27 The histogram of T cell proliferation of experiment N1 is shown in FIG. The histograms of experiments N2 and N3 are shown in FIG. Figure 32 and 33Proliferation statistics include the division index (average number of cells produced per dividing cell), the proliferation index (average number of cells relative to the number of cells at the initial passage 0), and the division percentage (the percentage of cells in the initial population that underwent division). By performing this allogeneic functional assay, we sought to answer two questions: (i) Does the MO seeding density affect the ability of iDCs to induce T cell proliferation? (ii) At a given MO seeding density, how do iDCs in the cell culture system of the present invention compare to iDCs in 6-well plates?

[0195] (i) There is a clear correlation between the MO seeding density of iDCs generated in the cell culture system of the present invention and the ability of those iDCs to induce T cell proliferation; in contrast, the MO seeding density appears to have little effect on the function of iDCs generated in the well plate. Compared with iDCs generated from a 600k MO seeding density, iDCs in the cell culture system of the present invention generated from low MO seeding densities (200k and 400k) exhibit a greater ability to induce T cell proliferation. For the cell culture system iDCs of the present invention, T cell proliferation decreases with increasing MO seeding density used to generate iDCs, and when generated at a 600k MO seeding density, the cell culture system iDCs of the present invention have similar functions to well plate iDCs.

[0196] (ii) Compared to iDCs grown in 6-well plates, IDCs generated in the cell culture system of the present invention at low MO seeding densities (200k and 400k) were significantly better at inducing T cell proliferation. This effect was reduced at a high MO seeding density (600k), with iDCs grown in the cell culture system of the present invention performing slightly better than iDCs grown in 6-well plates. These results were consistent across all three experiments.

[0197] Expectedly, when 500k iDC is seeded into T cell assay, T cell proliferation is larger compared to 200k iDC. Data from this assay show that the iDC generated by the cell culture system of the present invention can induce T cell proliferation without the addition of IL-2 (a common cytokine for T cell expansion). Compared to the iDC generated by 6-well plates, the iDC generated by the cell culture system of the present invention also induces larger T cell proliferation, regardless of MO seeding density. It is important to note that allogeneic T cell assays are direct benchmarks for determining DC function, and the results observed in this study may not be extended to specialized allogeneic and other mixed lymphocyte reaction (MLR) functional assays.

[0198] Relationship between iDC phenotype and T cell proliferation

[0199] To determine why the cell culture system iDC of the present invention has a greater ability to induce T cell proliferation, phenotypic data were compared. Figure 20 、 28 and 30 (cell culture system of the present invention) and Figure 21 、 29 Two important trends were observed: (1) phenotypic differences in the cell culture system of the present invention were strongly correlated with T cell proliferation; and (2) the correlation between the phenotype of iDCs in the 6-well plate and T cell proliferation was very weak (or even absent).

[0200] (1) The phenotype of iDCs in the cell culture system of the present invention depends on the MO seeding density. Compared with the 600k MO seeding density, the cell culture system of the present invention generates significantly more CD80 + / 83 + / 86 + . and CD80783786 - Compared to iDCs, these CD80 + / 83 + / 86 + iDCs were more differentiated and exhibited a phenotype more similar to mature DCs (mDCs). This may be due to the lower MO / cytokine activity ratio at lower MO seeding densities, and these iDCs therefore have a greater ability to induce T cell proliferation. For MO / cytokine activity values, see Table 1. These results are consistent with previous studies that CD80 + / 83 + / 86 + iDCs have greater functionality even when most cells in the sample are negative for these markers. + / 83 + / 86 + The presence of iDCs indicates greater functional capacity.

[0201] (2) The phenotype of iDCs in 6-well plates does not depend on the MO seeding density. All three MO seeding densities in 6-well plates mainly produced CD83786 - iDC and a large number of CD80 + Furthermore, no discernible phenotypic differences were observed in plate-derived iDCs generated from different MO seeding densities. This suggests that the MO / cytokine activity ratio did not affect plate-derived iDC generation within the range studied, likely because the MO / cytokine activity ratio was sufficient to achieve any reasonable MO seeding density in static culture. Because the cytokines available to the MOs were sufficient for differentiation and no phenotypic differences were observed, T cell proliferation induced by plate-derived iDCs was similar under all conditions studied.

[0202] When fewer iDCs are CD80 + / 83+ / 86 + When the T cell proliferation is reduced, this is confirmed by the cell culture system data of the present invention and the lower T cell proliferation of iDC in the well plate. - / 83V86 - iDCs also induce T cell proliferation, but to a lesser extent than iDCs that are CD80 + / 83 + / 86 + This indicates that CD209 alone is insufficient to predict the ability of iDCs to induce T cell proliferation and that the degree of CD80 / 83 / 86 expression is a better indicator.

[0203] Compared with well plate iDCs generated under the same conditions, iDCs generated from the cell culture system of the present invention at a seeding density of 600k MO generally induced greater T cell proliferation ( Figure 26 ). This difference may be due to the perfusion in the cell culture system of the present invention, because all other conditions remain the same. Perfusion may affect the dynamics of MO to iDC. The perfusion in the cell culture system of the present invention also removes the culture medium from the box, which simultaneously removes the toxic byproducts (CO2 and lactic acid) dissolved in the culture medium due to cellular respiration. Compared with the well plate in which the toxic byproducts are not removed, the continuous removal of the culture medium can maintain a lower pH in the cell culture system of the present invention. In addition, on the third day, 1mL / well of differentiation medium was added to the well plate to supplement the cytokines. This may have an impact on the total cytokine concentration in the well, which is different from the cell culture system of the present invention. Detailed analysis of cytokine dynamics (e.g., consumption during MO differentiation and cytokine degradation) and the dynamics of lactic acid and CO2 production is needed to better understand the specific reasons for these results.

[0204] Another factor that may explain the functional differences between the cell culture system of the present invention and the well-plate iDCs is the exact nature of the polystyrene surface in contact with the cells. The cell culture system of the present invention uses polystyrene treated with O2 plasma; whereas the 6-well plates are tissue culture treated. The type of surface treatment and the exact nature of the polystyrene may influence iDC generation. Despite these differences, the iDCs generated by the cell culture system of the present invention are phenotypically similar to those generated in standard well-plate cultures and are functionally capable of proliferating allogeneic T cells.

[0205] iDC yield

[0206] Table 2: Per cm for experiments N1-N3 2 Harvest iDC

[0207]

[0208]

[0209] Table 2 shows the results of experiments N1-N3, in particular the cell culture system of the present invention (39.7 cm 2 ) or 6-well plate (9.5cm 2 / hole) per cm 2 iDCs were harvested. The data of well N2 were ignored. 2 Average of harvested iDCs; mean ± SD.

[0210] Table 3: Mean (± standard deviation) iDC yields from experiments N1-N3

[0211]

[0212] Allograft functional assay

[0213] Table 4-6 shows Figure 26 Proliferation statistics for allogeneic functional assays. iDCs were co-cultured with 1 million allogeneic T cells for 5 days. Proliferation histograms are shown in Figure 2. Figure 27 (Experiment N1), Figure 32 (Experiment N2) and Figure 33 (Experiment N3) shows. Figures 34-36 Allogeneic functional assay T cell controls are shown.

[0214] Table 4: Proliferation statistics for allograft functional assays in experiment N1

[0215]

[0216]

[0217] Table 5: Proliferation statistics for allograft functional assays in experiment N2

[0218]

[0219] Table 6: Proliferation statistics for allograft functional assays in experiment N3

[0220]

[0221]

[0222] The cell culture system of the present invention is developed as a closed sterile cell culture system for improving the process of generating dendritic cells from precursor PBMC or monocytes. This study indicates that the iDC generated by the cell culture system of the present invention is phenotypically and functionally comparable to the iDC generated by standard well plates. The optimal MO seeding density of the cell culture system of the present invention and the effect of seeding density on the ability of iDC to induce T cell proliferation were systematically determined. The data indicate that there is a strong correlation between the iDC phenotype (particularly the degree of CD80 / 83 / 86 iDC expression) and its ability to induce T cell proliferation. Due to the larger CD80 / 83 / 86 expression of iDC, the cell culture system of the present invention increases from a low MO seeding density (200kMO / cm 2 ) showed the greatest ability to induce T cell proliferation. Within the studied MO seeding density range of 200k-600k, iDCs in the cell culture system of the present invention also performed better in allogeneic T cell assays compared to iDCs in 6-well plates. In addition, although the cell culture system of the present invention produces fewer iDCs than 6-well plates on a standardized basis at lower MO seeding densities, it generates a similar number of iDCs as 6-well plates at higher MO seeding densities. The decision to generate iDCs at low or high seeding densities should be carefully considered and will depend on the downstream applications of the iDCs, as it is more important to consider whether to generate a larger number of iDCs or to generate iDCs with greater functional capacity. These trade-offs are common in standard static cultures and naturally extend to the cell culture system of the present invention.

[0223] Examples

[0224] EDEN cell culture cassettes and fluidics systems

[0225] EDEN was developed to generate therapeutically relevant numbers of iDCs in a single cell culture cassette that is completely enclosed and not open to the external environment. Fresh differentiation medium is perfused into the cassette, and the spent medium is removed. iDCs generated by EDEN exhibit similar phenotypic expression and iDC yield to iDCs generated in 6-well plates. iDCs matured in the cassette according to the present invention exhibit standard upregulation of CD80 / 83 / 86 and downregulation of CD209. Computational fluid dynamics simulations aided the design of the EDEN cassette to ensure that the perfused medium flows appropriately throughout the cassette and is adequately replenished with cytokines. These results demonstrate that EDEN successfully generated approximately 25 million iDCs at a 20-35% iDC yield under the conditions tested.

[0226] EDEN system in Figure 10EDEN cell culture cassettes are made from commercially available polystyrene and acrylic (cut using an Epilog Zing 16 laser system and assembled using 3M adhesive transfer tape). The polystyrene base is plasma treated. The inner surface area of ​​the cassette is 383.6 cm 2 , with a volume of 122 mL and dimensions of 21.0 cm x 21.0 cm x 0.317 mm (length x width x height). Eight inlet ports around the perimeter allow fresh differentiation medium to be perfused into the cassette, and a single outlet port in the center allows spent medium to be removed from the cassette.

[0227] The fluid system consists of an inlet bottle of fresh differentiation medium, a peristaltic pump, and an outlet bottle for collecting the effluent from the box. An Ismatec IPC-N peristaltic pump was used together with a PharMED BPT tubing to maintain the continuous perfusion of fresh differentiation medium at 8.0 / iL / min / inlct. A silicone tubing was connected between the peristaltic tubing and the box inlet to promote gas exchange between the culture medium and the surrounding environment (maintained at 37°C and 5% CO2 in a Thermo Forma incubator). Silicone tubing was also used at the outlet port where the perfusion flow rate was estimated to be 64 μL / min. The effluent collected in the waste reservoir was centrifuged to determine whether the cells were washed out of the box due to perfusion; no cells were observed in the effluent, indicating that the generated iDCs remained in the box, and the perfusion flow rate was not high enough to resuspend the cells resident at the polystyrene base. At startup (day 0) and day 3, 285 mL of fresh differentiation medium was added to the inlet reservoir to maintain perfusion throughout the 6-day differentiation process. The cells were harvested by collecting the cell solution and washing each well twice with cold DPBS. The adherent cells after the two DPBS washes were not collected.

[0228] Differentiation medium

[0229] RPMI 1640 (Gibco 11875119) was supplemented with 10% fetal bovine serum (FBS; heat inactivated; MilliporeSigma F2442), 1% penicillin-streptomycin (P / S; Gibco 15140122), 500 U / mL IL-4 (R&D Systems 204IL) and 500 U / mL GM-CSF (R&D Systems 215GM).

[0230] PBMC isolation and monocyte enrichment

[0231] Peripheral blood mononuclear cells (PBMC) were isolated from whole blood StemExpress using Ficoll-Paque (GE Healthcare). Whole blood was extracted and processed on the same day. PBMC was isolated and stored at 50-60 million PBMC / mL cryogenic temperature in CryoStor CS10, and kept cryogenically for at least 7 days before recovery. Mononuclear cells (MO) were enriched from the recovered PBMC using Miltenyi CD 14 microbeads, and two LS columns were passed through to obtain MO purity > 95%. The enriched MO from a single donor was suspended in 122 mL differentiation medium and inoculated into EDEN boxes. Each experiment used MO from different donors.

[0232] 6-well plate control

[0233] Corning Costar 6-well plates (3516) are used as the static control for iDC generation. Each well contains 2.5 mL differentiation medium, and the empty wells are filled with 3.0 mL DPBS. On the 3rd day, 1 mL fresh differentiation medium is added to each well. Cells are harvested by collecting cell solution and washing each well 2 times with cold DPBS. Adherence cells after the DPBS washing twice are not collected.

[0234] IDC matures

[0235] Use 17.4cm 2 A small box containing 5.5 mL of maturation medium was perfused at 3.5 μL / min for maturation on the system according to the present invention. The maturation medium consisted of RPMI 1640 supplemented with 10% HI-FBS, 1% P / S, 2 ng / mL IL-1β (BD Biosciences 554602), 1000 U / mL IL-6 (BD Biosciences 550071), 10 ng / mL TNF-a (MilliporeSigma 11088939001) and 1 μg / mL PGE2 (MilliporeSigma P6532). At 422,200 iDC / cm 2IDCs from the EDEN 1 experiment were seeded and allowed to mature for 1 or 3 days in an incubator at 37° C. and 5% CO 2 . Cells were harvested using two washes of cold PBS as described in Kozbial, 2018, Automated generation of immature dendritic cells in a single-use system, Journal of Immunological Methods, 457:53-65, which is incorporated herein by reference in its entirety.

[0236] Immunophenotyping

[0237] Immunophenotyping of iDCs was performed using an ACEA Biosciences NovoCyte flow cytometer. Group A tested viability (live / dead fixed green dead cell staining; Invitrogen L34970), CD209 (R&D Systems FAB161P100), CD14 (Abeam abl57312), and CD45 (R&D Systems FAB1430A). Group B tested CD80 (BD Biosciences 557226), CD83 (BD ​​Biosciences 556855), CD86 (BD Biosciences 561128), and CD45; due to limited detection channels, they were not included. Group C tested CD80, CD83, CD86, and CD209 (R&D Systems FAB161A). The CD209 isotype control (R&D Systems IC0041P) and fluorescence minus one (FMO) control of Group A were used to set the gate.

[0238] Flow cytometry gating strategies

[0239] Large cells were gated on the SSC-A / FSC-A plot, followed by single cells on the FSC-A / FSC-H plot. Panel A: Live / CD45+ cells were gated, followed by plotting CD14 / CD209 to determine the percentage of MO or iDC. Panel B: Lymphocytes were gated on the CD45 histogram. CD80 / 83 and CD80 / 86 were then plotted to determine the iDC phenotype. Panel C: DCs were gated on the CD209 / 80 plot, followed by gates on the CD83 / 86 plot for either CD209+ / 80+ or ​​CD209+ / 80- cells.

[0240] IDC generation

[0241] Two iDC generation experiments were carried out, in which 114.3 million and 78.3 million MO were seeded into EDEN boxes. After differentiation for 6 days, 25.5 million and 24.8 million iDC were harvested from each box. Live harvest iDC was calculated by multiplying the total cells of live / CD45+ cell harvests by iDC (CD209+ / 14-). The IDC productive rate (standardized relative to the quantity of inoculated MO) was calculated as the quantity of harvested iDC divided by the quantity of inoculated MO, and the IDC productive rates of two EDEN experiments were respectively 22.3% and 31.7%. 6-well plate controls showed that iDC productive rate was similar to EDEN (wherein the productive rate of orifice plate in experiment 1 was higher, and the productive rate in experiment 2 was lower).

[0242] The data are shown in Table 7. The phenotypic data are Figure 16 Shown in.

[0243] Table 7: Differentiation data for iDC generation in EDEN and 6-well plates

[0244]

[0245] IDC phenotype

[0246] Immunophenotyping of generated iDCs Figure 16 Shown in . After 6 days of differentiation, iDCs generated by EDEN and 6-well plates were phenotypically similar. iDCs were CD209 (DC-SIGN) positive, CD 14 negative, and exhibited low CD80 / 83 expression, as expected for MO-derived iDCs. CD86 expression on EDEN 2iDCs was unexpectedly high, as this expression level is usually expected on mature DCs. Soluble proteins in fetal bovine serum (FBS) supplemented to the basal culture medium may be a possible explanation for this irregular expression, as FBS is of animal origin and its composition cannot be strictly controlled. In addition, since the box was built manually in the laboratory, contamination of proteins in the box may also explain this high expression. In the histogram of panel B (not shown), greater than 99.7% of the cells were CD45+. This protein expression profile of iDCs generated by EDEN demonstrates the efficacy of EDEN in generating clinically relevant numbers of DCs (which are phenotypically similar to static culture in well plates).

[0247] IDC matures

[0248] The iDCs generated in EDEN 1 were then matured in the cassettes according to the present invention for 1 or 3 days. 7.31 million iDCs (422,200 iDC / cm 2), and harvested 6 million (1 day maturation) and 4.8 million (3 days maturation) mature DC (mDC), with yields of 81.9% and 66.2%, respectively. Yield was calculated as the number of inoculated iDCs divided by the number of harvested mDCs. MDC counts were strictly determined by counting viable CD45+ / 209+ cells, so yield values ​​less than 100% indicated that the degree of cell death in each experiment was 18.1% (1 day maturation) and 33.8% (3 days maturation) in the two sub-experiments, respectively.

[0249] The ripening results are listed in Table 8. Maturation was performed in mini-boxes. Phenotypic data are given in Figure 17 In particular, the immunophenotypic analysis of EDEN 1mDC was Figure 17 As shown in Figure 2 . For mDCs, CD209 expression is low and decreases with maturation length. CD80 expression increases from approximately 11% in iDCs to 48% and 55% in 1-day and 3-day mature DCs, respectively. CD80 expression is generally low in iDCs but upregulated in mDCs, indicating successful maturation. CD83 / 86 expression is clearly dependent on CD80 expression, as shown in Figure 2 . Figure 17 As shown in the last two columns of , CD80+ mDCs showed higher expression of CD86 compared to CD80- mDCs; whereas CD83 expression remained unchanged.

[0250] Table 8: Maturation data of iDCs generated by EDEN 1

[0251]

[0252] Computational fluid dynamics (CFD) simulation

[0253] CFD simulations in COMSOL Multiphysics were used in the EDEN design to understand how the culture medium flows through the cartridge. Differentiation medium was simulated using water at 37°C. The cartridge was initially filled with water without cytokines. In practice, the cartridge was filled with differentiation medium containing cytokines. However, initially filling the cartridge with plain culture medium (water) allowed visualization of cytokine convection, as cytokine diffusion is extremely low (9216 μm). 2 / day), and convection is the driving force behind the cytokine gradient. 3 (500 U / mL) R&D Systems IL-4 in water was perfused into the cartridge at 8 / iL / min / inlct and discharged through the outlet at the center of the cartridge. Since we were interested in determining the optimal medium flow for fresh differentiation medium, cytokine consumption / depletion was not included in this analysis. Figure 11 The cartridge flow channels are shown, which describe the volume of medium flowing within the cartridge. Figure 12The concentration of IL-4 cytokine was modeled on the lower polystyrene surface of the flow channel where the cells reside at the base of the cartridge, as depicted by the purple surface in FIG. The streamlines and gauge pressure resulting from the perfusion are shown in FIG. Figure 13 and 14 The IL-4 concentration gradient perfused every 24 hours is shown in Figure 15 Shown in.

[0254] These CFD data are crucial in the design of a box that is sufficient to allow perfusion culture medium to diffuse into the entire box. Cytokine concentration and streamline data show that under 8 / iL / min / inlct laminar flow, the box is divided into eight zones. After approximately 4 days, each zone is supplemented with fresh differentiation culture medium. Initial CFD simulations indicate that flow dead zones or dead spots are formed at the positions of the v-shaped notches, so these notches are added to eliminate flow dead zones or dead spots and promote desired fluid flow. The 8 cylindrical posts in the box support the upper acrylic surface. Before adding these, a slight sagging of acrylic was observed, and acrylic was supported by the culture medium in the box, which could cause unnecessary pressure in the box, thereby possibly affecting cells. Therefore, these features (i.e., notches and posts) were added to alleviate flow dead zones or dead spots and pressure issues, thereby making final EDEN box design fully help perfusion culture medium flow in the box and not cause undesirable pressure gradients.

[0255] Incorporated by Reference

[0256] Throughout this disclosure, other documents, such as patents, patent applications, patent publications, journals, books, articles, web page content, have been referenced and cited. All of these documents are hereby incorporated herein by reference in their entirety for all purposes.

[0257] Equivalent content

[0258] While the invention has been described in conjunction with certain embodiments, those of ordinary skill, after reading the foregoing description, will be able to make various changes, substitutions of equivalents, and other modifications to the compositions and methods set forth herein.

Claims

1. A cell culture system comprising: A cell culture cassette comprising: top surface; bottom surface; an outlet provided on said top surface and located near the center of said top surface; a plurality of inlets symmetrically located around the periphery of the cell culture cassette; and one or more posts extending from the bottom surface to the top surface; a culture medium reservoir connected to the plurality of inlets; Waste storage connected to the outlet; and A pump is provided for moving culture medium from the culture medium reservoir to the cell culture cassette and / or from the cell culture cassette to the waste reservoir. 2 . The system of claim 1 , wherein the plurality of inlets and outlets are arranged to provide symmetrical fluid flow as the culture medium passes through the cell culture cassette. The cell culture system according to claim 1 , wherein the inlet is provided on a top surface of the cell culture box. 4 . The cell culture system according to claim 1 , wherein a periphery of the cell culture box includes a plurality of corners, each corner including one of the inlets. 5 . The cell culture system of claim 1 , wherein the cell culture system further comprises one or more sensors in operable communication with the cell culture cassette and a central processing unit.

6. The cell culture system of claim 5, wherein the one or more sensors measure one or more parameters selected from the group consisting of: pH, dissolved oxygen, total biomass, cell diameter, glucose concentration, lactate concentration, and cellular metabolite concentration.

7. The cell culture system according to claim 6, wherein In response to measurements from the one or more sensors, the central processing unit executes instructions to adjust an operating state of a pump. The cell culture system according to claim 1 , wherein the cell culture system maintains a sterile environment for culturing cells.

9. The cell culture system of claim 1, wherein the bottom surface is configured to allow dendritic cells to adhere thereto.

10. The cell culture system of claim 1, wherein the pump delivers fluid from the culture medium reservoir to the cell culture cartridge at a rate of less than 10 microliters per minute.

11. A method for culturing cells, comprising: A cell culture box is provided, comprising: top surface; bottom surface; an outlet provided on said top surface and located near the center of said top surface; a plurality of inlets symmetrically located around the periphery of the cell culture cassette; and one or more posts extending from the bottom surface to the top surface; and a plurality of regions designed to provide symmetrical fluid flow, wherein each of the plurality of regions includes an inlet; seeding cells into the cell culture chamber; and Culture medium is perfused into the cell culture cassette through the inlet, and spent culture medium is removed to a waste reservoir through the outlet on the cell culture cassette, thereby culturing cells in the cell culture cassette.

12. The method of claim 11, wherein the cells comprise dendritic cells. The method according to claim 11 , wherein the inlet is provided on a top surface of the cell culture cassette. The method according to claim 11 , wherein the culture medium is perfused into the cell culture cassette by a pump.

15. The method of claim 14, wherein the pump is operatively connected to a central processing unit.

16. The method of claim 12, further comprising harvesting the dendritic cells, wherein harvesting the cells comprises cooling the cartridge. 17 . The method of claim 12 , further comprising transferring the immature dendritic cells to a second cassette, wherein the second cassette has a size that is the same as or smaller than the cell culture cassette.

18. The method of claim 17, wherein the immature dendritic cells undergo maturation and antigen pulse in the second cartridge.

19. The cell culture system of claim 1, wherein the cell culture cassette comprises one or more notches arranged along an outer periphery thereof.

20. The method of claim 11, wherein the cell culture cassette comprises one or more notches disposed along an outer periphery thereof.

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