Apparatus and method for generating dendritic cells

Abstract

To provide a dendritic cell generating apparatus and method.SOLUTION: A cell culture cartridge comprises a plurality of zones geometrically configured to provide a symmetrical fluid flow with each of the plurality of zones to avoid dead areas in a flow within each of the plurality of zones. In certain embodiments, at least eight inlets are provided, with an inlet positioned at each corner of the cell culture cartridge. In certain embodiments, a shared outlet is positioned on a top surface of the cell culture cartridge. The present invention provides an optimal and further efficient approach to automated generation of dendritic cells (DCs).SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - References to Related Applications This application claims the benefit and priority of U.S. Patent Application No. 16 / 192,062, filed on November 15, 2018, the content of which is hereby incorporated by reference in its entirety. Government Support

[0002] 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. Field of the Invention

[0003] The present invention generally relates to cell culture chambers and methods of using them.

Background Art

[0004] Background Cell - based cancer immunotherapy is a method of treating cancer that uses immune - active cells including dendritic cells (DCs). Since DCs cannot be harvested in sufficient numbers by other methods, they are typically generated by the differentiation of monocytes extracted from peripheral blood. However, generating clinically appropriate numbers of monocyte - derived dendritic cells for therapeutic use can be difficult. Conventional generation techniques, such as standard well - plate and T - flask culturing, involve a cumbersome process with many manual steps that expose the cell culture to the external environment and require highly trained technicians.

[0005] Conventional generation techniques have a number of safety and contamination concerns, such as misidentification and confusion of patient samples, exposure to unknown contaminants inside the laminar flow hood (e.g., microparticles and bacteria / fungi resistant to standard sterilization techniques such as 70% ethanol), and accidental exposure to a decaying environment of the culture. Furthermore, scaling up manual DC generation techniques is generally not feasible, except for adding more culture vessels to the workflow. An automated system that continuously perfuses fresh medium into the culture vessel while simultaneously removing depleted medium is an alternative to conventional manual generation techniques.

[0006] Automated systems generally have fewer safety and contamination concerns than prior art, but automated systems are plagued by scaling up and other problems. For example, many commercially available automated systems are not scalable for DC research or clinical-level generation. Also, automated systems are plagued by non-uniform flow or dead spots in the flow within the cell culture vessel. A dead spot (dead region) in the flow is a region in the cell culture vessel where a uniform flow is not maintained when fresh medium is provided and depleted medium is removed, thereby affecting DC generation. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0007] Abstract The present invention provides an automated cell culture cartridge and system for the generation of dendritic cells having a uniform and symmetric flow within a cell culture cartridge. Aspects of the present invention are achieved by designing a cell cartridge chamber having a plurality of zones geometrically configured to provide a symmetric fluid flow and avoid dead regions in the flow within the cell culture chamber. The geometric design provides a uniform flow and avoids dead regions or dead spots in the flow. In this manner, the present invention provides an optimal and more efficient approach to the automated generation of dendritic cells (DC).

[0008] In certain embodiments, the cell culture chamber includes multiple corners. Inlets are located at each of the multiple corners, and outlets are located on the top surface of the cell culture chamber. The placement of inlets in the cell culture chamber allows for symmetrical fluid flow channels within the cell culture chamber. In some examples, the cell culture chamber includes an octagon with eight corners, each containing an inlet. The outlet is located in the center of the top surface of the cell culture chamber.

[0009] The cell culture chamber also incorporates various technical features that enable the automation of manual processes, dramatically reducing user intervention in the process and thereby significantly lowering the risk of contamination. The cell culture chamber allows for the perfusion of culture medium and cytokines into the chamber, enabling the maintenance of more consistent levels. Achieving consistent nutrient and cytokine levels is crucial for efficient cell culture and processing, and therefore for predictable and effective scale-up. Furthermore, a vertical channel is provided as the fluid exits the chamber, ensuring that DCs, antigen-specific T cells, and other cells involved in the culture process remain in the chamber during perfusion.

[0010] Furthermore, the present invention includes additional features aimed at achieving uniform flow. For example, the cell culture cartridge further includes one or more struts extending between the bottom and top surfaces. In another example, the bottom surface includes one or more notches on its outer circumference. Some embodiments of the present invention further include one or more stopcocks operably connected to the cell culture chamber.

[0011] The cell culture chamber of the present invention may 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 bottom surface material is treated with air or oxygen plasma under glow discharge or corona discharge. In some embodiments, the bottom surface material 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 examples, one or more materials are selected from the group consisting of polystyrene and acrylates. In some embodiments of the present invention, the cell culture cartridge is transparent. In some embodiments, the height of the cell culture cartridge is one-tenth or less of the maximum length or width dimension.

[0012] The cell culture cartridge and system may further include one or more stopcocks. One or more stopcocks may be operably connected to the cell culture chamber. When attached to a filter, the stopcocks in the cartridge allow air exchange when the cartridge is seeded with cell solution or harvested. When attached to a Luer-operated transfer valve, the stopcocks allow the aseptic transfer of differentiation medium to fill the inlet bottle and remove waste from the outlet bottle. This setup allows the tubing and cartridge system to remain sterile from setup to harvest without the need to break the system's sterile seal.

[0013] Furthermore, the present invention provides a fully sealed, sterile iDC generation system for generating immature DCs (iDCs) on a clinical scale, effectively eliminating the need for numerous well plates (or T-flasks / bags), ensuring a sterile, particulate-free culture system, and reducing the time technicians spend maintaining cell cultures. The present invention is an automated cell culture system for sterilely generating a therapeutically appropriate number of iDCs in a single cell culture cartridge. The system can also further process the iDCs to mature them by adding maturation reagents and to stimulate them by adding one or more antigens to the cell culture chamber. The cell culture system includes a cell culture cartridge that includes multiple zones geometrically configured to provide symmetrical fluid flow channels within the cell culture chamber and to avoid dead areas in the flow within the cell culture chamber. The cell culture system further includes one or more pumps operably attached to the cell culture chamber. In some embodiments, the peristaltic pump provides continuous perfusion of fresh medium into the culture vessel at a specific flow rate per inlet, e.g., 8 μL / min, along with the removal of depleted medium into a waste reservoir. The transfer of new culture medium, removal of depleted medium, cell seeding, and retrieval of iDCs are performed aseptically.

[0014] In some embodiments, the cell culture system further includes at least one fluid connector configured to fluidly connect a cell culture chamber to a second vessel, which may be a second cell culture chamber. For example, the cell culture chambers are configured to fluidly connect to one another to allow for the concentration of cells into smaller volumes when such concentrations are desired for maturation and antigen stimulation (also known as pulsed) steps. If the system is used to stimulate T cells in DCs, those T cells may be automatically moved between chambers to allow for further culture and proliferation of the T cells in new cell culture chambers. In some embodiments, the movement is caused by introducing a gas flow into the first cell culture chamber, which then moves the supernatant containing the first cell product to the second cell culture chamber through the fluid connector.

[0015] In certain embodiments, the cell culture chamber of the exemplary embodiment provides T cell proliferation and stimulation using antigen-presenting cells derived 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. These cell culture systems and methods greatly reduce the number of manual steps compared to conventional protocols. This method greatly reduces the risk of contamination and greatly increases the robustness and reproducibility of the manufacturing technique, both of which are important considerations for the safe and reliable manufacture of therapeutic products such as personalized T cell therapies that enable precise targeting.

[0016] In other embodiments, the cell culture chamber further includes one or more fluid reservoirs operably connected to one or more pumps. The fluid reservoirs are configured to supply the chamber with a culture medium containing nutrients and cytokines.

[0017] In some embodiments, the present invention further includes one or more sensors operably coupled to a cell culture cartridge. The one or more sensors can measure any preferred parameter. For 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 cell metabolite concentration.

[0018] The cell culture chamber may further include a central processing unit (CPU). The CPU may be communicatively connected to one or more sensors and may be configured to adjust the operating state of one or more pumps as a function of one or more measured parameters. In embodiments in which a flow-generating mechanism such as an electrohydrodynamic mechanism rather than a pump is used, the central processing unit may change the operating state of the flow-generating mechanism to adjust the flow rate of the first cell product as a function of one or more parameters.

[0019] In this embodiment, the central processing unit executes a command to cause the system to receive first input data, which includes the size of the cell culture chamber. Second input data is then received, which includes a first concentration of a first cell type and a second concentration of a second cell type in one or more fluids introduced into the cell culture chamber. Based on the first and second inputs, the perfusion rate of the perfusion fluid introduced into the cell culture chamber is calculated. The calculated perfusion rate maximizes the likelihood that the first and second cell types will come into contact with each other within the cell culture chamber. The first cell type is a peripheral blood mononuclear cell, and the second cell type is a dendritic cell.

[0020] In the embodiment, the system further includes one or more pumps operably connected to one or more perfusion fluid reservoirs and operably connected to a central processing unit, the central processing unit controlling the perfusion velocity of the perfusion fluid by controlling one or more pumps.

[0021] In certain embodiments, the present invention provides a method for culturing dendritic cells. The method includes the step of providing a cell culture cartridge. The cell culture cartridge includes a plurality of zones geometrically configured to provide a fluid flow symmetrical to each of the plurality of zones in order to avoid dead areas in the flow within each of the plurality of zones. In some embodiments, the cell culture cartridge includes a cell culture chamber including a plurality of corners, inlets located at each corner of the plurality of corners, and an outlet located on the top surface of the cell culture chamber. The fluid flows symmetrically through the cell culture chamber.

[0022] Monocyte cells are seeded into a cell culture cartridge. Dendritic cells are generated by seeding the monocytes into a cell culture chamber, providing continuous perfusion of culture medium to the cell culture cartridge via an inlet, and removing the depleted medium into a waste reservoir via an outlet. In some embodiments, the method further includes a step of harvesting the dendritic cells, the cell harvesting step including cooling the cartridge.

[0023] In embodiments, the method further includes the step of transferring immature dendritic cells to a second cartridge, the second cartridge being smaller than the cell culture cartridge. The immature dendritic cells mature and undergo antigen pulsation in the second cartridge. In embodiments, maturation and antigen pulsation may occur in the cell cartridge without using the second cartridge.

[0024] In some embodiments, the method of the present invention further includes maturation of dendritic cells and pulsation of the cells with antigens.

[0025] In certain embodiments, the cell culture chamber is sized and configured to fit within an incubator in order to help maintain a desired environment within and around the chamber. In some embodiments, one or more pumps are located inside the incubator. In other embodiments, one or more pumps are located outside the incubator and are operably connected to the cell culture chamber inside the incubator.

[0026] In certain embodiments, at least a portion of the system includes disposable components, some or all of which may be housed within a non-disposable frame. In other embodiments, all components of the system are disposable. Furthermore, in some embodiments, the system includes a sample tracking component for tracking and describing patient material.

[0027] The system and method are designed so that any number of additional cartridges or cell culture chambers may be provided. In some embodiments, the system includes two or more cell culture cartridges for generating T cells.

[0028] In certain embodiments, the system of the present invention has the ability to automatically calculate and set a desired perfusion rate of a perfusion fluid that gives various inputs, such as the size of the cell culture chamber and the concentrations of two or more cell types, including dendritic cells and peripheral blood mononuclear cells. In an exemplary configuration, a cell culture system is provided, which includes one or more cell culture chambers, and a central processing unit including a memory that includes instructions to be received by the central processing unit, which include a first input data including the size of the cell culture chambers, 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 introduced into the cell culture chambers, and which causes the system to calculate a perfusion rate of the perfusion fluid introduced into the cell culture chambers that maximizes the likelihood that the first and second cell types will come into contact with each other within the cell culture chambers, based on the first and second inputs. In certain embodiments, the first cell type is peripheral blood mononuclear cells, and the second cell type is dendritic cells. [Brief explanation of the drawing]

[0029] [Figure 1] Figure 1 shows an embodiment of the cell culture chamber of the cell culture cartridge according to the present invention.

[0030] [Figure 2] Figure 2 shows a front view of the cell culture cartridge and system.

[0031] [Figure 3] Figure 3 shows a top view of the cell culture cartridge and system.

[0032] [Figure 4]Figure 4 shows a left side view of the cell culture cartridge and system.

[0033] [Figure 5] Figure 5 shows a right-side view of the cell culture cartridge and system.

[0034] [Figure 6] Figure 6 shows an embodiment of the system 100 of the present invention.

[0035] [Figure 7] Figure 7 shows an embodiment of the present invention having two cartridges.

[0036] [Figure 8] Figure 8 shows an embodiment of the present invention illustrating the transfer from a smaller cartridge to an infusion bag.

[0037] [Figure 9] Figure 9 shows the disposable and non-disposable components of the present invention.

[0038] [Figure 10] Figure 10 shows an embodiment of the EDEN automatic fluid system.

[0039] [Figure 11] Figure 11 shows the design of the cell culture cartridge with a flow channel for the cell culture cartridge.

[0040] [Figure 12] Figure 12 shows the design of a cell culture cartridge with a polystyrene surface (shared) at the bottom of the cell culture cartridge where the cells reside.

[0041] [Figure 13] Figure 13 shows the design of a cell culture cartridge with streamlines resulting from perfusion within the cartridge.

[0042] [Figure 14]Figure 14 shows the design of a cell culture cartridge with gauge pressure due to perfusion within the cell culture cartridge.

[0043] [Figure 15] Figure 15 shows cytokine perfusion into the cell culture cartridge.

[0044] [Figure 16-1] Figure 16 shows the phenotypes of iDCs generated in cell culture cartridges and 6-well plates, differentiated from MO over 6 days. [Figure 16-2] Figure 16 shows the phenotypes of iDCs generated in cell culture cartridges and 6-well plates, differentiated from MO over 6 days.

[0045] [Figure 17-1] Figure 17 shows the phenotypes of iDCs and mDCs from a cell culture cartridge. iDCs were generated in the cell culture cartridge and then seeded in the cell culture system of the present invention for 1 or 3 days of maturation. The labels at the top of the figure indicate the gate from which the plot originated. [Figure 17-2] Figure 17 shows the phenotypes of iDCs and mDCs from a cell culture cartridge. iDCs were generated in the cell culture cartridge and then seeded in the cell culture system of the present invention for 1 or 3 days of maturation. The labels at the top of the figure indicate the gate from which the plot originated.

[0046] [Figure 18] Figure 18 shows an exemplary method for producing an immunotherapy product according to an embodiment of the present invention.

[0047] [Figure 19] Figure 19 shows a system of the present invention according to a particular embodiment.

[0048] [Figure 20-1] Figure 20 shows the phenotype of MicroDEN N3 iDC. Data for experiments N1 and N2 are shown in Figures 28 and 30. [Figure 20-2] Figure 20 shows the phenotype of MicroDEN N3 iDC. Data for experiments N1 and N2 are shown in Figures 28 and 30.

[0049] [Figure 21-1] Figure 21 shows the phenotype of N3 iDCs in a 6-well plate. Data for experiments N1 and N2 are shown in Figures 29 and 31. [Figure 21-2] Figure 21 shows the phenotype of N3 iDCs in a 6-well plate. Data for experiments N1 and N2 are shown in Figures 29 and 31.

[0050] [Figure 22] Figure 22 shows differentiation data for iDCs generated in the cell culture system and 6-well plate of the present invention, in particular, the recovered iDCs normalized to the surface area of ​​the cartridge (39.7 cm²) or 6-well plate (9.5 cm² / well) of the present invention.

[0051] [Figure 23] Figure 23 shows differentiation data for iDCs generated in the cell culture system and 6-well plate of the present invention, in particular, the average recovered iDCs normalized to the surface area of ​​the cartridge or 6-well plate of the present invention. The data are expressed as the mean ± standard deviation of the experiments shown. The data are listed in Tables 1-3.

[0052] [Figure 24] Figure 24 shows the differentiation data for iDCs generated in the cell culture system and 6-well plates of the present invention, in particular, the iDC yield for each experiment at seeding densities of 200k to 600k MO / cm2.

[0053] [Figure 25]Figure 25 shows differentiation data for iDCs generated in the cell culture system and 6-well plates of the present invention, particularly the average iDC yield for each experiment at seeding densities of 200k to 600k MO / cm2. The data are expressed as the mean ± standard deviation of the experiment shown. The data are listed in Tables 1-3.

[0054] [Figure 26-1] Figure 26 shows the proliferation statistics of an allogeneic functional assay for iDCs generated in the cell culture system or 6-well plate of the present invention at differentiation seeding densities of 200k–600k MO / cm2. The legend indicates the source of iDCs (the cell culture system or 6-well plate of the present invention) and the number of iDCs co-cultured with 1 million allogeneic T cells from a single donor. Tabular data are shown in Tables 4–6. [Figure 26-2] Figure 26 shows the proliferation statistics of an allogeneic functional assay for iDCs generated in the cell culture system or 6-well plate of the present invention at differentiation seeding densities of 200k–600k MO / cm2. The legend indicates the source of iDCs (the cell culture system or 6-well plate of the present invention) and the number of iDCs co-cultured with 1 million allogeneic T cells from a single donor. Tabular data are shown in Tables 4–6. [Figure 26-3] Figure 26 shows the proliferation statistics of an allogeneic functional assay for iDCs generated in the cell culture system or 6-well plate of the present invention at differentiation seeding densities of 200k–600k MO / cm2. The legend indicates the source of iDCs (the cell culture system or 6-well plate of the present invention) and the number of iDCs co-cultured with 1 million allogeneic T cells from a single donor. Tabular data are shown in Tables 4–6.

[0055] [Figure 27-1]Figure 27 shows the proliferation histogram of the allogeneic functional assay for Experiment N1. The columns represent the MO seeding density for iDCs generated in the cell culture system or 6-well plate of the present invention. The rows represent the source of iDCs (the cell culture system or 6-well plate of the present invention) and the number of iDCs co-cultured with 1 million allogeneic T cells for 5 days. The green vertical lines represent the peak positions of the stained, unstimulated control, which are also the positions of non-dividing cells. Thicker curves represent the overall fit, and thinner curves represent the generation of individual T cells. Histograms for Experiments N2–N3 are shown in Figures 32 and 33. Unstimulated T cell controls are shown in Figures 34–36. [Figure 27-2] Figure 27 shows the proliferation histogram of the allogeneic functional assay for Experiment N1. The columns represent the MO seeding density for iDCs generated in the cell culture system or 6-well plate of the present invention. The rows represent the source of iDCs (the cell culture system or 6-well plate of the present invention) and the number of iDCs co-cultured with 1 million allogeneic T cells for 5 days. The green vertical lines represent the peak positions of the stained, unstimulated control, which are also the positions of non-dividing cells. Thicker curves represent the overall fit, and thinner curves represent the generation of individual T cells. Histograms for Experiments N2–N3 are shown in Figures 32 and 33. Unstimulated T cell controls are shown in Figures 34–36. [Figure 27-3] Figure 27 shows the proliferation histogram of the allogeneic functional assay for Experiment N1. The columns represent the MO seeding density for iDCs generated in the cell culture system or 6-well plate of the present invention. The rows represent the source of iDCs (the cell culture system or 6-well plate of the present invention) and the number of iDCs co-cultured with 1 million allogeneic T cells for 5 days. The green vertical lines represent the peak positions of the stained, unstimulated control, which are also the positions of non-dividing cells. Thicker curves represent the overall fit, and thinner curves represent the generation of individual T cells. Histograms for Experiments N2–N3 are shown in Figures 32 and 33. Unstimulated T cell controls are shown in Figures 34–36.

[0056] [Figure 28-1]Figure 28 shows the phenotype of iDCs in N1 of the cell culture system of the present invention. [Figure 28-2] Figure 28 shows the phenotype of iDCs in N1 of the cell culture system of the present invention.

[0057] [Figure 29-1] Figure 29 shows the phenotype of iDCs in N1 of a 6-well plate. [Figure 29-2] Figure 29 shows the phenotype of iDCs in N1 of a 6-well plate. [Figure 29-3] Figure 29 shows the phenotype of iDCs in N1 of a 6-well plate.

[0058] [Figure 30-1] Figure 30 shows the phenotype of iDCs in N2 of the cell culture system of the present invention. [Figure 30-2] Figure 30 shows the phenotype of iDCs in N2 of the cell culture system of the present invention.

[0059] [Figure 31-1] Figure 31 shows the phenotype of N2 iDCs in a 6-well plate. [Figure 31-2] Figure 31 shows the phenotype of N2 iDCs in a 6-well plate.

[0060] [Figure 32-1] Figure 32 shows the histogram for Experiment N2: Allogeneic Functional Assay. T cell controls are shown in Figures 34-36. [Figure 32-2] Figure 32 shows the histogram for Experiment N2: Allogeneic Functional Assay. T cell controls are shown in Figures 34-36. [Figure 32-3] Figure 32 shows the histogram for Experiment N2: Allogeneic Functional Assay. T cell controls are shown in Figures 34-36.

[0061] [Figure 33-1] Figure 33 shows the histogram for Experiment N3: Allogeneic Functional Assay. T cell controls are shown in Figures 34-36. [Figure 33-2]Figure 33 shows the histogram for Experiment N3: Allogeneic Functional Assay. T cell controls are shown in Figures 34-36. [Figure 33-3] Figure 33 shows the histogram for Experiment N3: Allogeneic Functional Assay. T cell controls are shown in Figures 34-36.

[0062] [Figure 34] Figure 34 shows the T cell control for the allogeneic functional assay for N1. One million T cells (from the same donor as N1-N3) were cultured for 5 days without iDCs.

[0063] [Figure 35] Figure 35 shows the T cell control for the allogeneic functional assay for N2. One million T cells (from the same donor as N1-N3) were cultured for 5 days without iDCs.

[0064] [Figure 36] Figure 36 shows the T cell control for the allogeneic functional assay for N3. One million T cells (from the same donor as N1-N3) were cultured for 5 days without iDCs. [Modes for carrying out the invention]

[0065] Detailed explanation Dendritic cells (DCs) are antigen-presenting cells found in both the circulating blood and other parts of the body. DCs are a vital component of the immune system. Antigen presentation by these cells drives the mobilization of the immune system against all types of infection, as well as the development and persistence of immunological memory. Vaccines specifically designed to target DCs are currently being developed for a wide range of diseases, including cancer, and major attempts are underway to develop personalized DC vaccines for infectious diseases, cancer, and transplant rejection. In these disease categories, cell-based therapies using T cells grown in vitro represent another untapped area where major progress is currently being made. DCs are the most potent antigen-presenting cells (APCs), and only APCs can induce naive T cells. DCs play a crucial role in the in vivo proliferation of T cells and can be used to proliferate T cells in vitro. From a mechanistic standpoint, DCs are an essential part of the study of human responses that are important for protective immunity against cancer and infectious diseases, as well as for preventing autoimmunity and transplant rejection.

[0066] Despite the crucial role of DCs in both clinical and basic research contexts, methods for obtaining these cells from individuals remain in development and inefficient processes. Because DCs are present in blood at very low concentrations (<1%), these cells must be generated from monocytes through a time-consuming and laborious process involving static culture and stimulation with cytokines (IL-4 and GM-CSF) contained in the culture medium. In particular, numerous manual steps are required to advance patient-derived whole blood, leukocyte ferresis products, or peripheral blood mononuclear cell (PBMC) samples to a sufficient number of DCs usable for vaccine development, T-cell therapy, or mechanistic studies. Scaling is cumbersome, even at the level of dozens of samples for studies involving one or two conditions or separate blood collections, due to the resource requirements regarding staff time and the number of manual steps. Given the existing and planned uses of these cells on a larger scale, such as in autologous DC-based cell therapies and vaccines, conventional approaches to DC generation pose a significant burden, most importantly in terms of supply and labor costs as well as efficiency and reliability of the manufacturing process.

[0067] The present invention provides an automated cell culture cartridge and system for generating dendritic cells with a uniform and symmetrical flow within the cell culture cartridge. The provided cell culture cartridge includes a cell culture chamber formed between the top and bottom surfaces of the cell culture cartridge. The cell culture chamber includes multiple zones geometrically configured to provide a symmetrical fluid flow channel within the cell culture chamber and to avoid dead spots or dead regions within the cell culture chamber. A dead spot (dead region) in the flow is a region within the cell culture vessel where a uniform flow is not maintained when new medium is provided and depleted medium is removed, thereby affecting DC generation. By providing multiple zones within the cell culture cartridge, the present invention provides a symmetrical flow channel with no dead spots or dead regions with respect to fluid flow. In addition, the cell culture chamber of the exemplary embodiment provides features that enable a uniform flow of new medium and removal of depleted medium.

[0068] Figure 1 shows a top view of the cell culture chamber 1000. The cell culture chamber 1000 is formed between the top and bottom surfaces of the cell culture cartridge. Multiple fluid flow inlets 1130 are provided within the chamber. The embodiment shown in Figure 1 includes eight inlets, one of which is indicated by a very thin line 1135. The inlets 1130 are located at each corner 1120 of the cell culture cartridge. The inlets 1130 may also be located on the top surface of the cell culture cartridge. One outlet 1150 is located in the center of the cell culture chamber on the top surface of the cell culture cartridge. The chamber 1000 includes multiple zones 1160 that are geometrically configured to provide symmetrical fluid flow channels within the cell culture chamber 1000. Notches 1110 are located on the outer periphery of the cell culture chamber to help avoid dead regions or dead spots where uneven fluid flow exists within the cell culture chamber. The support column 1140 extends from the bottom to the top, so that the top does not sag or bend, and as a result, it creates increased pressure in the chamber. The embodiment shown in Figure 1 is an exemplary non-limiting embodiment of the present invention. Other non-limiting embodiments may include a different number of inlets. In some examples of non-limiting embodiments, the cartridge according to the present invention may include 2 inlets, 5 inlets, 10 inlets, 13 inlets, 14 inlets, 20 inlets, 30 inlets, and 100 inlets. Non-limiting embodiments may further include a different number of corners. In some examples of non-limiting embodiments, the cartridge according to the present invention may include 5 corners, 10 corners, 17 corners, 25 corners, 50 corners, and 100 corners.

[0069] In this invention, fluid flow symmetry is achieved within the cell culture cartridge. For example, the cartridge is composed of individual zones, each of which is a space between two fluid inlets. As shown in Figure 1, each zone has a triangular base that tapers in the middle, and each zone is symmetrical with respect to the other zones. In some examples, the number of fluid inlets in the cartridge may be more than or less than eight. In a preferred embodiment, the cartridge is divided into eight individual regions or zones, each having an inlet and a shared outlet (center). This ensures that the entire cartridge is perfused with fresh differentiation medium and that no dead regions or dead spots are formed in the flow. Furthermore, four triangular notches 1110 are positioned around the periphery to avoid dead regions or dead spots in the flow that may occur within these regions. The cartridge includes eight supports that support the top surface of the cell culture cartridge, which may be constructed of poly(methyl methacrylate) (PMMA or acrylate). Without support, the top surface of the PMMA would sag, and the culture medium would support the top of the cartridge, causing pressure to build up inside the cartridge.

[0070] The cartridge may be constructed from any suitable material. In some examples, the cartridge is constructed from polystyrene, acrylate, or a combination thereof. For example, the bottom or base may contain polystyrene, while the top and sides are made of acrylate. In another example, for high-volume production, the cartridge may be made entirely of polystyrene.

[0071] In one exemplary embodiment, the bottom surface comprises polystyrene and / or acrylate. One advantage of using polystyrene for the bottom surface where culture takes place is that this material plays a useful role in the process of generating dendritic cells from PBMCs. In particular, the polystyrene surface can be used to enrich monocytes from a heterologous suspension of PBMCs. This is the first step in a culture process utilized, for example, to generate DCs by differentiating monocytes by culturing in a medium containing IL4 and GM-CSF. Using the same polystyrene surface for dendritic cell generation throughout one cycle of T cell stimulation is highly valuable from a bioprocess perspective, as it eliminates numerous migration steps that would otherwise be required, thereby enabling a closed system for the production of DC-stimulated therapeutic T cells.

[0072] Furthermore, any suitable material may be treated in the cartridge. In some embodiments, the bottom polystyrene surface may be modified to facilitate cell adhesion. For example, the bottom polystyrene surface may be treated with air or oxygen plasma, also known as glow discharge or corona discharge. For example, the bottom polystyrene surface may be modified with proteins or polyamino acids known to facilitate cell adhesion, including, but not limited to, fibronectin, laminin, and collagen.

[0073] The base is for 6-well and 24-well plates (9.5 cm each). 2 and 1.9cm 2 ) or T flask (25cm 2 ~225cm 2 It may have a surface area equivalent to that of conventional well plates such as ). It is also understood that the surface area may be smaller or larger than that of conventional well plates (e.g., those with a surface area equivalent to that of standard cell culture dishes and flasks), for example, about 2.0 cm². 2 ~about 500cm 2For example, approximately 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 100.0, 125.0, 150.0, 175.0, 200.0, 400.0, 500.0 cm 2 It has a surface area and any surface area between them.

[0074] The surfaces of cell culture cartridges can be joined to one another using any method known in the art, such as mechanical fixation, adhesive and solvent bonding, and welding. However, given that the cell immunotherapy products produced using the systems and methods of the embodiments of the present invention are administered to human patients, regulatory issues may prevent the use of certain or all adhesives when assembling the 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, such as the bottom wall, side walls, and top wall, consist of a first material (e.g., polystyrene) and are joined to one another using ultrasonic welding. It should be understood that the above arrangements are merely examples, and that other arrangements for joining surfaces are also embodiments intended for the present invention.

[0075] The height of one or more cell culture chambers can be varied. For example, but are not limited to, examples of cell culture chamber height ranges from 0.5 mm to 100 mm, e.g., 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0 mm or higher, or any height in between. In certain embodiments, the chamber height may be equivalent to the liquid height in cultures typically performed in 6 and 24-well plates, such as 2 to 6 mm, with a volume of approximately 0.8 mL to 6 mL. In other embodiments, the cell culture chamber may be approximately 50 cm 2 They have a culture surface and are large in size, ranging from 10mm to 50mm.

[0076] In some embodiments of the present invention, the cartridge is optically clear or transparent. Such optical clarity, combined with appropriately separated fluid ports, allows the user to view cells on any vertical plane within the cartridge. As shown in Figures 2–5, embodiments of the present invention include an optically clear or transparent cell culture cartridge.

[0077] Figure 2 shows a front view of the cell culture cartridge and system. Figure 3 shows a top view of the cell culture cartridge and system. Figure 4 shows a left side view of the cell culture cartridge and system. Figure 5 shows a right side view of the cell culture cartridge and system.

[0078] Furthermore, as shown in Figures 2-5, stopcocks may be installed on the cartridge or on the reservoir bottle. In particular, stopcocks are installed on specific ports on the cartridge, each performing a specific function. The installation is specific to each function, and the work was done to determine the optimal position to ensure the process is successful and the workflow is easy. For example, a front stopcock is for seeding and harvesting, and the Luer operating valve (LAV) on top of the stopcock allows for aseptic connection of a syringe. A front right stopcock is for seeding and harvesting (adding cold buffer for washing), and air inside the cartridge flows out through the filter at this stopcock as the cell solution is seeded into the cartridge. As another example, a rear left stopcock is for harvesting, and air inside the cartridge flows into the cartridge as the cell solution is removed. Filters attached to the stopcocks prevent the accumulation of pressure or depressurization within the cartridge as liquid is added to or removed from the cartridge.

[0079] In this invention, LAV may be used in a bottle for adding and / or removing culture medium. Traditionally, LAV is sold and marketed for use in anesthesia and IV lines. Therefore, the use of LAV for adding or removing culture medium deviates from conventional use.

[0080] Computational fluid dynamics (CFD) was helpful in the design of the current EDEN cartridge. In particular, CFD was useful in designing the cartridge size, the placement of the supports, and the placement and sizing of the triangular notches.

[0081] In some embodiments, a perfusion rate of 8 μL / min can be maintained. Since this is the same perfusion rate as cell culture systems such as MicroDEN, a linear scale-up of MicroDEN using the system according to the present invention (EDEN) is likely. Each of the eight subsections of the EDEN cartridge is slightly larger than that of a single MicroDEN cartridge, and therefore the effect of perfusion on cells should be similar in EDEN as in MicroDEN. Thus, the present invention makes it possible to easily scale MicroDEN experiments to EDEN without unknown factors such as different fluid flow rates.

[0082] Figure 6 shows an embodiment of system 100 of the present invention. It comprises a peristaltic pump 110. The pump 110 is used to pump fluid to and from a cell culture cartridge 120. The cell culture cartridge 120 has a bottom surface 125 to which cells adhere. In other embodiments, cells do not adhere to the bottom surface. The cell culture cartridge 120 has eight fluid inlets 145 located at the corners of the cell culture cartridge 120. One fluid outlet 135 is located in the center of the cell culture cartridge 120. Connecting tubes 140 connect the fluid inlets to a differentiation medium reservoir (perfusion source) 180 containing differentiation medium 182. The differentiation medium reservoir 180 contains differentiation medium 182 which is pumped to the cell culture cartridge 120. Connecting tubes 140 also connect the fluid outlet 135 to a waste reservoir 184. The depleted culture medium exits the cell culture cartridge 120 through outlet 135 and is pumped to the waste reservoir 184. The lids 170 and 175 on the differentiation medium reservoir 180 and waste reservoir 184 are not removable, thereby maintaining a sterile system. In other embodiments, the lids 170 and 175 can be removed. Stopcocks and / or LAVs 160 and 165 on the reservoir bottles 180 and 184 allow for sterile transfer of differentiation medium to fill the inlet bottle and waste to be removed from the outlet bottle. The console 190 features design space for the arrangement of the previously described components and also includes a display / user face 192, connections 194 and an on / off switch 196.

[0083] Figure 7 shows an embodiment of the present invention having two cartridges. Cell culture cartridge 200 is provided for dendritic cell differentiation from monocytes. A smaller cartridge 220 is provided for maturation and antigen pulsation. In other embodiments, maturation and antigen pulsation may be performed in the main cell culture cartridge without using the second cartridge.

[0084] Figure 8 shows an embodiment of the present invention having a smaller cartridge 320 for maturation and antigen pulsed generation. The smaller cartridge 320 is fluidly connected to an infusion bag 330 containing the final product that is transferred from the smaller cartridge 320.

[0085] Figure 9 shows the disposable and non-disposable components of the present invention. The EDEN console 410 is non-disposable and has a length L. In this embodiment, the length L is 14 inches. The smaller cartridge 420 is for maturation and antigen pulsed generation. The connecting tube 430 connects the inlet and outlet to the reservoir and cartridge. The smaller cartridge 420 and connecting tube 430 are single-use and disposable.

[0086] Figure 10 shows an embodiment of the EDEN automated fluid system. The EDEN system generates monocytes derived from iDCs while continuously perfusing a fresh differentiation medium into a cell culture cartridge.

[0087] Figures 11-14 show the design of a cell culture cartridge according to an embodiment of the present invention. Figure 11 shows the design of a cell culture cartridge with a flow channel. Figure 12 shows the design of a cell culture cartridge with a polystyrene surface (shared) at the bottom of the cell culture cartridge where cells are present. Figure 13 shows the design of a cell culture cartridge with streamlines due to perfusion within the cell culture cartridge. Figure 14 shows the design of a cell culture cartridge with gauge pressure due to perfusion within the cell culture cartridge.

[0088] Figure 15 shows cytokine perfusion into a cell culture cartridge. In this embodiment, the cartridge is initially filled with water (culture medium) without cytokines. Cytokines are perfused into the cartridge at 1.16 mol / m3 (IL-4) through eight inlet ports, and the flow through the cartridge is driven by the perfusion and flows out through the outlet port in the center. In practice, the cell culture cartridge is filled with culture medium containing cytokines. Data are acquired at the lower or bottom face of the flow channel, as shown in Figure 12.

[0089] Figure 16 shows the phenotypes of iDCs generated in cell culture cartridges and 6-well plates, differentiated from MO over 6 days. The labels at the top of the figure indicate the gate from which the plot originated.

[0090] Figure 17 shows the phenotypes of iDCs and mDCs from a cell culture cartridge. iDCs were generated in the cell culture cartridge and then seeded in the cell culture system of the present invention for 1 or 3 days of maturation. The labels at the top of the figure indicate the gate from which the plot originated.

[0091] Figure 18 illustrates an exemplary method for producing an immunotherapy product according to embodiments of the present invention. Figure 18 outlines a method for producing a cell-based immunotherapy product using the system described herein. Briefly, the step of producing a cell therapy product according to a particular embodiment of the present invention includes co-culturing stimulated antigen-presenting cells with cells containing T cells in a bioreactor containing cell culture chambers. A supernatant containing the proliferated therapeutic T cell product is produced during the culture. In a particular embodiment, the T cells must undergo additional culture in one or more additional cell culture chambers to produce a sufficient quantity of antigen-specific T cells to produce a therapeutic response in a patient. To achieve this additional culture, a transfer of the supernatant must occur from the culture chamber in which the supernatant was produced to a subsequent cell culture chamber containing a new supply of antigen-presenting cells. The transfer of the supernatant between cell culture chambers may include introducing a gas flow into the first cell culture chamber, which transfers the supernatant containing the first cell product to the new cell culture chamber through a fluid connector. Furthermore, between each culture step, a perfusion fluid containing, for example, culture medium and cytokines may be perfused into the chamber. In certain embodiments, the perfusion fluid flows through the chamber along a vertical channel to ensure that cells remain in the chamber during culture. The only manual step involved in using the system of the present invention is providing one or more subsequent cell culture cartridges to the system, each cell culture cartridge containing a cell culture chamber, each chamber containing a fresh batch of antigen peptide pulsed autoantigen-presenting cells. The use of gases to facilitate movement may also involve manual steps to operate the system setup, but without compromising the sterility of the system.

[0092] In certain embodiments of the present invention, cells are recovered. Cell recovery is typically achieved by injecting a cold buffer into the cartridge. In some embodiments of the present invention, a Peltier element may be incorporated beneath the cartridge to cool it to somewhere between approximately 20°C and approximately 30°C, which allows for the release of cells without the need to dilute them to a larger volume of fluid.

[0093] In some embodiments, dendritic cells generated in an octagonal cartridge may be moved to a smaller cartridge. When manufacturing dendritic cell-based immunotherapy, immature dendritic cells generated from monocyte differentiation (first step) are typically subjected to additional steps (maturation and antigen pulsation). Conventionally, this is achieved by performing the first step in multiple flasks or wells and then combining the immature dendritic cells into a single flask or well. This type of concentration / combination allows for less use of expensive reagents used for maturation and antigen pulsation, and subsequent waste. In this invention, immature dendritic cells from the octagonal cartridge in which the first step is performed are moved to a smaller cartridge for maturation and antigen pulsation. In some embodiments of this invention, maturation and antigen pulsation are performed in the main cell culture cartridge, and the use of a second cartridge is not required.

[0094] Some embodiments of the present invention may use a Luer-operated valve (LAV) to seed and harvest monocytes (MOs) and immature dendritic cells (iDCs), respectively. This improves the workflow so that the cell solution is not lost during seeding / harvesting. A syringe may be connected to an LAV such as a MicroDEN system. The syringe may be used as a funnel to add MO solution (for seeding) and cold buffer (for harvesting). The syringe may be used to “pipette” up and down to de-adhere and resuspend the iDCs by turbulence caused by the “pipette” action. This “pipette” up and down is actually the pushing and pulling of the syringe plunger.

[0095] The present invention describes exemplary configurations of the system and method utilizing one or more cell culture cartridges, each containing a cell culture chamber configured to be fluidly coupled to one another for processing patient cell material to produce immunotherapy products. It should be understood that, in certain embodiments, the cell culture cartridges are provided in a closed environment. Scaling up this exemplary embodiment is within the knowledge of those skilled in the art by adding modules (e.g., cell culture cartridges) to enable series and / or parallel processing. Those skilled in the art will also recognize that different or alternative configurations may be desired based on the products to be produced.

[0096] In certain embodiments, one or more pumps are operably connected to the cell culture chamber to perfuse the cell culture chamber with perfusion medium. The perfusion medium includes any suitable medium. In some embodiments, the perfusion medium is a differentiation medium. The cell culture cartridge may also include one or more fluid reservoirs. The fluid reservoirs are fluid-connected to the cell culture chamber and may be operably connected to one or more pumps. The fluid reservoirs also include one or more tubes for connecting the pumps and the cell culture chamber. In certain embodiments, one or more pumps are configured to pump fluid from the fluid reservoirs, through the cell culture chambers, and to a waste collection reservoir. In embodiments, the fluid moves from the fluid reservoirs through the tubes to the pumps, and through the inlet to the cell culture chambers, and through the outlets to the outlets, through the tubes, and to the waste collection reservoirs.

[0097] In certain embodiments, the fluid reservoir and / or waste collection reservoir may each be provided as one or more lidded bottles, either contained within the cell culture chamber or fluid-coupled to the chamber. Each reservoir includes an inlet port and an outlet port, or an outlet port and a vent fluid-coupled to the inlet of one or more cell culture chambers. In certain embodiments, for example, a Luer connector and a silicone gasket cut to fit around the Luer connector can be used to prevent leakage through either or both the inlet and outlet.

[0098] In certain embodiments, one or more cell culture cartridges are sized and configured to fit inside an incubator, so that the process takes place within the incubator. Conditions within the incubator include maintaining a temperature of 37°C and a relative humidity of 95–100%. Therefore, the selected material must possess the integrity to withstand these conditions, given that materials (including fluids and organisms) tend to grow under such conditions. Furthermore, in some situations, the conditions within the incubator remain stable, and automatic temperature recording can provide knowledge of temperature fluctuations correlated with any anomalies in the reactions taking place within the incubator. Thus, any power supply should not alter the environment within the incubator. For example, certain pumps generate heat.

[0099] Therefore, in one embodiment, the pump is housed separately from the cell culture cartridge but is still operably fluidly connected to the cell culture cartridge. In another embodiment, the pump is directly attached to the cell culture cartridge and located within the incubator, but is either free from heat or operably connected to a heat sink and / or fan to dissipate heat. Regardless of this configuration, the pump is operably connected to the cell culture cartridge, and then to the cell culture chamber. Additional details relating to perfusion-based automated cell culture systems, e.g., a large-scale culture system made possible by an onboard disposable peristaltic pump for the generation of dendritic cells from monocytes using a chamber with a polystyrene bottom, and a small-scale culture system for endothelial cell culture with onboard reagent storage and perfusion, can be found in International Patent Applications PCT / US2016 / 040042 and PCT / US2016 / 60701, both of which are incorporated herein by reference in their entirety.

[0100] This system may also include a heater for controlling the temperature of the cell culture reservoir, and, if necessary, a fluid reservoir. In such a configuration, an incubator is not required, and the system can operate autonomously with only a power source. If the system lacks a heater, this can be operated inside a cell culture incubator. Some embodiments of the present invention include a carbon dioxide (CO2) environment for the culture medium buffer.

[0101] In yet another embodiment, the cell culture chamber includes one or more sensors (not shown) operably connected to the cell culture chamber. The sensors may be capable of measuring any preferred parameter. For example, the sensors 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, one or more sensors may be connected to one or more cell culture chambers. In a particular embodiment, one or more sensors are connected to one or more cell culture chambers, but not to all chambers in the system. In another embodiment, one or more sensors are connected to all cell culture chambers in the system. In a system having multiple chambers operably connected to one or more sensors, the sensors may be the same in each chamber to which they are connected, they may all be different, or some sensors may be the same and some may be different. In a particular embodiment, one or more sensors are operably connected to a computer system having a central processing unit for issuing commands, so that automatic monitoring and parameter adjustment are possible. Additional details regarding a computer system for implementing the method of the present invention using a cell culture chamber are provided below.

[0102] In certain embodiments, the cell culture chamber has an inlet and an outlet, both of which can be used to fluidly connect the chamber to one or more additional containers via fluid connectors. In certain embodiments, the additional containers include one or more additional cell culture chambers. The system of the present invention may include, for example, any number of cell culture chambers between 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or up to 100, or more than 100, configured to fluidly connect to one another in series for the production of immunotherapy products. Alternatively, one or more cell culture chambers may be arranged parallel to one another to allow the production of immunotherapy products for two or more individuals at a time. In preferred embodiments, the cell culture chambers of the cell culture cartridge are connected via sterile connections.

[0103] Some or all of the system and its components can be designed using CAD software and then transferred to a laser cutter that allows cutting plastic to a specific size and shape. Various connections, such as inlets and outlets, can be generated by laser cutting through holes and then manually drilled to provide threads for receiving male luer fittings. Fluid can then be introduced into the system by connecting the luer adapter to a blunt dispensing needle having a tube pushed into the blunt needle portion. Additional details relating to the construction of the components of the fluid system can be found in international patent applications PCT / US2016 / 040042 and PCT / US2016 / 60701, both of which are incorporated herein by reference in their entirety. Some or all of the system and its components can also be produced using injection molding.

[0104] The above description focuses on the components of the system and various possible configurations. The following description focuses on the process carried out using the system of an exemplary embodiment of the present invention. To stimulate and proliferate antigen-specific T cells, the process begins in a cell culture chamber with co-culture of T cell-containing cells and APCs obtained from the same organism. In certain embodiments, T cell-containing cells include peripheral blood mononuclear cells (PBMCs), and APCs include DCs. The ratio of T cell-containing cells to APCs is approximately 1000:1 to 1:1000 (T cell-containing cells:APCs), for example, but not limited to approximately 1000:1, 900:1, 800:1, 700:1, 600:1, 500:1, 400:1, 300:1, 200:1, 100:1, 75:1, 50:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1 It can be supplied to the cell culture chamber in ratios such as 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:50, 1:75; 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, or any ratio between these. In one embodiment, a ratio of 10:1 is preferred.

[0105] To initiate T cell stimulation and proliferation through interaction between APCs and T cell-containing cells, the APCs need to be stimulated. This can be done by using one or more stimulating molecules. In certain embodiments, the stimulating molecules are non-tumor-specific. In other embodiments, the stimulating molecules are tumor-specific. For example, the stimulating molecules can be selected from one or more characteristics of an organism's tumor, such as different antigenic peptides. In some embodiments, the stimulating molecules are preferably added only at the start of the culture cycle. The stimulating molecules can be added for a period of about a few minutes, an hour, several hours, or longer. In one preferred embodiment, the stimulating molecules are added for a period of about one hour.

[0106] During the culture of the two cell materials, a supernatant is formed containing lighter, non-adherent T cells, while heavier, mature APCs (e.g., dendritic cells) adhere to or reside at the bottom. In these embodiments where DCs are used as APCs, the proliferated T cells must be removed from the cell culture chamber by the end of the 7-day period, as primary DCs cannot be maintained in the culture for more than 7 days. Therefore, if additional T cell proliferation is desired, a fresh supply of dendritic cells is required. It should also be understood that the cell culture using one batch of dendritic cells can be for any period shorter than 7 days. For example, cells can be cultured for any period from less than 1 minute to 7 days, and the culture period depends on the desired degree of stimulation.

[0107] In the exemplary embodiment, after culturing for 7 days, the proliferated T cells are extracted and transferred to a new cell culture chamber containing new DCs pulsed with, for example, the same antigen peptide used in the initial cell culture chamber. The stimulation process can be repeated many times as needed to generate a sufficient number of cells for a therapeutic dose of T cells. When using a culture surface area equivalent to that of a typical well plate, the stimulation process is typically repeated four times to generate a sufficient supply of T cells.

[0108] Co-culture of APCs and T cells is carried out in a culture medium. Examples of culture media include, but are not limited to, RPMI medium and DC medium sold under the trademark CELLGENIX by CellGenix Inc. (Portsmouth, NH). Any other suitable culture media known in the art may be used according to embodiments of the present invention. Cytokines such as IL-4 and GM-CSF may also be added to the culture medium.

[0109] In one embodiment, perfusion of culture medium and cytokines may be provided to a cell mixture in a cell culture chamber to aid in the formation of cell-based immunotherapy products. In plate-based protocols for stimulating T cells with DCs, a culture volume of approximately 2 mL is maintained from the start by cytokine injections occurring twice, each within a 7-day stimulation period. The main advantage of perfusion is its ability to maintain a consistent local concentration profile of culture medium and cytokines, ensuring higher yields and the potential to accelerate the process of monocyte differentiation into DCs compared to conventional plate-based protocols. However, the combination of adherent (DC) and non-adherent (T cell) types, along with the high sensitivity of DCs to mechanical forces, presents challenges for the stimulation and proliferation of antigen-specific T cells, particularly with respect to fluid flow through the cell culture chamber. Therefore, in these embodiments where culture medium and cytokines are provided by perfusion, the system of the present invention must be able to supply nutrients and cytokines to cells without removing cells from the cell culture cartridge, while also taking into account the shear sensitivity of certain antigen-presenting cells, such as DCs. In particular, some embodiments of the present invention aim to optimize the maintenance of favorable autocrine / paracrine signals for T cell proliferation while restoring growth factors and maintaining minimal physical stimulation of DCs. To achieve this, both the direction and velocity of the perfusion flow through the cell culture chamber must be taken into consideration. For example, some embodiments of the present invention may include non-unidirectional flow media configurations, such as counter-flow media configurations.

[0110] In certain embodiments, the fluid flow rate is maintained lower than the sedimentation velocity of the antigen-presenting cells. In this way, the antigen-presenting cells are retained in the culture chamber due to their mass. In other words, the antigen-presenting cells sink towards the bottom of the cell culture chamber and therefore remain in the cell culture chamber.

[0111] In other embodiments, the multiple inlets and outlets of the cell culture chamber are arranged so that a fluid, such as a perfusion fluid, moves within the cell culture chamber along a vertical channel. This configuration is particularly useful in preventing cells (e.g., both DCs and T cells) from leaving the chamber when the flow rate through the chamber is in the range of 2–10 mL / min. The configuration with symmetrical inflow and vertical outflow prevents cells from leaving the chamber. Certain embodiments of the cell culture cartridge of the present invention have eight inlets and one vertical outlet, as shown at least in Figure 1.

[0112] Although Figure 1 shows a chamber with eight inlets and one vertical outlet, it can have any number of inlets and outlets, as long as the fluid flowing out of the chamber flows vertically from the top of the chamber and through symmetrical fluid channels within the chamber. For example, a chamber may have any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more inlets and / or outlets for perfusion fluid.

[0113] In one particular embodiment, perfusion of the culture medium occurs at specific points in time over the period that the cells are cultured in any one of the cell culture chambers, for example, one, two, three, four, five, six, seven, eight, nine, ten times, or more, each day or week. In other embodiments, the culture medium is perfused continuously throughout the culture. Continuous perfusion helps maintain a nearly constant culture volume throughout the process.

[0114] In certain embodiments, cytokines are injected at one or more points during culture, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more. Alternatively, cytokines may be continuously perfused with the culture medium. In these embodiments, continuous perfusion helps maintain a consistent local concentration profile of cytokines, which can help ensure higher yields compared to static cell culture methods and has the ability to increase the rate at which T cells are stimulated and proliferate.

[0115] The perfusion parameters can be changed at any time during the culture cycle. Examples of parameters include, but are not limited to, the median flow rate, cytokine concentration, and duration of the culture cycle. Each of these parameters can have an impact on the effectiveness of T stimulation. For example, in recent studies designing culture chambers for the differentiation of monocytes into DCs, as described in International Patent Applications No. PCT / US2016 / 040042 and No. PCT / US2016 / 60701, a perfusion rate of the medium corresponding to a wall shear stress level of 0.1 dyn / cm 2 was determined to be able to generate DCs that are phenotypically identical to those generated using a protocol based on conventional 6- or 24-well plates. Thus, by measuring one or more of the phenotypic and functional metrics described above during the culture cycle, the effect of one or more perfusion parameters on effectiveness can be monitored, allowing for appropriate adjustments.

[0116] According to certain embodiments, the effectiveness of the stimulation can be evaluated at any point during the culture, preferably after 7 days. Both phenotypic and functional metrics can be used to evaluate effectiveness. For example, the cell number (fold expansion) can be calculated using the indicated cell counting method. Cell phenotypes, including the evaluation of antigen specificity by tetramer staining, can be characterized by flow cytometry. Functional assays can also be used to evaluate the ability of expanded T cells to recognize target cells and autologous tumor cells loaded with antigen. The results can be evaluated according to criteria for DC-based T cell stimulation performed in both 24-well plate and G-Rex® formats.

[0117] As described above, since certain APCs, such as dendritic cells, cannot survive in culture for more than 7 days, certain embodiments of the present invention include multiple cycles of T cell stimulation using two or more cell culture cartridges in a semi-batch configuration. Each cycle is performed using newly generated autoantigen-presenting cells. In certain embodiments, the antigen-presenting cells are pulsed with the same set of antigens for each stimulation cycle. In other embodiments, different sets of antigens are used for one or more stimulation cycles.

[0118] Generally, multiple cycles of T cell stimulation involve culturing cells in a first cell culture chamber in a manner that generates a supernatant containing a first cell product, providing a second cell culture chamber, and the subsequent transfer of 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.

[0119] For example, in one particular embodiment, a cell culture system is provided which includes a cell culture chamber and a central processing unit including a memory containing instructions to be executed by the central processing unit. In one particular embodiment, the instructions are to be received by the system as first input data including the size of the cell culture chamber, as 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 introduced into the cell culture chamber, and, based on the first and second inputs, to calculate the perfusion rate of the perfusion fluid introduced into the cell culture chamber that maximizes the likelihood that the first and second cell types will come into contact with each other within the cell culture chamber.

[0120] In some embodiments, the system also includes one or more pumps operably connected to one or more perfusion fluid reservoirs and operably connected to the central processing unit, such that the central processing unit controls one or more pumps to control the perfusion rate of the perfusion fluid.

[0121] In certain embodiments, the systems and methods of the present invention utilize fluid-coupled modules (e.g., cell culture cartridges and systems including cell culture chambers) for processing individual cell material to produce immunotherapy products. The systems or devices of the present invention are modular and can be fluid-coupled in series (i.e., fluid flows from one device to another) and / or in parallel with other similar devices, and may be configured to be physically stacked with each other or to be physically placed within associated devices such as incubators. The modular design of the system allows for flexible switching of modules, in particular, depending on the desired process to be included in the system.

[0122] The fluid device of the present invention, which includes a cell culture cartridge containing cell culture chambers, may be provided in either a microfluidic embodiment (i.e., one or more channels or chambers within them having dimensions in the range of about 1 μm to about 999 μm) or a macrofluidic embodiment (all channels or chambers within them having dimensions of about 1 mm or larger).

[0123] The fluid device may further include additional fluid channels or compartments, gaskets or seals, mixing zones, valves, pumps, vents, channels for pressurized gas, conductors, reagents, ports, and tubing, as required by the specific design. They may also include one or more control modules, transmitters, receivers, processors, memory chips, batteries, displays, buttons, control devices, motors, pneumatic actuators, antennas, electrical connectors, etc. The device preferably contains only materials that are nontoxic to mammalian cells and suitable for sterilization by the use of alcohol and / or heat or other means, such as exposure to gamma rays or ethylene oxide gas.

[0124] The materials for the equipment are selected based on their appropriate chemical compatibility under the different temperature and pressure ratings specific to each process. In addition, the selection of pumps implemented in devices such as syringes, peristaltic pumps, pressure pumps, and rotary pumps is based on the flow and pressure requirements for different functions, ranging from nL to mL in flow rate and 10 to 10,000 psi in pressure.

[0125] The system of the present invention may also include one or more sample solution reservoirs, wells, or other devices for introducing samples into the device at various inlets of a module that are in fluid communication with an inlet channel. Reservoirs and wells used to load one or more samples into the fluid device of the present invention include, but are not limited to, syringes, cartridges, vials, Eppendorf tubes, and cell culture materials (e.g., 96-well plates).

[0126] Where useful, the surface of the device can be made more hydrophilic, for example, by exposure to plasma, or coated with one or more gels, chemically functionalized coatings, proteins, antibodies, proteoglycans, glycosaminoglycans, cytokines, or cells. In embodiments, the cell culture cartridge and system are located in a centralized area. The device is single-use, i.e., patient material is processed in bags, tubes, and cell culture vessels, used only for a single patient's cells.

[0127] The fluid device of the present invention preferably does not leak fluid under operating conditions and allows for sterile operation over a period of several days to several weeks. The fluid device of the present invention also includes a sampling mechanism that allows the fluid to be removed from the system for testing without introducing new materials or contaminants into the system.

[0128] In certain embodiments, at least a portion of the cell culture system includes disposable components, some or all of which may be housed in a non-disposable frame or console. In other embodiments, all components of the system are disposable. Furthermore, in some embodiments, the cell culture system includes a sample tracking component for tracking and describing patient material. In embodiments, the cell culture cartridge and system are located in a centralized area. The equipment is single-use, i.e., patient material is processed in bags, tubes, and cell culture vessels used only for a single patient's cells.

[0129] During the manufacturing process, at least one step, and sometimes multiple or all steps, the characteristics of the product (e.g., purity and polymorphism) are monitored using various in-line process analysis tools (PTA) or miniaturized micro-total analysis systems (micro-TAS).

[0130] As described above, the cell culture system of the present invention can control the direction and flow of fluids and entities within the system. The system of the present invention can manipulate the flow of cells, reagents, etc., in one or more directions and / or into one or more channels of a fluid device, for example, using a pressure-driven flow control device utilizing valves and pumps. However, other methods may also be used, either alone or with electroosmotic flow control devices, electrophoresis and dielectrophoresis (Fulwyer, Science 156, 910 (1974); Li and Harrison, Analytical Chemistry). It can be used in combination with pumps and valves such as 69, 1564 (1997); Fiedler, et al. Analytical Chemistry 70, 1909-1915 (1998); and U.S. Patent No. 5,656,155 (each of which is incorporated herein by reference).

[0131] The system of the present invention may also include, or be operably linked to, one or more control systems for controlling the movement of fluids through the system, monitoring and controlling various parameters such as temperature within the system, and detecting the presence, quantity (direct or indirect), and conversion rate of cell-based immunotherapy products. The system may also include a number of classes of software, such as advanced real-time process monitoring and control processes that enable feedback control, and processes that enable integration and scale-up considering the reaction and purification results obtained using the system.

[0132] In certain embodiments, the system includes combinations of micro, milli, or macrofluidic modules and tubes that are interchangeable in terms of channel dimensions, flow geometry, and interconnections between different modules of the device. Each module and tube may be designed for a specific function. In one embodiment, all modules in the system are designed for cell culture and T cell stimulation. In other embodiments, modules in the system are designed for different functions such as tissue processing, dendritic cell generation, cell culture, concentration, and / or purification, all integrated for the continuous production of immunotherapy products. Both homogeneous and heterogeneous processes suitable for flow applications are considered. These processes are designed and optimized with respect to the handling of starting materials, as well as conditions such as temperature, pressure, and flow rate, so as not to easily clog the system during the flow process.

[0133] The device is scaled up by adding modular reactors in parallel or expanding modular channels, while maintaining the set of dimensionless parameters characteristic of each process constant, as well as the dimensional parameters within upper and lower limits. During process integration and optimization, process determination variables, including temperature, pressure, flow rate, and channel dimensions, are modified to achieve the desired trade-off between yield, purity, and capacity. Throughout the optimization process, the aforementioned set of dimensionless parameters undergoes algebraic optimization due to operational constraints, which are lower and upper bounds on the determination variables. The objective function considers the combination of operational variables for purity, yield, and capacity. While the dimensionless parameters determine the steady-state quality of the device, the quality of the device's startup is also useful, as it determines the device's productivity in the form of the time required to reach a steady state, and subsequently delay time and waste. The startup dynamics are analyzed using both simulation and experiment, and the results are used to optimize the startup through the implementation of real-time feedback control.

[0134] Aspects of the disclosure described herein, such as controlling the movement of fluids through the systems described above, and monitoring and controlling various parameters, can be carried out using any type of computer device, such as a computer or a programmable logic controller (PLC), which may include a processor, such as a central processing unit, or any combination of computer devices, each of which performs at least a part of a process or method. In some embodiments, the systems and methods described herein may be carried out using a portable device, such as a smart tablet, a smartphone, or a specific device made for the system.

[0135] The methods of this disclosure can be carried out using software, hardware, firmware, hardwiring, or a combination thereof. Features that implement the functionality may also be physically located in various locations, and include being distributed so that some of the functionality is implemented in different physical locations (for example, wirelessly or wired, with the imaging device in one room, the host workstation in another room, or in separate buildings).

[0136] Suitable processors for executing computer programs include, for example, one or more general-purpose and dedicated microprocessors, as well as any type of processor in a digital computer. Generally, a processor receives instructions and data from read-only memory, 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.

[0137] Generally, a computer also includes, or is operablely connected to, one or more non-temporary mass storage devices for storing data, such as magnetic disks, magneto-optical disks, or optical disks, or to receive data from them, transfer data to them, or both. In some embodiments, sensors on the system send process data via Bluetooth® to a central data acquisition unit located outside the incubator. In some embodiments, data is sent directly to the cloud rather than to physical storage. Suitable information carriers for embodying computer program instructions and data include, by example, all forms of non-volatile memory, including semiconductor memory devices (e.g., EPROM, EEPROM, solid-state drives (SSDs), and flash memory devices); magnetic disks (e.g., internal hard disks or removable disks); magneto-optical disks; and optical disks (e.g., CDs and DVDs). The processor and memory may be complemented by or integrated with dedicated logic circuits.

[0138] To provide user interaction, the subject matter described herein may be implemented in a computer having I / O devices, such as CRTs, LCDs, LEDs, or projection devices for presenting information to the user, as well as input or output devices such as keyboards and pointing devices (e.g., mice or trackballs), thereby allowing the user to provide input to the computer. Other types of devices can similarly be used to provide user interaction. For example, feedback provided to the user may be in any form of sensory feedback (e.g., visual, auditory, or tactile feedback), and input from the user may be received in any form, including acoustic, voice, or tactile input.

[0139] The subject matter described herein may be implemented in a computer system including backend components (e.g., data servers), middleware components (e.g., application servers), or frontend components (e.g., a client computer having a graphical user interface or web browser that allows a user to interact with the implementation of the subject matter described herein), or any combination of such backend, middleware, and frontend components. The components of the system may be interconnected through a network by any form or medium of digital data communication, such as a communication network. Examples of communication networks include cell networks (e.g., 3G or 4G), local area networks (LANs), and wide area networks (WANs), such as the Internet.

[0140] The subject matter described herein may be implemented as one or more computer programs explicitly embodied on an information carrier (e.g., a non-temporary computer-readable medium) for execution by or control of one or more computer program products, such as data processing devices (e.g., programmable processors, one computer, or more computers). A computer program (also known 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 can be deployed as a standalone program or module, in any form including components, subroutines, or other units suitable for use in a computer environment. The systems and methods of the present invention may include, but are not limited to, instructions written in any suitable programming language known in the art, including C, C++, Perl, Java®, ActiveX, HTML5, Visual Basic, or JavaScript®.

[0141] Computer programs do not necessarily correspond to files. A program may be stored in a file or part of a file that holds other programs or data, in a single file dedicated to the target program, or in multiple collaborative files (for example, a file containing one or more modules, subprograms, or parts of code). Computer programs may be deployed to run on one computer or multiple computers in one location, or they may be distributed across multiple locations and interconnected by communication networks.

[0142] A file can be a digital file stored on, for example, a hard drive, SSD, CD, or other tangible, non-temporary medium. A file can be sent from one device to another over a network (for example, through a network interface card, modem, wireless card, etc., as a packet sent from a server to a client).

[0143] Writing a file according to embodiments of the present invention involves transforming a tangible, non-temporary computer-readable medium by, for example, adding, removing, or rearranging particles (e.g., by net charge or dipole moment on a magnetization pattern by a read / write head), the pattern then representing a new collocation of information about a physical phenomenon of interest that is desired and useful to the user. In some embodiments, writing involves a physical transformation of a substance in a tangible, non-temporary computer-readable medium (e.g., by certain optical properties, so that an optical read / write device can then read the new, useful collocation of information, e.g., by burning a CD-ROM). In some embodiments, writing a file involves transforming a physical flash memory device, such as a NAND flash memory device, and storing information by transforming physical elements in an array of memory cells made of floating-gate transistors. Methods for writing to a file are well known in the art and can be invoked manually or automatically, for example, by a program, or by save commands from software or write commands from a programming language.

[0144] A suitable computer device typically includes mass memory, at least one graphical user interface, at least one display device, and typically includes communication between devices. Mass memory describes a type of computer-readable medium, i.e., computer storage medium. Computer storage mediums may include volatile, non-volatile, removable, and non-removable media implemented in any way or technique for storing information such as computer-readable instructions, data structures, program modules, or other data. Examples of computer storage media include RAM, ROM, EEPROM, flash memory, or other memory technologies; CD-ROM, digital versatile disk (DVD), or other optical storage devices; magnetic cassettes, magnetic tapes, magnetic disk storage devices, or other magnetic storage devices; wireless automatic identification tags or chips; or any other media that can be used to store desired information and can be accessed by a computer device.

[0145] A computer system or machine used in embodiments of the present invention may include one or more processors (e.g., a central processing unit (CPU), a graphics processing unit (GPU), or both), main memory, and static memory, communicating with each other via a bus, as a person skilled in the art would recognize as necessary or optimal for carrying out the methods of the present invention.

[0146] In the exemplary embodiment shown in Figure 19, the system 600 may include a computer 649 (e.g., a laptop, desktop, or tablet). The computer 649 may be configured to communicate across the entire network 609. The computer 649 includes one or more processors 659 and memory 663, as well as an input / output mechanism 654. If the method of the present invention uses a client / server architecture, the operation 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 can obtain data, instructions, etc., or provide results via an interface module 625, or provide results as a file 617. The server 613 may operate across the entire network 609 through the computer 649 or terminal 667, or the server 613 may be directly connected to terminal 667, which includes one or more processors 675 and memory 679, as well as an input / output mechanism 671.

[0147] A system 600 or machine according to an exemplary embodiment of the present invention may further include a video display unit (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)) for any of I / O 649, 637, or 671. A computer system or machine according to some embodiments may also include an alphanumeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse), a disk drive unit, a signal generating device (e.g., a speaker), a touchscreen, an accelerometer, a microphone, a cellular radio frequency antenna, and a network interface device which may be, for example, a network interface card (NIC), a Wi-Fi card, or a cellular modem.

[0148] Memory 663, 679, or 629 according to exemplary embodiments of the present invention may include a machine-readable medium in which one or more sets of instructions (e.g., software) embodying one or more of the methods or functions described herein. The software may also reside, all or at least partially, in main memory and / or the processor during its execution by the computer system, and the main memory and processor also constitute the machine-readable medium. The software may be further transmitted or received over the network via a network interface device. Effect of monocyte seeding density on DC generation

[0149] Dendritic cells (DCs) are becoming increasingly important for research and clinical use, but obtaining a sufficient number of dendritic cells is becoming a challenge. The effect of monocyte (MO) seeding density on the generation of monocyte-derived immature DCs (iDCs) was investigated in the 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 examined. The data show a strong relationship between iDC phenotype, particularly CD80 / 83 / 86 expression, and T cell proliferation. The cell culture system for generating iDCs of the present invention was studied at 200k–600k MO / cm². 2 The system demonstrated superior induction of T cell proliferation within a given seeding density range compared to iDCs produced in well plates. This may be due to the perfusion in the cell culture system of the present invention, which continuously supplies fresh differentiation medium to differentiating MOs while simultaneously removing depleted medium and toxic byproducts of cellular respiration. The cell culture system of the present invention produced fewer iDCs than well plates on a normalized basis at lower MO seeding densities, but produced a comparable number of iDCs at an MO seeding density of 600k. These results demonstrate that the cell culture system of the present invention can produce more iDCs with less manual work than standard well plate culture, and that the cell culture system producing iDCs of the present invention has a higher capacity to induce T cell proliferation.

[0150] Dendritic cells are antigen-presenting cells that are primarily found in solid tissues and play an essential role in activating both adaptive and humoral immune responses. The main functions of dendritic cells (DCs) are to identify and capture foreign antigens that pose a threat to the body, process them into smaller peptides, and present these peptides to naive T cells or naive B cells. During antigen presentation, DCs use CD4 + Helper T cells and CD8 + DCs can activate cytotoxic T cells, as well as naive B cells and memory B cells. In addition, DCs activate natural killer (NK) cells and natural killer T (NKT) cells. Given their ability to elicit responses from various immune cells, DCs are attractive targets for therapeutic manipulation. Vaccines containing antigen-loaded DCs for in vivo activation and proliferation of T and B cells are used to treat infectious diseases and have been developed in several clinical and preclinical trials to specifically target cancer cells. Furthermore, DCs also play a significant role in the emerging field of T cell-based immunotherapy and are used to proliferate activated T cells in vitro.

[0151] Direct isolation of patient-specific dendritic cells (DCs) is challenging because they reside in solid tissues and are present in human blood at very low concentrations (<1%). Therefore, DCs are often generated ex vivo from monocytes or stem cell precursors, which can be readily isolated from circulating blood. A standard method for generating DCs for therapeutic operations involves isolating peripheral blood mononuclear cells (PBMCs) from peripheral blood leukocyte-removed products, enriching CD14+ monocytes (MOs) by plastic adhesion, eluting, or positive selection by magnetic beads, followed by culture with IL-4 and GM-CSF for 5–10 days. Traditionally performed in well plates and T-flasks, this method requires numerous manual operations, including differentiation medium replenishment, throughout the culture period. The number of immature DCs (iDCs) generated ranges from approximately 9–15 million and 6–20 million in 6-well plates and T-175 flasks, respectively, although treatment may require approximately 150 million DCs per single dose. Scaling up current DC generation technologies to produce the appropriate number of DCs required for clinical immunotherapy is a challenge due to the need for numerous manual operations, a large number of well plates / T flasks, and considerable labor costs. In addition, identifying the optimal monocyte seeding density is a substantial challenge associated with scaling up the differentiation process from MOs to iDCs, and such information is difficult to elucidate from the literature.

[0152] To overcome the aforementioned shortcomings of manual DC generation, we designed a closed, automated cell culture system that generates DCs from monocytes with similar functionality to iDCs generated in well plates. The cell culture system of this invention incorporates a closed-tube and cell culture cartridge system that continuously perfuses the cartridge with fresh differentiation medium while simultaneously removing depleted medium and waste (CO2 and lactate). This setup also reduces the manual steps required for initiation and medium replenishment.

[0153] The effects of MO seeding density on iDC yield, phenotype, and functionality were investigated. Three seeding densities were studied in the cell culture system and 6-well plate control of the present invention: 200,000 MO / cm³. 2 400,000 MO / cm 2 and 600,000 MO / cm 2 As the iDC seeding density increased, the iDC yield increased in the cell culture system of the present invention and remained constant in well plates. The iDC yield in the cell culture system of the present invention was lower in well plates at MO seeding densities of 200k and 400k, and comparable to that in well plates at MO seeding density of 600k. The iDC phenotype showed a strong dependence on MO seeding density in the cell culture system of the present invention, where iDCs produced from lower seeding densities induced higher T cell proliferation. The iDCs produced in the cell culture system of the present invention were phenotypically similar to those produced in 6-well plates, thereby supporting previous studies that iDCs produced in the cell culture system of the present invention induce more T cell proliferation than iDCs produced in well plates.

[0154] Three identical experiments (N1, N2, and N3) were systematically performed to evaluate the performance of the cell culture system of the present invention. Each experiment consisted of three cartridges of the present invention (one cartridge per seeding density) and one or two 6-well plates (2-3 wells per seeding density). MO cells from a single donor were used for each individual experiment (N1, N2, or N3), and a total of three donors were required for iDC generation. T cells from a fourth donor were used for all allogeneic functional assays. CellGenix GMP DC medium was used as the basal medium for differentiation from MO cells to iDCs. The medium was supplemented with 1% penicillin-streptomycin (Gibco 15140122) and 350 U / mL of preclinical IL-4 and GM-CSF (CellGenix) for iDC generation.

[0155] Each differentiation experiment from MO to iDC lasted 6 days. All experiments were performed in a standard cell culture incubator maintained at 37°C and 5% CO2, with near-saturation humidity. All procedures were carried out under sterile conditions in a laminar flow hood. All cell counting was performed using a Countess II automated cell counter. Cell culture system of the present invention

[0156] Experiments using the cell culture system of the present invention were performed using an automated cell culture system previously described by the inventors' group. The polystyrene surface of each cell culture cartridge was treated with O2 plasma at a power output of 50 W for 90 seconds. Each cartridge had a polystyrene surface area of ​​39.7 cm² and a volume of approximately 12.7 mL. The perfusion rate of differentiation medium was 8.0 μL / min throughout the entire duration of the experiment. For each experiment, one cartridge of the present invention was used, for a total of three cartridges for each seeding density. This required two pump devices, as each device holds two cartridges. Previous experiments, verified by centrifugation of the effluent and subsequent cell counting, showed that cells were not removed from the cartridges by a medium perfusion of 8.0 μL / min throughout the experiment.

[0157] On day 0, differentiation medium was added to the inlet bottle to allow for 3 days of medium perfusion. On day 3, when the inlet bottle was almost empty, new differentiation medium was added to allow for another 3 days of medium perfusion. Fluid was removed from the outlet bottle on day 3. On day 6, cells were collected by aspirating the cell medium and washing the cartridge twice with cold DPBS (4°C). Adherent cells remaining after the two DPBS washes were not collected. 6-well plate

[0158] Corning Costar 6-well plates (3516) were used as controls for each experiment. 2.5 mL of differentiation medium was added to each well. Empty wells were filled with 3.0 mL of DPBS to minimize evaporation. On day 3, 1 mL of fresh differentiation medium was added to each well. On day 6, cells were collected by aspirating the cell medium and washing each well twice with cold DPBS (4°C). Adherent cells remaining after two DPBS washes were not collected. Isolation of PBMCs and MO / T cells

[0159] Four units of whole blood (approximately 470 mL / unit) were collected from a normal, healthy donor and purchased from StemExpress. The blood was collected by venipuncture and processed on the same day. PBMCs were isolated using Ficol density gradient medium and suspended in CryoStor CS10 cryopreservation medium at a concentration of approximately 50 million PBMCs per mL. The cells were cooled in Mr.Frosty containers at -80°C for 12–24 hours and then transferred to cryogenic LN2 storage for at least one week before resuscitation. MO or T cells were enriched from PBMCs using Miltenyi Biotec CD14 or CD3 microbeads and passed through two LS columns. Allogeneic T-cell function assay

[0160] Allogeneic T cell function assays were performed in Corning Costar 24-well plates (3526) using 0, 200,000 (200k), or 500,000 (500k) iDCs generated in MicroDEN or 6-well plates. Each well contained 1 million allogeneic T cells. CellGenix DCM was used as the base medium and supplemented with 1% penicillin-streptomycin (Gibco 15140122) and 5% human AB serum (Sigma Aldrich H4522). T cells were stained with CellTrace Far Red to assess proliferation. The 24-well plates were covered with foil to protect them from light and placed in an incubator (37°C and 5% CO2) for 5 days. Upon harvesting, the cell solution was aspirated, and the wells were washed twice with cold DPBS (4°C) to collect any remaining cells. The data was analyzed using FCS Express 6 Flow software. Immunophenotypic testing

[0161] Flow cytometry was performed using an ACEA Biosciences NovoCyte instrument with 488 nm and 640 nm laser beams and four fluorescence channels. Cells were first stained with Fc Block (BD Biosciences 564220) for 10 minutes after viable cell staining and before antibody staining. Panel A tested viability (Live / Dead Fixable Green; Invitrogen L34970), CD209 / DC-SIGN (R&D Systems FAB161P100), CD14 (Abcam abl57312), and CD45 (R&D Systems FAB1430A). Panel B tested CD80 (BD Biosciences 557226), CD83 (BD ​​Biosciences 556855), CD86 (BD Biosciences 561128), and CD45. Panel C includes survival rates, HLA-DR (R&D Systems FAB4869P), CD11c (BD Biosciences 565227), and CD45 (R&D The Systems FAB1430A was tested. Panel D measured survival rate, CD3 (BD). Biosciences 555333), CD45(BD Biosciences 340953) and CellTrace Far Red were tested. Gating was set using a CD209 isotype control (R&D Systems IC0041P) and a fluorescence-minus-one (FMO) control for panel A. Data were analyzed using FlowJo software. Flow cytometry gating strategy

[0162] Large cells were gated using an SSC-A / FSC-A plot, followed by single cells using an FSC-A / FSC-H plot. Panel A: Survival / CD45 + The cells were gated, and then CD14 / CD209 was plotted. - / CD209 + The percentage of iDCs was determined based on the population. Panel B: Lymphocytes were gated in the CD45 histogram, and then CD80 / 83 and CD80 / 86 were plotted to determine the iDC phenotype. Panel C: Survival / CD45 + Cells were gated, and then HLA-DR / CD11c was plotted to determine the iDC phenotype. Panel D: Viable cells were gated, followed by CD3 / CD45 plotting to isolate T cells, and then CellTrace Far Red histograms were performed to untangle T cell proliferation. Three experiments (N1, N2, N3) were conducted consecutively (starting on different days), each containing one cartridge of the present invention and 2-3 wells of a 6-well plate at a different MO seeding density. Viability and iDC immunophenotyping were performed by flow cytometry. The number of iDCs generated and the iDC yield were calculated as follows:

[0163] The number of recovered viable iDCs = [recovered cells] × [viable / CD45+ cells] × [CD209+ / CD14- cells]. iDC yield = recovered viable iDCs ÷ seeded MO iDC phenotype

[0164] iDCs generated using the cell culture system and 6-well plates of the present invention were phenotypically similar, with only subtle differences in CD209(DC-SIGN) / 80 / 83 / 86 expression, which depended on the MO seeding density. MO-derived iDCs were similar in their CD209 expression. + Therefore, depending on the differentiation conditions, it may have low CD14 expression. Regarding this study, CD209 + CD14 - Only cells were considered as iDCs. Figures 20 and 21 show the phenotypic expression for iDCs generated in the cell culture system and 6-well plates of the present invention, respectively, for experiment N3. Data from N1 and N2 are shown in Figures 28-32.

[0165] The viability of the recovered cells was >90%, which was comparable between the cell culture system and the 6-well plate of the present invention for experiments N1 and N3. Experiment N2 had significantly lower viability, approximately 70–90% for the cell culture system and approximately 77% for the well plate. There was no correlation between MO seeding density and viability, and all recovered cells were CD45+ leukocytes. CD209 expression in iDCs did not show any dependence on MO seeding density in either the cell culture system or the 6-well plate, however, iDCs in the cell culture system had slightly lower CD209 expression than iDCs in the well plate for experiments N1 and N3 (indicated by a left shift in CD209 fluorescence). iDCs in experiment N2 had similar CD209 expression in both the cell culture system and the well plate.

[0166] A considerable population of viable CD45+ cells recovered from the cell culture system of the present invention is CD209 - This CD209 -The population accounted for approximately 20–40% of the cells recovered for experiment N1 / N3 and approximately 2–7% for experiment N2. The 6-well plates contained approximately 2–4% CD209 in all three experiments. - Cells were generated. There was no clear trend between the CD209- population and MO seeding density, however, MO seeding densities of 200k and 400k produced fewer CD209- cells than MO seeding density of 600k. The dichotomy between the CD209- cells recovered from the cell culture system of the present invention and those recovered from 6-well plates may be due to perfusion in the cell culture system of the present invention during differentiation under the tested conditions. Perfusion may play a role in delaying the kinetics from MO to iDC, which may require a longer differentiation period or higher cytokine concentrations for this population to further differentiate into CD209+ iDCs. The cell culture system of the present invention produces fewer differentiated iDCs under certain conditions, suggesting that further optimization of the differentiation conditions in the cell culture system of the present invention (i.e., differentiation period and cytokine concentrations, particularly IL-4 concentration) is needed.

[0167] There is a striking dependence of CD80 / 83 / 86 iDC expression on MO seeding density in the cell culture system of the present invention. iDCs generated in well plates showed relatively constant CD83 / 86 expression, while CD80 expression was highest at an MO seeding density of 200k and decreased with increasing seeding density. All iDCs in both the cell culture system and the 6-well plate of the present invention were HLA-DR + and CD11c + In summary, the phenotypic expression of cells recovered from the cell culture system and well plates of the present invention shows iDCs derived from MO. The iDCs produced in the cell culture system of the present invention are phenotypically similar to iDCs produced in a 6-well plate under similar conditions, with only slight differences at low MO seeding densities. Recovered iDC

[0168] A direct comparison of the total number of recovered cells between the cell culture system and the 6-well plate of the present invention is not useful because the cell culture system of the present invention had a larger number of seeded MOs. Therefore, the number of recovered iDCs is normalized to the surface area of ​​either the cell culture system or the well plate of the present invention, and the iDC yield is plotted in Figures 22-25 to allow for a direct comparison. There was variability between each experiment (N1-N3) in both the cell culture system and the well plate of the present invention, as is expected when using different donor cells for each experiment. CD209 (DC-SIGN) expression is typically high for iDCs derived from MOs, and the relatively high percentage of CD209-cells generated in the cell culture system of the present invention negatively impacts the number of recovered iDCs. Table 1 shows experimental data for iDC generation experiments in the cell culture system and the 6-well plate of the present invention. Normalized recovered iDC

[0169] Figures 22 and 23 show the number of recovered iDCs and average data normalized to the cell culture surface area for each experiment. Both the cell culture system and the 6-well plate of the present invention are "1 cm 2 A positive correlation was observed between MO seeding density and recovered iDCs on a "per" basis, indicating that more MO seeding resulted in the generation of more iDCs. At lower MO seeding densities, the well plates were 1 cm larger than those in the cell culture system of the present invention. 2 Many iDCs were generated around 600k MO seeding density. Both the cell culture system and well plate of the present invention produced 1cm 2 A similar number of iDCs were generated in that area. iDC yield

[0170] Figures 24 and 25 show the iDC yield and average data for each experiment. The cell culture system of the present invention showed a slight positive correlation between MO seeding density and iDC yield when the data were averaged across three experiments; however, there was no clear trend within each individual experiment. At an MO seeding density of 600k, the average iDC yield was similar for both the cell culture system of the present invention and the 6-well plate. The 6-well plate had a relatively constant iDC yield with increasing MO seeding density in experiments N1 and N3; however, the iDC yield decreased sharply with increasing MO seeding density in experiment N2.

[0171] The N2 well plate showed a trend inconsistent with the data generated in this study and other experiments conducted in the inventors' laboratory. The experimental procedure was exactly the same for this well plate, and the inventors are unaware of any specific problem causing this outlier trend. The survival rate for experimental N2 was lower than expected, which may be related to the inconsistent iDC yield in this experiment. Interestingly, the phenotype was normal for the iDCs generated in these well plates. The mean iDC data were plotted with and without data for the 6-well plate N2.

[0172] The cell culture system and 6-well plate of the present invention exhibit similar iDC yields at the highest seeding density, which vary as the seeding density decreases. 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 highest at higher seeding densities, similar to that of the 6-well plate. Furthermore, increasing the MO seeding density above 600k may 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. Similarly, the iDC yield at an MO seeding density of 600k between the cell culture system and well plate of the present invention indicates that the cell culture system of the present invention produces phenotypically similar iDCs with similar yields to those of the well plate. Moreover, more MOs can be seeded into a single cartridge of the present invention, allowing for the recovery of a greater number of iDCs from a single cartridge of the present invention compared to the use of multiple well / well plates. This ultimately reduces user time and minimizes potential errors and contamination. Table 1: Differentiation data for the cell culture system and 6-well plate of the present invention. [Table 1-1] [Table 1-2]

[0173] As shown above in Table 1, for the 6-well plate, N1 used 3 wells, and N2-N3 used 2 wells. Phenotypic data are shown in Figure 20 (cell culture system of the present invention) and Figure 21 (6-well plate). Similar Functional Assays

[0174] The ability of the generated iDCs to induce T cell proliferation was investigated by an allogeneic functional assay. One million T cells derived from a single donor were co-cultured with 200k or 500k iDCs derived from different MO donors for each experiment (N1, N2, N3). Figure 26 shows the proliferation statistics, and Figure 27 shows the T cell proliferation histogram for experiment N1. Histograms for experiments N2 and N3 are shown in Figures 32 and 33. The proliferation statistics include the mitotic index (average number of cells resulting from each divided cell), the proliferation index (average number of cells compared to the number of initial generation 0 cells), and the mitotic percentage (percentage of cells in the initial population that had not undergone division). By performing this allogeneic functional assay, the inventors attempted to answer two questions: (i) does MO seeding density affect the ability of iDCs to induce T cell proliferation, and (ii) how do the iDCs in the cell proliferation system of the present invention compare to iDCs in a 6-well plate at a given MO seeding density?

[0175] (i) A clear correlation exists between the MO seeding density used for iDCs produced in the cell culture system of the present invention and the ability of those iDCs to induce T cell proliferation; however, the MO seeding density appears to have only a very slight effect on the functionality of iDCs produced in well plates. iDCs produced in the cell culture system of the present invention from low MO seeding densities (200k and 400k) exhibit a higher ability to induce T cell proliferation than iDCs produced from an MO seeding density of 600k. T cell proliferation decreases as the MO seeding density used to produce iDCs increases for iDCs in the cell culture system of the present invention, and iDCs in the cell culture system of the present invention have similar functionality to iDCs in well plates when produced at an MO seeding density of 600k.

[0176] (ii) iDCs generated from low MO seeding densities (200k and 400k) in the cell culture system of the present invention significantly outperform iDCs in inducing T cell proliferation compared to iDCs in a 6-well plate. This effect is reduced at higher MO seeding densities (600k), where iDCs in the cell culture system of the present invention perform slightly better than iDCs generated in a well plate. These results were consistent across all three experiments.

[0177] As expected, T cell proliferation was higher when 500k iDCs were seeded in the T cell assay compared to 200k iDCs. Data from this assay demonstrate that iDCs produced in the cell culture system of the present invention can induce T cell proliferation without the addition of IL-2, a conventional cytokine used for T cell proliferation. iDCs produced in the cell culture system of the present invention also induce higher T cell proliferation compared to iDCs produced in 6-well plates, regardless of MO seeding density. It is important to note that allogeneic T cell assays are a standard benchmark used to elucidate the functionality of DCs, and the results observed within this study may not extend to specialized syngeneic and other mixed lymphocyte reaction (MLR) functional assays. Relationship between iDC phenotype and T cell proliferation

[0178] To elucidate why the iDCs in the cell culture system of the present invention have a higher ability to induce T cell proliferation, phenotypic data were compared and shown in Figures 20, 28, and 30 (cell culture system of the present invention) and Figures 21, 29, and 31 (6-well plate). Two important trends were observed: (1) differences in the phenotype of iDCs in the cell culture system of the present invention strongly correlate with T cell proliferation, and (2) there is a very weak to no correlation between the phenotype of iDCs in the 6-well plate and T cell proliferation.

[0179] (1) The phenotype of iDCs in the cell culture system of the present invention depends on the MO seeding density. The cell culture system of the present invention produces substantially more CD80 at lower MO seeding densities compared to an MO seeding density of 600k. + / 83 + / 86 + These CD80 were generated. + / 83 + / 86 + iDC is more differentiated, CD80 - / 83 - / 86 - Compared to iDCs, mature DCs (mDCs) exhibit a more similar phenotype. This is sometimes a result of a lower MO-to-cytokine activity ratio at lower MO seeding densities, and these iDCs consequently have a higher ability to induce T cell proliferation. See Table 1 for MO-to-cytokine activity values. These results indicate that CD80 has higher functionality even when the major cells in the sample are negative for these markers. + / 83 + / 86 + This is consistent with previous studies involving iDCs. Therefore, CD80 generated in the cell culture system of the present invention + / 83 + / 86 + The presence of an iDC demonstrates higher functional capabilities.

[0180] (2) The phenotype of iDC in the 6-well plate is independent of the MO seeding density. The well plate showed a considerable amount of CD80 at all three MO seeding densities. + Along with the group, mainly CD83 - / 86 - iDCs were generated. Furthermore, there were no discernible phenotypic differences in iDCs from well plates generated from different MO seeding densities. This suggests that the MO-to-cytokine activity ratio does not affect iDCs from well plates generated within the scope of this study. This is likely because the MO-to-cytokine activity ratio is sufficient for any reasonable MO seeding density in static culture. Since there were sufficient cytokines available for differentiation relative to the MO and no phenotypic differences were observed, T cell proliferation induced by iDCs from well plates was similar under all conditions studied.

[0181] T cell proliferation occurs when a small number of iDCs are CD80 + / 83 + / 86 + This is reduced in the case of CD80, and is evidenced by lower T cell proliferation for iDCs in the cell culture system of the present invention and in well plates. - / 83 - / 86 - iDC is also iDC is CD80 + / 83 + / 86 + It induces T cell proliferation, albeit to a lower degree than in the case of CD209. This indicates that CD209 itself is not sufficient to predict the ability of iDCs to induce T cell proliferation, and that the degree of CD80 / 83 / 86 expression is a good indicator.

[0182] iDCs in the cell culture system of the present invention, generated from an MO seeding density of 600k, typically induce higher T cell proliferation compared to iDCs in well plates generated under the same conditions (Figure 26). This difference may be a result of perfusion in the cell culture system of the present invention, as all other conditions remain equivalent. Perfusion can affect the dynamics from MO to iDCs. Perfusion in the cell culture system of the present invention also removes the medium from the cartridge, which simultaneously removes toxic by-products (CO2 and lactate) dissolved in the medium due to cellular respiration. Sequential removal of the medium can maintain a lower pH in the cell culture system of the present invention compared to well plates where toxic by-products are not removed. In addition, 1 mL / well of differentiation medium is added to the well plates on day 3 to replenish cytokines. This has an effect on the overall cytokine concentration in the wells, which differs from the cell culture system of the present invention. Detailed analysis of cytokine dynamics (e.g., consumption during MO differentiation and cytokine degradation) along with lactate dynamics and CO2 production is necessary to better understand the specific causes of these results.

[0183] Another factor that can explain the functional differences between the iDCs produced in the cell culture system and well plates of the present invention is the precise properties of the polystyrene surface in contact with the cells. The cell culture system of the present invention used polystyrene treated with O2 plasma, while the 6-well plates were tissue culture treated. The type of surface treatment and the precise properties of the polystyrene can affect iDC generation. Despite these differences, the iDCs produced in the cell culture system of the present invention are phenotypically similar to those produced in standard well plate cultures and are functionally qualified for the proliferation of allogeneic T cells. iDC yield Table 2: 1cm for experiments N1-N3 2 Recovered iDCs [Table 2]

[0184] Table 2 shows the results of experiments N1-N3, particularly the cell culture system of the present invention (39.7 cm²). 2 ) or 6-well plate (9.5cm) 2 1cm about / well) 2 This shows the recovered iDC per unit area. N2 data for the well plate is omitted (1 cm). 2 The average iDC recovered per unit; mean ± standard deviation. Table 3: Mean (± standard deviation) iDC yield for experiments N1-N3 [Table 3] Similar Functional Assays

[0185] Tables 4-6 show the proliferation statistics for the allogeneic functional assays for the data in Figure 26. iDCs were co-cultured with 1 million allogeneic T cells for 5 days. Proliferation histograms are shown in Figures 27 (Experiment N1), 32 (Experiment N2), and 33 (Experiment N3). Figures 34-36 show the T cell controls for the allogeneic functional assays. Table 4: Growth statistics for the allogeneic functional assay for Experiment N1 [Table 4] Table 5: Growth statistics of the allogeneic functional assay for Experiment N2 [Table 5-1] [Table 5-2] Table 6: Growth statistics for the allogeneic functional assay for Experiment N3 [Table 6]

[0186] The cell culture system of the present invention was developed as a sealed, sterile cell culture system to improve the process of generating dendritic cells from precursor PBMCs or monocytes. This study showed that iDCs produced in the cell culture system of the present invention are phenotypic and functionally equivalent to iDCs produced in standard well plates. The optimal MO seeding density for the cell culture system of the present invention and the effect of seeding density on the ability of iDCs to induce T cell proliferation were systematically determined. The data show a strong correlation between iDC phenotype, particularly the degree of CD80 / 83 / 86 iDC expression, and their ability to induce T cell proliferation. Low MO seeding density (200 kMO / cm²) 2iDCs from the cell culture system of the present invention, generated from the above, exhibited the highest ability to induce T cell proliferation, due to the higher CD80 / 83 / 86 expression of the iDCs. The iDCs from the cell culture system of the present invention also performed well in allogeneic T cell assays compared to iDCs from 6-well plates within the studied MO seeding densities range of 200k–600k. Furthermore, the cell culture system of the present invention produced a similar number of iDCs as 6-well plates at higher MO seeding densities, but produced fewer iDCs than 6-well plates at lower MO seeding densities on a normalized basis. Decisions to produce iDCs at lower or higher seeding densities should be carefully considered and depend on the downstream application of the iDCs, taking into account whether it is more important to produce a larger number of iDCs or iDCs with higher functional capacity. These trade-offs are common in standard static culture and, naturally, extend to the cell culture system of the present invention. [Examples]

[0187] EDEN Cell Culture Cartridge and Fluid System EDEN was developed to generate a therapeutically appropriate number of iDCs in a single, completely sealed cell culture cartridge, isolated from the external environment. Fresh differentiation medium was perfused into the cartridge, and depleted medium was removed. iDCs generated in EDEN exhibited similar phenotypic expression and iDC yield to iDCs generated in a 6-well plate. Matured iDCs in the cartridge according to the present invention showed standard upregulation of CD80 / 83 / 86 and downregulation of CD209. Computational fluid dynamics simulations aided in the design of the EDEN cartridge to ensure proper flow of the perfusing medium throughout the cartridge and adequate cytokine replenishment. These results demonstrate that EDEN successfully generated approximately 25 million iDCs with an iDC yield of 20–35% under the tested conditions.

[0188] The EDEN system is shown in Figure 10. The EDEN cell culture cartridge was fabricated from commercially available polystyrene and acrylate cuts using an Epilog Zing 16 laser system and assembled using 3M adhesive transfer tape. The polystyrene substrate was plasma-treated. The cartridge is 383.6 cm². 2 It has an internal surface area, a volume of 122 mL, and dimensions of 21.0 cm × 21.0 cm × 0.317 mm (length × width × height). Eight inlet ports around the outer perimeter allow fresh differentiation medium to be perfused into the cartridge, while a single central outlet port allows depleted medium to be removed from the cartridge.

[0189] The fluid system consisted of an inlet bottle for new differentiation medium, a peristaltic pump, and an outlet bottle for collecting effluent from the cartridge. An Ismatec IPC-N peristaltic pump was used with a PharMED BPT tube to maintain continuous perfusion of new differentiation medium at 8.0 μL / min / inlet. Silicone tubing was connected between the peristaltic tube and the cartridge inlet to facilitate gas exchange between the medium and the ambient environment, where the Thermo Forma incubator interior was maintained at 37°C and 5% CO2. Silicone tubing was also used at the outlet port, where the perfusion rate was estimated to be 64 μL / min. The effluent collected in the waste reservoir was centrifuged to determine whether cells had been washed out of the cartridge by perfusion. No cells were observed in the effluent, and the generated iDCs remained inside the cartridge, indicating that the perfusion rate was not high enough to resuspend the cells present on the polystyrene substrate. 285 mL of fresh differentiation medium was added to the inlet reservoir at the start (day 0) and on day 3, and perfusion was maintained for 6 days of differentiation. Cells were collected by collecting the cell solution and washing each well twice with cold DPBS. Adhering cells after two DPBS washes were not collected. Differentiation medium

[0190] 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). PBMC isolation and monocyte enrichment

[0191] Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood StemExpress using Ficoll-Paque (GE Healthcare). Whole blood was collected and processed on the same day. Isolated PBMCs were cryopreserved in CryoStor CS10 at 50-60 million PBMCs / mL and kept cryopreserved for at least 7 days before resuscitation. Monocytes (MOs) were concentrated from resuscitated PBMCs using Miltenyi CD14 microbeads and passed through two LS columns to obtain MOs with >95% purity. Concentrated MOs from a single donor were suspended in 122 mL of differentiation medium and seeded into EDEN cartridges. Each experiment used MOs from a different donor. 6-well plate control

[0192] Corning Costar 6-well plates (3516) were used as a static control for iDC generation. Each well contained 2.5 mL of differentiation medium, and the empty wells were filled with 3.0 mL of DPBS. 1 mL of fresh differentiation medium was added to each well on day 3. Cells were collected by collecting the cell solution and washing each well twice with cold DPBS. Adhering cells after two DPBS washes were not collected. iDC maturation

[0193] Maturation: 17.4cm 2The process was carried out in the system according to the present invention using a small cartridge holding 5.5 mL of mature medium with a perfusion rate of 3.5 μL / min. The mature 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-α (MilliporeSigma 11088939001), and 1 μg / mL PGE2 (MilliporeSigma P6532). iDCs from the EDEN1 experiment were measured at 422,200 iDCs / cm³. 2 The cells were seeded and matured in an incubator at 37°C and 5% CO2 for either 1 or 3 days. The cells were harvested using two cold PBS washes 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 entirely incorporated herein by reference. Immunophenotypic testing

[0194] An ACEA Biosciences NovoCyte flow cytometer was used for immunophenotyping of recovered iDCs. Panel A tested for viability (LIVE / DEAD Fixable Green Dead Cell Stain; Invitrogen L34970), CD209 (R&D Systems FAB161P100), CD14 (Abcam abl57312), and CD45 (R&D Systems FAB1430A). Panel B tested for CD80 (BD Biosciences 557226), CD83 (BD ​​Biosciences 556855), CD86 (BD Biosciences 561128), and CD45, with viability not included due to channel detection limitations. Panel C tested for CD80, CD83, CD86, and CD209 (R&D Systems FAB161A). The gates were set using a CD209 isotype control (R&D Systems IC0041P) and a fluorescence-minus-one (FMO) control for panel A. Flow cytometry gating strategy

[0195] Large cells were gated in the SSC-A / FSC-A plot, followed by single cells in the FSC-A / FSC-H plot. Panel A: Surviving / CD45+ cells were gated, then CD14 / CD209 was plotted to determine the percentage of MO or iDCs. Panel B: Lymphocytes were gated in the CD45 histogram. Then CD80 / 83 and CD80 / 86 were plotted to determine the iDC phenotype. Panel C: DCs were gated in the CD209 / 80 plot, followed by the CD83 / 86 plot for CD209+ / 80+ or ​​CD209+ / 80- cells. iDC generation

[0196] Two iDC generation experiments were conducted, in which 114.3 million and 78.3 million MO cells were seeded in EDEN cartridges. After 6 days of differentiation, 25.5 million and 24.8 million iDCs were recovered from each cartridge. The recovered viable iDCs were calculated by multiplying the total recovered cells by the number of viable / CD45+ cells and iDCs (CD209+ / 14-). The iDC yield (normalized to the number of seeded MO cells) was calculated as the number of recovered iDCs divided by the number of seeded MO cells, and was 22.3% and 31.7% for the two EDEN experiments. The 6-well plate control shows that the iDC yield was similar to that of EDEN, with the well plate having a higher yield in Experiment 1 and a lower yield in Experiment 2.

[0197] The tabular data is shown in Table 7. The phenotypic data is shown in Figure 16. Table 7: Differentiation data on iDC generation in EDEN and 6-well plates [Table 7] iDC phenotype

[0198] The immunophenotyping of the generated iDCs is shown in Figure 16. iDCs generated in EDEN and 6-well plates were phenotypically similar 6 days after differentiation. The iDCs were CD209 (DC-SIGN) positive, CD14 negative, and showed low CD80 / 83 expression, as expected for MO-derived iDCs. CD86 expression in EDEN2 iDCs was unexpectedly high. This level of expression is typically expected in mature DCs. Since FBS is animal-derived and its composition cannot be strictly controlled, lysed proteins in fetal bovine serum (FBS) supplemented to the basal medium may explain this irregular expression. Additionally, protein contamination of the cartridges, as they were manually incorporated in the laboratory, may also explain this high expression. More than 99.7% of cells were CD45+ in the panel B histogram (not shown). This protein expression profile for iDCs generated with EDEN demonstrates the effectiveness of EDEN in generating a clinically appropriate number of DCs that are phenotypically similar to those produced in static well-plate culture. iDC maturation

[0199] iDCs generated in EDEN1 were then matured in the cartridge according to the present invention for either one or three days. 7.31 million iDCs were seeded into each cartridge (422,200 iDCs / cm2), and 6 million (1-day matured) and 4.8 million (3-day matured) mature DCs (mDCs) were recovered with yields of 81.9% and 66.2%, respectively. The yield was calculated as the number of seeded iDCs divided by the number of recovered mDCs. The number of mDCs was strictly determined by calculating the number of viable CD45+ / 209+ cells, so yield values ​​less than 100% indicate the degree of cell death in each experiment, which was 18.1% (1-day matured) and 33.8% (3-day matured) for the two sub-experiments.

[0200] List the results of maturation in Table 8. Maturation was performed in small-sized cartridges. Phenotypic data are shown in Figure 17. In particular, the immunophenotypic examination of mDCs of EDEN1 is shown in Figure 17. CD209 expression was lower for mDCs and decreased with the duration of maturation. CD80 expression increased from approximately 11% for iDCs to 48% and 55% for DCs matured for 1 day and 3 days, respectively. CD80 expression is generally low in iDCs and upregulated in mDCs, indicating successful maturation. CD83 / 86 expression clearly depends on CD80 expression and is shown in the last two rows of Figure 17. CD80+mDCs showed higher expression of CD86 compared to CD80-mDCs, but CD83 expression remained unchanged. Table 8: Maturation data for iDCs generated in EDEN1

Table 8

[0201] CFD simulations in COMSOL Multiphysics were used in the design of EDEN to understand how the medium flows within the cartridge. Water at 37°C was used to simulate the differentiation medium. The cartridge was first filled with just water without cytokines. In reality, the cartridge is filled with a differentiation medium containing cytokines. However, filling the cartridge first with just the medium (water) made it possible to visualize cytokine convection. The diffusion of cytokines is extremely low (9216 μm 2 / day), because convection serves as the driving force for the cytokine gradient. 1.16 mol / m 3Water containing 500 U / mL of R&D Systems' IL-4 was perfused into the cartridge at 8 μL / min / entrance and exited out through the outlet at the center of the cartridge. Since the inventors were interested in determining the optimal media flow for the new differentiation media, cytokine consumption / depletion was not incorporated into this analysis. Figure 11 shows the flow channels of the cartridge depicting the volume within the cartridge through which the media flows. The IL-4 cytokine concentration was modeled on the lower polystyrene surface of the flow channels where cells are present on the cartridge substrate, represented by the purple surface in Figure 12. The streamlines and gauge pressures due to perfusion are shown in Figures 13 and 14, respectively. The IL-4 concentration gradients are shown in Figure 15 for each 24-hour period of perfusion.

[0202] These CFD data were important in the design of the cartridge for the perfused media to be well-diffused throughout the cartridge. The cytokine concentration and streamline data indicate that with a laminar flow of 8 μL / min / entrance, the cartridge is divided among eight regions. Each region is replenished with fresh differentiation media approximately four days later. Initial CFD simulations indicated that dead regions or dead spots in the flow were formed at the location of the v-shaped notch, so these notches were added to facilitate the desired fluid flow except for the dead regions or dead spots in the flow. The eight cylindrical struts within the cartridge support the upper acrylic surface. Before these were added, slight sagging of the acrylic was observed and the acrylic was supported by the media within the cartridge, which would cause unnecessary pressure within the cartridge that could affect the cells. Thus, adding these features, namely the notches and struts, to improve the dead regions or dead spots in the flow and concerns about pressure led to the final EDEN cartridge design that sufficiently supports the perfused media flowing through the cartridge without causing undesirable pressure gradients. Incorporation by reference

[0203] Throughout this disclosure, references and citations have been made to other documents, including patents, patent applications, patent publications, journals, books, articles, and web content. All such documents are incorporated herein by reference in their entirety for all purposes. Evenly

[0204] Although the present invention has been described in conjunction with certain specific embodiments, those skilled in the art will be able to make various transformations, substitutions of equivalents, and other modifications to the compositions and methods described herein after reading the above specification. The present invention provides, for example, the following items: (Item 1) The multiple zones are geometrically configured to provide fluid flow symmetric to each of the multiple zones in order to avoid dead areas in the flow within each of the multiple zones. A cell culture cartridge containing [the specified components]. (Item 2) The cell culture cartridge according to item 1, wherein the cell culture chamber includes a plurality of corners, an inlet located at each of the plurality of corners, and an outlet located on the top surface of the cell culture chamber. (Item 3) The cell culture cartridge according to item 2, wherein the cell culture chamber includes an octagon having eight corners, each containing an entrance. (Item 4) The cell culture cartridge according to item 2, wherein the outlet is located in the center of the upper surface of the cell culture chamber. (Item 5) A cell culture cartridge as described in item 1, wherein the cell culture chamber includes a bottom surface composed of a material to which cells adhere. (Item 6) The cell culture cartridge according to item 5, wherein the material on the bottom surface is treated with air or oxygen plasma under glow discharge or corona discharge. (Item 7) The cell culture cartridge according to item 5, wherein the material of the bottom surface is modified with proteins or polyamino acids such as fibronectin, laminin, and collagen. (Item 8) The cell culture cartridge according to item 5, further comprising one or more support columns extending between the bottom and top surfaces. (Item 9) The cell culture cartridge according to item 5, wherein the bottom surface includes one or more notches on the outer circumference of the bottom surface. (Item 10) The cell culture cartridge according to item 1, wherein the cell culture cartridge is transparent and made of one or more materials selected from the group consisting of polystyrene and acrylate. (Item 11) The cell culture cartridge described in item 1 further comprises one or more stopcocks operably connected to a cell culture chamber. (Item 12) A cell culture cartridge comprising a plurality of zones, which are geometrically configured to provide fluid flow symmetrical to each of the plurality of zones in order to avoid dead areas in the flow within each of the plurality of zones; and One or more pumps operably attached to a cell culture chamber A cell culture system including... (Item 13) The cell culture system according to item 12, wherein the cell culture chamber includes a plurality of corners, an inlet located at each of the plurality of corners, and an outlet located on the top surface of the cell culture chamber. (Item 14) The cell culture system according to item 13, wherein the cell culture chamber comprises an octagon having eight corners, each containing an inlet, and the placement of the inlets allows for symmetrical fluid flow channels within the cell culture chamber. (Item 15) The cell culture system according to item 13, wherein the outlet is located in the center of the upper surface of the cell culture chamber. (Item 16) The cell culture system according to item 15, wherein the cell culture chamber includes a bottom surface made of a material to which cells adhere. (Item 17) The cell culture system according to item 16, further comprising one or more support columns extending between the bottom surface and the top surface. (Item 18) The cell culture system according to item 16, wherein the bottom surface includes one or more notches on the outer circumference of the bottom surface. (Item 19) The cell culture system according to item 12, further comprising one or more stopcocks operably connected to the cell culture chamber. (Item 20) The cell culture system according to item 12, further comprising at least one fluid connector configured to fluidly connect the cell culture chamber to a second vessel. (Item 21) The cell culture system according to item 12, further comprising one or more sensors operably connected to the cell culture cartridge. (Item 22) The cell culture system according to item 21, wherein 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 cell metabolite concentration. (Item 23) The system further includes a central processing unit, the central processing unit causing the system to receive first input data, including the size of the cell culture chamber; The cell culture chamber receives 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 introduced into the cell culture chamber; and Based on the first and second inputs, the perfusion rate of the perfusion fluid introduced into the cell culture chamber is calculated to maximize the likelihood that the first and second cell types will come into contact with each other within the cell culture chamber. The cell culture system according to item 12, which executes a command. (Item 24) The system according to item 23, wherein the first cell type is peripheral blood mononuclear cells and the second cell type is dendritic cells. (Item 25) The system according to item 23, further comprising one or more pumps operably connected to one or more perfusion fluid reservoirs and operably connected to the central processing unit, wherein the central processing unit controls the perfusion rate of the perfusion fluid by controlling the one or more pumps. (Item 26) A method for culturing dendritic cells, comprising: Providing a cell culture cartridge comprising a plurality of zones geometrically configured to provide a fluid flow symmetric to each of the plurality of zones to avoid dead zones in the flow within each of the plurality of zones; Seeding the cell culture cartridge with monocyte cells; and Incubating the monocyte cells in a cell culture chamber to provide continuous perfusion of a medium into the cell culture cartridge through an inlet and removing depleted medium to a waste reservoir through an outlet to generate dendritic cells. A method comprising the above steps. (Item 27) The method according to item 26, further comprising the step of recovering the dendritic cells, wherein the step of recovering the cells comprises cooling the cartridge. (Item 28) The method according to item 26, further comprising the step of moving immature dendritic cells to a second cartridge, wherein the second cartridge is smaller than the cell culture cartridge. (Item 29) The method according to item 28, wherein the immature dendritic cells mature in the second cartridge and receive antigen pulsing. (Item 30) The method according to item 26, wherein fluid flows symmetrically through the cell culture chamber.

Claims

1. A cell culture system, wherein the cell culture system is A cell culture cartridge, top surface, Bottom, An outlet provided on the upper surface and positioned near the center of the upper surface, Multiple inlets are symmetrically arranged around the cell culture cartridge, and One or more support columns extending from the bottom surface to the top surface Cell culture cartridges containing; A culture medium reservoir fluidized to the aforementioned multiple inlets; A waste reservoir fluidized at the outlet; and A pump fluidly connected to one or more of the culture medium reservoir, the waste reservoir, and the cell culture cartridge, wherein the pump is configured to move culture medium from the culture medium reservoir to the cell culture cartridge and / or from the cell culture cartridge to the waste reservoir. A cell culture system comprising, wherein the bottom surface and the top surface together define at least partially a plurality of zones, each zone comprising one of the plurality of inlets, and each of the plurality of zones provides a symmetrical fluid flow between its respective inlet and the outlet, at least partially due to the position of the inlet in each zone relative to the outlet, thereby avoiding dead areas in the flow within each of the plurality of zones as the culture medium moves through the cell culture cartridge.

2. The cell culture system according to claim 1, wherein the inlet is located on the upper surface of the cell culture cartridge.

3. The cell culture system according to claim 1, wherein the periphery of the cell culture cartridge includes a plurality of corners, each including one of the entrances.

4. The cell culture system according to claim 1, further comprising one or more sensors operably connected to the cell culture cartridge and a central processing unit.

5. The cell culture system according to claim 4, wherein one or more sensors measure one or more parameters including pH, dissolved oxygen, total biomass, cell diameter, glucose concentration, lactate concentration, or cell metabolite concentration.

6. The cell culture system according to claim 5, wherein the central processing unit executes a command to adjust the operating state of the pump in response to a measurement performed by the one or more sensors.

7. The cell culture system according to claim 1, wherein the cell culture system maintains a sterile environment for culturing cells.

8. The cell culture system according to claim 1, wherein the bottom surface is configured so that dendritic cells can adhere to the bottom surface.

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

10. A method for cell culture, A cell culture cartridge, top surface, Bottom, An outlet provided on the upper surface and positioned near the center of the upper surface, Multiple inlets are symmetrically arranged around the cell culture cartridge, and One or more support columns extending from the bottom surface to the top surface, the bottom surface and the front The upper surface together, at least partially, defines a plurality of zones, each zone including one of the plurality of inlets, and each of the plurality of zones provides a symmetrical fluid flow between its respective inlet and outlet, at least partially due to the position of the inlet in each zone relative to the outlet, thereby avoiding dead areas in the flow within each of the plurality of zones as the culture medium moves through the cell culture cartridge, one or more supports Cell culture cartridges containing; A culture medium reservoir fluidized to the aforementioned multiple inlets; A waste reservoir fluidized at the outlet; and A pump fluidly connected to one or more of the culture medium reservoir, the waste reservoir, and the cell culture cartridge, wherein the pump is configured to move culture medium from the culture medium reservoir to the cell culture cartridge and / or from the cell culture cartridge to the waste reservoir. A step of providing a cell culture system including; The step of seeding cells into the cell culture cartridge; and The step of culturing cells in the cell culture cartridge by providing continuous perfusion of culture medium to the cell culture cartridge via the inlet via the pump, and simultaneously removing the depleted culture medium into the waste reservoir via the outlet of the cell culture cartridge. Methods that include...

11. The method according to claim 10, wherein the cultured cells include immature dendritic cells.

12. The method according to claim 10, wherein the inlet is located on the upper surface of the cell culture cartridge.

13. The method according to claim 10, wherein the pump is operably connected to a central processing unit.

14. The method according to claim 11, further comprising the step of collecting the immature dendritic cells, wherein the step of collecting the cells includes the step of cooling the cartridge.

15. The method according to claim 11, wherein the system further comprises a second cell culture cartridge fluidly connected to the cell culture cartridge, and the method further comprises the step of transferring the immature dendritic cells to the second cell culture cartridge, wherein the second cell culture cartridge is the same size as or smaller than the cell culture cartridge.

16. The method according to claim 15, wherein the immature dendritic cells undergo maturation and antigen pulsation in the second cell culture cartridge.

17. The cell culture system according to claim 1, wherein the cell culture cartridge includes one or more notches arranged along the outer circumference of the cell culture cartridge.

18. The method according to claim 10, wherein the cell culture cartridge includes one or more notches arranged along the outer circumference of the cell culture cartridge.

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