Systems and methods for large-scale immune cell expansion and activation

Abstract

Three dimensional (3D) bioreactors for the large-scale expansion of immune cells and methods of use thereof are provided.SOLUTION: 3D bioreactors comprise at least one packed bed chamber comprising at least one microporous scaffold, at least one microporous scaffold coated with one or more extracellular matrix proteins (ECMs), at least one vessel comprising a fluidic media, wherein the fluidic media is configured to flow through the packed bed chamber having the at least one microporous coated scaffold, and at least one population of immune cells suspended in the fluidic media. The at least one porous scaffold coated with ECM forms an anchoring niche with low shear forces that mimics the natural growth environment of immune cells. This allows for large scale expansion of the immune cell population flowing through the coated porous scaffold.SELECTED DRAWING: FIG. 2 (1)

Description

[Technical Field]

[0001] The present invention relates to systems and methods for culturing and / or activating immune cells on a large scale, more particularly to the large scale culturing and / or activation of immune cells in packed bed bioreactors. [Background technology]

[0002] Mammalian cell culture is inherently complex due to their high sensitivity, relatively slow proliferation, complex differentiation process, and the fundamental requirement of complete sterility. Large-scale cell culture is always a challenging procedure. Serious problems are often encountered, and even small-scale cell culture requires careful and specific knowledge. For example, cell cultures can be stressed or damaged during cell culture preparation and testing procedures, and analyses based on cultured cells may show results that are at least partially the result of such damage. Furthermore, applying conclusions obtained with damaged cells to in vivo conditions or the use of cells in immunotherapy can have potentially fatal consequences. Furthermore, damage and stressful conditions are not reproducible between individual cell cultures and can affect cell proliferation. These challenges become even more significant when working on large-scale cell cultures. Various attempts have been made over the past few decades. Patent Document 1 describes an apparatus and method for culturing cells and / or tissues that mimic the cellular structure and immune function of immunoactive tissues, but this system and method is limited to a 4 ml volume. Further patents and patent applications related to the field of the present invention are Patent Documents 2 and 3.

[0003] Additionally, the use of bioreactors for cell and tissue culture is well known. Detailed overviews on bioreactor design, prototyping, and process control for reproducible 3D tissue culture are provided in the following links: https: / / www.minerva-kg.de / libraryonline / upload / files / file6400.pdf

[0004] In vitro cultivation of immune cells is even more challenging. In recent years, the human immune system has been harnessed as a platform for therapeutic technologies capable of recognizing and killing tumor cells, becoming a central goal of anti-cancer immunotherapy. Therefore, there is growing interest in improving the efficacy and accessibility of this technology, making it widely applicable to adoptive cell therapy (ACT), including chimeric antigen receptor T (CAR-T) cells, tumor-infiltrating lymphocytes (TILs), dendritic cells (DCs), natural killer (NK) cells, and many others. However, to implement this technology, currently available immune cell culture systems fall short due to cell damage, low efficacy, limited scalability, and prohibitive costs (see Non-Patent Document 1). In this paper, the major culture methods known to date for immune cell culture are reviewed, along with their advantages and disadvantages. More specifically, a comparison is made between shaker flasks, G-Rex flasks, rocking bioreactors, stirred tank bioreactors, hollow fiber bioreactors, and the CliniMACS Prodigy. Thus, there is an urgent need for scalable, cost-effective, and GMP-compliant bioreactors for the cultivation of immune cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent Application No. 2006 / 0194320 [Patent Document 2] U.S. Patent No. 8,911,995 [Patent Document 3] U.S. Patent No. 10,472,612 [Non-patent literature]

[0006] [Non-Patent Document 1] Ganeeva I. et al. 2022 "Recent Advances in the Development of Bioreactors for Manufacturing of Adoptive Cell Immunotherapies", Bioengineering 2022,9,808. https: / / doi.org / 10.3390 / bioengineering9120808 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide systems and methods for culturing and / or activating immune cell populations on a large scale. [Means for solving the problem]

[0008] In one main aspect, the present invention relates to a large-scale system and method for culturing and / or activating immune cells. The challenges to be overcome for effective large-scale culture of immune cells are to avoid or minimize high shear stress that would damage cells in order to obtain a large-scale homogeneous system, and to create conditions that allow interaction between cells, activating agents, and transfection agents to create physical niches and conditions that allow high cell-cell interaction.

[0009] In one aspect, the present invention discloses a method and system for culturing immune cells, comprising a porous stationary phase positioned within a flow medium. The stationary phase may be disposed within the bioreactor or in a separate chamber operatively connected to the bioreactor. The porous elements, described in detail below, are disposed within the basket of the packed-bed bioreactor and are not mobile, flowing through the medium without moving with the flow of the liquid surrounding them. The stationary phase can be implemented in various forms and can be intended to create an environment of low flow rate and low shear force.

[0010] As used herein, the terms "porous stationary phase," "porous scaffold," "porous element," and "porous coated scaffold" all refer to the same thing and may be used interchangeably in the following description. In one embodiment, the porous stationary phase is coated with an extracellular matrix (ECM) and may be further coated with an immune system cell activator to activate and expand immune cells. The combination of all components creates a niche for immune system cells that mimics their natural environment within tissues and lymph nodes.

[0011] As used herein, the terms "activator" and "immune cell activator" all refer to the same thing and may be used interchangeably in the following description. In some embodiments, an "activator" and an "immune cell activator" are antigen-presenting cells loaded with an antigen presented on their surface. In another embodiment, the activator is an antibody directed against an activating receptor on the surface of an immune cell. In yet another embodiment, the activator is an antigen bound to a molecule presentable to an activating receptor on the surface of an immune cell.

[0012] As used herein, the term "niche" refers to a stationary phase, such as, but not limited to, a scaffold, bead, or carrier, that has pores that allow fluids and particles to pass through. Particles may be cells or other components, and may be synthetic or natural.

[0013] The created niche mimics the lymph node / tissue in terms of the microenvironment that immune cells naturally grow in, resulting in optimal cell proliferation. Furthermore, because the created niche mimics the cells' natural environment, it allows for activation of cells in a way that only specific cells respond to activation, resulting in specific selection for desired cells.

[0014] Furthermore, the formed niche allows immune cells to proliferate extensively while maintaining relatively low shear forces, minimizing cellular damage.

[0015] The terms "media" and "medium" are synonymous and may be used interchangeably below.

[0016] The terms "packed bed chamber," "packed bed basket," "basket," and "growth basket" are all intended to be synonymous and may be used interchangeably below.

[0017] References herein to "growth" of cells or cell populations are intended to be synonymous with proliferation of the cell population, culturing of the cell population, with or without activation of the cells.

[0018] As used herein, the terms "immune cells," "immune cell populations," and "lymphoid cells" all refer to the same thing and may be used interchangeably in the following description.

[0019] In certain embodiments, lymphoid cells are expanded without significant differentiation, and in various embodiments, this expansion is performed on a 2D substrate, a 3D substrate, or a 2D substrate followed by a 3D substrate.

[0020] In some embodiments, lymphoid cells are incubated in a bioreactor, non-limiting examples of which are suspension culture and culture on 3D carriers. The term "bioreactor culture" refers to culture in a device (bioreactor), typically sterile, in which cells are maintained under controlled conditions as described below in connection with FIG. 1.

[0021] As referred to herein, "activation" of an immune cell or population of immune cells is intended to be synonymous with exposure of the immune cell to an antigen, which results in changes in cell morphology and triggers an immune response as detected by rapid proliferation and secretion of various cytokines and chemokines.

[0022] Thus, in one main aspect, the present invention provides a three-dimensional (3D) bioreactor for large-scale immune cell expansion, comprising at least one packed bed chamber, at least one porous scaffold coated with one or more extracellular matrix proteins (ECMs) surrounded by said at least one packed bed chamber, at least one container surrounding said at least one packed bed chamber, and a flow medium having at least one suspended immune cell population contained in said at least one container, wherein said flow medium having at least one suspended immune cell population flows through said packed bed chamber and said at least one porous scaffold coated with said one or more ECMs, and the flow medium ... the at least one porous scaffold coated with one or more ECMs is configured to form a fixed niche with low shear forces that mimics the natural growth environment of the immune cells, and has porosity to allow large-scale expansion of the immune cell population that flows through the at least one porous scaffold coated with the one or more ECMs, wherein the immune cells are any of Jurkat cells, T2 cells, K562 cells, Raji, U937, THP-1, HL-60, peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs), conventional and non-conventional T cells, B cells, B cell hybridomas, CAR-B, NKT cells, CAR-NKT, gamma delta T (gdT) cells, CAR-gdT, NK cells, CAR-NK, or combinations thereof.

[0023] The at least one porous scaffold may be further coated or bound with at least one immune cell activator. In some optional embodiments, the immune cell activator is an antigen-presenting cell (APC) loaded with or without an antigen, or an antigen directly bound to the coated porous scaffold. If the APC is not loaded with an antigen, the antigen may be presented at a later stage depending on the requirement for activating the immune cell.

[0024] In a further option, the expanded immune cells may be further activated within the packed bed chamber by exposing the immune cell population to the at least one porous coated scaffold bound to the immune cell activator.

[0025] Alternatively, the expanded immune cells may be further activated in the packed bed chamber by exposure to a suspended soluble immune cell activator and further expanded by at least one porous ECM-coated scaffold.

[0026] In some embodiments of the invention, the expanded and / or activated immune cell population is harvested or reactivated by exposing APCs bound to said at least one coated porous scaffold to an antigen to generate an additional activation signal for said immune cell population.

[0027] Furthermore, in some further embodiments, the immune cell population may be harvested or reactivated by transferring the expanded immune cells to a different bioreactor comprising at least one porous scaffold coated with a different or similar immune cell activator.

[0028] The porous scaffold may be a single porous scaffold matrix that extends into the packed bed chamber interior space, or may be multiple mini- or micro-porous scaffolds that fill the packed bed chamber.

[0029] In some further optional embodiments of the present invention, the immune cell population is genetically modified by using a genetic modifying agent incorporated into the bioreactor medium.

[0030] The present invention further provides a method for large-scale immune cell expansion in a three-dimensional (3D) bioreactor, comprising the steps of: a. inserting at least one porous scaffold into at least one packed bed chamber; b. coating the at least one porous scaffold with one or more extracellular matrix proteins (ECM); and c. circulating a flow medium having at least one suspended immune cell population contained in at least one container surrounding the at least one packed bed chamber, the flow medium being configured to flow through the at least one packed bed chamber and the at least one porous scaffold coated with the one or more ECMs, wherein the at least one porous scaffold coated with an ECM is configured to form a stationary niche with low shear forces that mimics the natural growth environment of the immune cells, and is porous and configured to allow large-scale expansion of the immune cell population that flows through the at least one porous scaffold coated with the one or more ECMs, wherein the immune cells are any of Jurkat cells, T2 cells, K562 cells, Raji, U937, THP-1, HL-60, peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs), conventional and non-conventional T cells, B cells, B cell hybridomas, CAR-B, NKT cells, CAR-NKT, gamma delta T (gdT) cells, CAR-gdT, NK cells, CAR-NK, and combinations thereof.

[0031] The method may further comprise the steps of coating the at least one porous scaffold with at least one immune cell activating agent after coating the scaffold with ECM, and exposing the at least one immune cell population to the at least one activating agent after suspending the at least one immune cell population in the circulated flow medium, to expand and activate the immune cell population within the packed bed chamber.

[0032] The method may further comprise genetically modifying immune cells within the 3D bioreactor using a genetic modifying agent added to the fluid medium.

[0033] In some optional embodiments, the method further comprises harvesting the immune cells or a portion of the cells and further expanding and reactivating the immune cell population in the same bioreactor or a different bioreactor.

[0034] Additionally or alternatively, the above-described methods may further comprise, after harvesting the immune cells, performing genetic modification outside the system and then reseeding the genetically modified immune cells into the same or a different bioreactor.

[0035] In yet a further aspect, the present invention provides a three-dimensional (3D) bioreactor for large-scale expansion and activation of immune cell populations, comprising at least one packed-bed chamber, at least one porous antigen-presenting cell-mimicking scaffold (APC-MS) coated with one or more extracellular matrix proteins (ECMs) surrounded by said at least one packed-bed chamber, at least one container surrounding said at least one packed-bed chamber, and a flow medium containing at least one suspended immune cell population contained in said at least one container, wherein said flow medium containing at least one suspended immune cell population flows through said at least one porous APC-MS coated with said one or more ECMs, and said one or more ECM-coated APC-MS is adapted to bind to said at least one ECM-coated APC-MS. the at least one porous APC-MS coated with one or more ECMs is configured to form a fixed niche with low shear forces that mimics the natural growth environment of the immune cell population, and has porosity to allow large-scale expansion of the at least one immune cell population that flows through the at least one porous APC-MS coated with the one or more ECMs, the immune cell population being any of Jurkat cells, T2 cells, K562 cells, Raji, U937, THP-1, HL-60, peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs), conventional and non-conventional T cells, B cells, B cell hybridomas, CAR-B, NKT cells, CAR-NKT, gamma delta T (gdT) cells, CAR-gdT, NK cells, CAR-NK, and combinations thereof.

[0036] The immune cell population may be reactivated by exposing the coated porous APC-MS to an antigen, generating an additional activation signal in the immune cell population.

[0037] In some optional embodiments, the immune cell population is reactivated by transferring the cells to a different bioreactor containing at least one coated porous APC-MS with a different or similar antigen.

[0038] According to an embodiment of the present invention, the at least one porous APC-MS may be composed of a single unit extending into the interior space of the packed bed chamber, or may be multiple mini / micro porous APC-MSs filling the packed bed chamber.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In carrying out or testing the present invention, methods and materials similar or equivalent to those described herein can be used, and suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will prevail. Furthermore, the materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0040] The present invention is described herein, by way of example only, with reference to the accompanying drawings. Referring now in detail to the drawings, it is emphasized that the details shown are exemplary and are for the purpose of illustrative explanation of embodiments of the invention, and are presented to provide a useful and understandable explanation of the principles and conceptual aspects of the invention. In this regard, no attempt has been made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, and the description, taken in conjunction with the drawings, will make apparent to those skilled in the art how to actually practice several forms of the invention. [Brief explanation of the drawings]

[0041] [Figure 1] FIG. 1 is a schematic diagram of an optional packed-bed bioreactor for expanding, activating, and harvesting immune cell populations, according to embodiments of the present invention. [Figure 2(1)] FIG. 1 is a flow chart diagram illustrating at a high level the sequence of major steps for preparing and using a packed-bed bioreactor for activating, expanding, and harvesting immune cells. [Figure 2(2)] FIG. 3 is a flowchart showing a continuation of FIG. 2(1). [Figure 3A]2A-2C are partial schematic front views of the packed-bed bioreactor 100 of FIG. 1 at different stages in the growth process of an immune cell population, showing the initial stage with an uncoated scaffold in the packed-bed basket. [Figure 3B] FIG. 1 shows a packed bed basket containing a coated scaffold and filled with an ECM protein solution. [Figure 3C] FIG. 3C shows a packed bed basket in which at least one immune cell activator is further linked to the coated scaffold of FIG. 3B. [Figure 3D] FIG. 1 shows a packed-bed bioreactor with a coated scaffold to which one or more activating agents are attached and through which immune cells flow in the medium. [Figure 4(1)] FIG. 1 is a flow cytometry diagram showing activation and proliferation of T cells cultured for 7 days in a packed bed bioreactor, with cell activation and proliferation measured on day 0. [Figure 4(2)] Flow cytometry diagram showing cell activation and proliferation measured on day 5. [Figure 4(3)] Flow cytometry diagram showing cell activation and proliferation measured on day 7. [Figure 4(4)] Flow cytometry diagram showing cell activation and proliferation measured on day 7 (post-harvest). [Figure 5A(1)] FIG. 1 is a flow cytometry diagram showing activation and proliferation of MAIT cells cultured for 10 days in a packed-bed bioreactor, with cell activation and proliferation measured on day 0. [Figure 5A(2)] Flow cytometry diagram showing cell activation and proliferation measured on day 5. [Figure 5A(3)] Flow cytometry diagram showing cell activation and proliferation measured on day 7. [Figure 5A(4)] Flow cytometry diagram showing cell activation and proliferation measured at 10 days. [Figure 5B(1)]FIG. 11 is a flow cytometry image showing the growth of MAIT cells after transferring the cells from the first bioreactor to the second bioreactor on day 10 and then growing in the second bioreactor for an additional 7 days. [Figure 5B(2)] Flow cytometry images on day 12. [Figure 5B(3)] Flow cytometry images on day 14. [Figure 5B(4)] Flow cytometry images on day 17. [Figure 6] FIG. 1 is a diagram of cell distribution inside and outside the packed bed of the bioreactor for three different immune cell types (Jurkat cells, PBMCs, MAIT cells). [Figure 7] FIG. 1 is a schematic diagram of different sizes of packed-bed bioreactors, illustrating the high scalability of the system of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0042] Before describing at least one embodiment of the present invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or illustrated by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0043] The present invention aims to provide systems and methods for the large-scale expansion and activation of various immune cell populations.

[0044] In one main aspect, the present invention provides a 3D bioreactor for large-scale immune cell expansion comprising: a) at least one packed-bed chamber comprising at least one porous scaffold; b) at least one porous scaffold coated with one or more ECM proteins; c) at least one vessel comprising a flow medium, said flow medium configured to flow through said packed-bed chamber with at least one porous coated scaffold; and d) at least one immune cell population suspended in the flow medium, wherein the at least one ECM-coated porous scaffold is configured to form a stationary niche with low shear forces that mimics the natural growth environment of the immune cells, allowing for large-scale expansion of the immune cell population flowing through the coated porous scaffold.

[0045] In a further aspect, the present invention provides a method comprising the steps of: a) inserting at least one porous scaffold into at least one packed bed chamber; b) coating the at least one porous scaffold with one or more extracellular matrix proteins (ECM); c) circulating a flow medium from at least one container, the flow medium configured to flow through the packed bed chamber comprising the at least one porous coated scaffold; and d) suspending at least one immune cell population in the circulated flow medium, wherein the at least one ECM-coated porous scaffold forms an anchoring niche with low shear forces that mimics the natural growth environment of the immune cells and is configured to allow large-scale expansion of the immune cell population flowing through the at least one porous scaffold.

[0046] Additionally, in a further aspect, the present invention provides a bioreactor comprising: a) at least one packed-bed chamber comprising at least one porous APC-MS; b) at least one APC-MS coated with one or more ECM proteins; c) at least one container comprising a flow medium, the flow medium configured to flow through the coated porous APC-MS; and d) at least one immune cell population suspended in the flow medium, wherein the at least one APC-MS forms a fixed microenvironment with low shear forces that mimics the natural growth environment of the immune cell population, allowing for large-scale expansion and / or activation of the immune cell population flowing therethrough.

[0047] The principal aspects of the present invention and options for practicing the invention will be better understood from the following detailed description of various illustrative, non-limiting figures and examples that follow. Reference is now made to the drawings.

[0048] FIG. 1 is a schematic cross-sectional view of an optional packed-bed bioreactor 100 for the expansion, activation, and harvesting of immune cell populations, according to an embodiment of the present invention.

[0049] In the illustrated embodiment, the growth and vibration chamber 116 (hereinafter referred to as the "basket") is loaded with at least one porous scaffold 10. The terms "carrier" and "scaffold" may be used interchangeably, and both refer to a porous element that is coated and configured to form a fixed niche within the basket, as described in detail with reference to Figures 2(1) and 2(2), reducing shear forces and mimicking the immune cell population's natural environment. The basket wall 1161 is preferably separated from the bioreactor inner wall 40 and can move up and down. The bioreactor 100 is configured and operable to supply various media to the bioreactor, followed by liquid media, optionally for autoclaving. In other embodiments, after sterilization, the liquid is replaced with growth medium that saturates the basket 116 and its contents. The basket 116 essentially divides the fluid within the bioreactor 100 into three main sections: an upper section 122 that primarily contains fresh medium inserted via the inlet pipe 120, a middle section 124 that contains medium within the basket 116 surrounding the coated scaffold, and a lower section 126 that primarily contains medium that has flowed through the basket. Compared to the fresh medium in the upper section 122, the medium flowing through the middle section typically contains fewer nutrients consumed by immune cells, has a reduced flow rate as it flows through at least one porous coated scaffold, interacts with and / or is activated by other cells, and is enriched with immune cell-secreted compounds and debris. In the embodiments described herein, the medium in the upper section 122 is agitated by an impeller 119, which creates fluid movement, as indicated by arrow 109. In yet further embodiments, various parameters, such as temperature, pH, and dissolved oxygen concentration, are set at the beginning of the process as part of a system setup procedure (see the flow diagrams in Figures 2(1) and 2(2) below) and are continually adapted to the suspension conditions as needed. In yet further embodiments, the initial agitation speed of the medium may be set slow to promote cell adhesion to the coated scaffold, and then increased. If desired, cells can be harvested from the medium for processing into a final product or for further immune cell expansion, as described in detail below (Figures 2(1) and 2(2)). In some embodiments, the rotation of the impeller 119 creates a negative pressure in the draft tube 150, drawing the medium, along with the cells, from the lower section 126 through the draft tube 150 and then through a port in the impeller 119 into the upper section 122, uniformly circulating the medium and immune cells in a continuous loop in the direction indicated by arrow 109. In still further embodiments of the present invention, adjustments to the medium may be made by monitoring various parameters through electrodes 106, thereby allowing various parameters of the medium to be controlled. In some optional embodiments, a ring sparger (not visible) may be placed within the impeller aeration chamber 11 to oxygenate the media flowing through the port in the impeller 119 via gas added from external port 103, which may be retained within the housing 5 and sparger line 7. In some other optional embodiments, gas may be added via inlet 120. Alternatively, the sparged gas may be trapped in a remote chamber and absorbed into the nutrient medium, flushing the entire system. In some optional embodiments, a water jacket 117 covers the media area within the bioreactor 100 and is provided with ports 13 and 14 for the inflow and outflow of jacket water. A removal pipe 110 is positioned along the bioreactor and has an opening in the lower media section 126 to allow immune cells to be harvested from the media below the basket 116, if desired. The removal pipe 110 can also be used to remove debris and refresh the media by removing a portion of the spent media and adding fresh media.

[0050] In some embodiments, a continuous stirred tank bioreactor may be used, in which culture medium is continuously fed into the bioreactor and product is continuously withdrawn, maintaining a constant steady state within the bioreactor over time. Stirred tank bioreactors equipped with fibrous bed baskets are available, for example, from New Brunswick Scientific Co. (Edison, NJ). Additional bioreactors that can be used include, but are not limited to, fixed-bed bioreactors, perfusion bioreactors using polyactive foam, radial-flow perfusion bioreactors containing tubular poly-L-lactic acid (PLLA) porous scaffolds, and other bioreactors known in the art that are suitable for the purposes of the present invention. A "fixed-bed bioreactor" refers to a bioreactor in which the cell growth substrate typically does not lift off the bottom of the incubation vessel in the presence of growth medium. For example, the substrate can be sufficiently dense to prevent lifting and / or packed with mechanical pressure to prevent lifting. The substrate may be a single mass or multiple masses. Typically, the substrate remains more or less in place during the standard agitation rate of the bioreactor. In some embodiments, a plurality of carriers are loosely packed, eg, forming a loose packed bed, and are submerged in nutrient medium.

[0051] Additionally, in certain embodiments, a perfusion bioreactor is used, and the perfusion chamber contains a 3D substrate. In certain embodiments, the 3D substrate is in the form of a porous scaffold 10. The porous scaffold may be made of a single large unit that encompasses the entire volume or most of the volume of the packed bed chamber. Alternatively, the porous scaffold in use may be multiple small particles. In some further optional embodiments, the porous scaffold may be, for example, a macrocarrier, a microcarrier, or a mixture thereof. Non-limiting examples of commercially available carriers include alginate-based (GEM, Global Cell Solutions), dextran-based (Cytodex®, GE Healthcare), collagen-based (Cultispher®, Percell Biolytica), and polystyrene-based (SoloHill Engineering) microcarriers.

[0052] In certain embodiments, the T cells are incubated in a bioreactor in what are referred to as "APC-MS," a term that refers to a scaffold (which in more specific embodiments may include any scaffold referred to herein) that is bound to or associated with a lymphocyte-activating component, more specific embodiments of which are fiber carriers or mesoporous silica microrods that are bound to or coated with the activating component.

[0053] Unless otherwise specified, the term packed-bed bioreactor refers to a bioreactor in which the cell growth substrate does not typically lift off the bottom of the incubation vessel in the presence of growth medium. For example, the substrate can be sufficiently dense to prevent lifting and / or packed with mechanical pressure to prevent lifting. The substrate can be a single mass or multiple masses. Typically, the substrate remains substantially in place during perfusion at the standard perfusion rate of the bioreactor. In certain embodiments, the definition does not exclude that abnormally high perfusion rates, for example, greater than 200 rpm, may cause substrate lifting.

[0054] In other embodiments, the biocontainers are used to grow cells, and in further embodiments, are adapted for suspension culture. In various embodiments, the biocontainers are used and / or adapted for batch, fed-batch, or continuous culture.

[0055] 2(1) and 2(2) are flow chart diagrams illustrating at a high level the sequence of major steps for preparing and using a packed-bed bioreactor system for the expansion, activation, and harvesting of immune cell populations, according to embodiments of the present invention.

[0056] The system setup step 310 includes assembling the system, filling the bioreactor basket with the desired amount of porous scaffold, connecting the desired electrodes for monitoring and controlling culture parameters (e.g., pH, dissolved oxygen, temperature) while the system is in use, connecting the necessary tubing for fresh nutrient supply and by-product removal, performing a system seal and integrity test, and sterilizing the system by steam sterilization in an autoclave. After sterilization, the system is connected to the bioreactor control station and the electrodes are calibrated.

[0057] Porous scaffolds, hereafter referred to as "carriers," may be made of natural or synthetic materials and may have a variety of dimensions. Some non-limiting examples of commercially available carriers include alginate-based (GEM, Global Cell Solutions), dextran-based (Cytodex®, GE Healthcare), collagen-based (Cultispher®, Percell Biolytica), and polystyrene-based (SoloHill Engineering) carriers. Alternatively, the porous scaffold may comprise a fibrous material, optionally an adhesive fibrous material, such as a woven or non-woven fibrous matrix. Non-limiting examples of fibrous scaffolds include those manufactured by Eppendorf, Germany. TM Eppendorf TM AG) and includes a polypropylene support. TMand polyester mesh-containing carriers such as the Fibra-Cel® discs available from CESCO BioProducts, Inc. (Atlanta, GA) and BioNOC, which is made of PET (polyethylene terephthalate). TM There is a II carrier. In particular embodiments, the referenced fiber matrix comprises polyester, polypropylene, polyalkylene, polyfluorochloroethylene, polyvinyl chloride, polystyrene, or polysulfone. In more particular embodiments, the fiber matrix is ​​selected from polyester and polypropylene.

[0058] An ECM coating step 312 is performed to create an environment that mimics the natural environment of immune cells. In this step, the hydrophilic end groups of the Fibra-Cel® discs allow the scaffold to be coated with various materials. This is done by simply immersing the scaffold in a solution containing ECM proteins to create electrostatic interactions. A detailed description of the effect of the extracellular matrix on immune cells is provided in Sutherland TE et al., "Extracellular Matrix and the Immune System: Interdependence," Science, February 17, 2023, Vol. 379 (No. 6633), PMID: 36795835. Note that other scaffolds can also be used, and the Fibra-Cel® discs described are a non-limiting example. Furthermore, the scaffold can be coated with natural or synthetic ECM components.

[0059] In this way, various proteins, including but not limited to albumin, fibronectin, fibrin, fibrinogen, collagen, hyaluronic acid, elastin, laminin, and selectins, can be used to coat scaffolds and create environments similar to the natural environment. For example, to mimic the structure of lymph nodes, scaffolds can be coated with type III collagen, a major component of reticular fibers, the primary component of the lymph node ECM. Furthermore, because different organs have different combinations of ECM proteins, different combinations and concentrations can be used for coating. In another embodiment, adhesion molecules can be attached to the scaffold or to proteins coating the scaffold to promote strong interactions between immune cells and the ECM-coated scaffold. For example, E-selectin, P-selectin, ICAM1, ICAM2, and VCAM1 are known in the art to promote interactions between leukocytes and the ECM and help direct leukocytes to inflamed tissues. Therefore, coating the scaffold with these molecules may increase interactions between leukocytes and the ECM-coated scaffold. Alternatively, ECM coating (step 312) is performed after system setup (step 310), followed by a scaffold activation coating step (step 314). In different embodiments (depending on the product and its activating ligand), an activation coating step (step 314) of the porous scaffold is performed after the ECM coating (step 312).

[0060] Step 314 describes the process where the scaffold is coated with immune cell activating ligands. Step 314 can be performed before or after step 312. Immune cells need to be activated by antigen presentation, which typically occurs in lymph nodes or infected tissue. By coating the scaffold with ECM components and providing antigen presentation in a low shear environment, the niche formed mimics the natural environment in which activation occurs. In one embodiment, after ECM coating with serum ECM proteins, adherent or semi-adherent cells can attach to the fibers comprising the scaffold. Because some immune cells require interaction with antigens presented by antigen-presenting cells (APCs) to become activated, antigen-loaded APCs are seeded in step 314 after coating step 312. The APCs can attach to the scaffold and present antigens to various immune cells, such as T cells, B cells, and all their subpopulations. In another embodiment, APCs can be seeded into the bioreactor and attached to the scaffold without antigen presentation. After the cells attach, antigen can be added to the media filling the bioreactor to present antigen to immune cells, such as T cells and B cells. A more specific example of this embodiment is the use of monocytes to activate mucosal-associated invariant T cells (MAIT). In this example, after the scaffold is coated with fetal bovine serum proteins, PBMCs are seeded into the bioreactor, the monocytes attach to the scaffold, and the remaining cells are suspended in the media. After initial cell seeding, 5-OP-RU (5-(2-oxopropylideneamino)-6-D-ribitylaminouracil), an activating antigen for MAIT, is added to the culture medium and is presented by monocytes to specifically activate MAIT cells. In another embodiment of the present invention, immune cell activation may be regulated by an antigen bound to the scaffold without an APC. The antigen may be an antibody against a specific activating receptor on immune cells, a protein recognized by an activating receptor such as a TCR, or the like. For example, the scaffold is coated with both monoclonal anti-CD3 (OKT3) and anti-CD28 (CD28.2) antibodies to provide costimulatory signals that engage the T cell receptor. After an incubation period, a blocking step is performed, followed by a washing step. After the washing step, the scaffold is coated with fetal bovine serum proteins. The medium is replaced, and PBMCs are seeded into the bioreactor; the cells are activated by interaction with the ECM and the antibody-coated scaffold.

[0061] Another example is the use of MR1 monomers, tetramers, and other forms, with or without 5-OP-RU, to activate MAIT cells. When MR1 without 5-OP-RU is used, blocking and washing steps are performed before the addition of 5-OP-RU.

[0062] Step 316 describes the seeding of cells containing target cells for activation and proliferation. In this step, various immune cell sources can be used to seed the target cells, such as PBMCs collected from apheresis or PBMCs isolated from specific organs or tumors. Immune cells can be used fresh or frozen. In this step, immune cells are seeded into the bioreactor. Different environmental parameters, such as, but not limited to, agitation speed, pH, dissolved oxygen, and temperature, can be used to allow the cells to adhere or remain suspended within the bioreactor. The adherent cells enter the packed-bed chamber and interact with ECM proteins on the coated scaffold, forming an organized cellular microstructure within the packed-bed. According to this embodiment, APCs are seeded into the bioreactor and adhere to the scaffold in a non-antigen-presenting format, creating an environment that mimics a lymph node until activation occurs. A more specific example of this embodiment is the use of monocytes to activate mucosal-associated invariant T cells (MAIT). In this example, after a scaffold is coated with fetal bovine serum proteins, PBMCs are seeded into the bioreactor, allowing the monocytes to adhere to the scaffold while the remaining cells are suspended in culture medium. Non-adherent cells remain suspended within the bioreactor in the mobile phase and can move in and out of the packed-bed chamber to create cell-to-cell interactions with adherent cells.

[0063] After seeding the immune cells in step 316, a decision is made as to whether to perform gene editing on the expanded cell population (step 318). If gene editing is not to be performed, proceed to step 326.

[0064] Step 326 describes the target cell expansion phase, in which environmental parameters such as agitation speed, pH, dissolved oxygen, and temperature are controlled. During this step, the bioreactor is constantly heated and a preset gas mixture is supplied to the system to maintain the desired conditions within a predetermined range. Once cells are seeded and activated by ligand-loaded APCs or by an activator (antibody)-coated scaffold, an infection condition is simulated within the bioreactor, triggering a series of mechanisms. This leads to clonal expansion of antigen-specific immune cells, during which they can grow to a large extent and potentially represent up to 90% of all immune cells. To support rapid immune cell expansion, adequate nutrient supply and removal of inhibitory metabolites are provided without disrupting the local microenvironment. In certain embodiments, the bioreactor may operate in batch mode, fed-batch mode, and / or perfusion mode. In another embodiment of the invention, a perfusion system (e.g., TFF, ATF, BioSep, etc.) may be connected to the bioreactor. The perfusion system allows for the exchange of medium from the bioreactor without extracting the cells from the system. The perfusion system allows for the removal of conditioned medium from the system, the separation of suspended cells from the conditioned medium, and simultaneously the supply of fresh medium to the bioreactor based on a level electrode, weight, a preset flow rate, or manual prompting. In this example, the conditioned medium is sampled daily, and the number of suspended cells and the concentration of essential substrates in the medium are determined based on the cell mass and nutrient concentration, and the amount of fresh medium to be supplied is calculated.

[0065] In step 328, the medium along with the immune cells is drained from the system to harvest the target cells, and a wash step preferably continues outside the bioreactor system. In this embodiment, the medium exchange step can be performed using a batch centrifuge or a continuous flow centrifuge (e.g., kSep, unifuge). After the medium exchange, the cells or a portion of the cells can be returned to the bioreactor in step 332 to continue growth in fresh growth medium. Alternatively, the harvested cells can be transferred in step 330 for downstream processing of the immune cells for final product manufacturing.

[0066] In step 322, when gene editing is performed on target cells, immune cells are genetically modified to add or remove specific characteristics. Modifications such as adding new target or activating receptors, deleting specific receptors for self-recognition, or deleting unnecessary target receptors are used to transform immune cells for better treatment of various indications. In this step (322), genetic modifiers may be inserted into the bioreactor using non-viral agents such as liposomes (e.g., lipofectamine), polymers (e.g., PEI), or electroporation processes (described below), or by viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or others. These genetic modifiers can be loaded with RNA or DNA constructs that can add or remove new data from the genome using methods such as CRISPR / Cas9, transposomes (sleeping beauty, PiggyBac), and DNA-binding domains (e.g., zinc finger domains). In these embodiments, a genetic modifier, such as a viral vector, is added to the bioreactor medium after activation of the scaffold coating (312, 314), cell seeding (316), and during cell growth (326). At this stage, agitation and other environmental parameters, such as pH and temperature, may be adjusted to better prepare the cells for penetration of the genetic modifier. After addition, an incubation period is allowed under appropriate conditions to allow the genetic modifier to penetrate the cells.

[0067] Additionally, in some optional embodiments, the activated immune cells can be harvested from the bioreactor according to step (328) and transferred under sterile conditions to an electroporation or other 2D flask device to allow penetration of the cells with genetic modifiers by electroporation or viral and non-viral methods in step 322. In this embodiment, after the incubation period, the immune cells can be replated in the bioreactor for a wash step, or the wash step can be performed in the 2D flask.

[0068] After the incubation period, a wash step 324 is initiated. During wash step 324, the medium in the bioreactor is exchanged several times to extract the genetic modifier from the system. In one embodiment of the present invention, a perfusion system (e.g., TFF, ATF, BioSep, etc.) is connected to the bioreactor. The perfusion system allows for medium exchange from the bioreactor without extracting cells from the system. The perfusion system drains the old medium while simultaneously injecting new medium into the bioreactor via a level electrode, weight, or manual prompt. This process may be performed for several chamber volumes until no genetic modifier is found in the extracted medium. Alternatively, after cell growth (step 326), the medium with the cells and genetic modifier is drained or harvested from the system in step 328, and the wash step continues outside the bioreactor. In this embodiment, the medium exchange step can be performed using a batch centrifuge or a continuous-flow centrifuge (e.g., kSep, Unifuge). After changing the medium, the cells may be reseeded into the starting bioreactor or a new bioreactor (332) for further growth.

[0069] Once the growth duration has been reached or the immune cell concentration required for the desired end product or for seeding the cells into a larger bioreactor has been reached, a cell harvesting step (328, 340) is performed. In one embodiment, the harvesting step is performed by simply draining the medium from the system, with or without agitation. In another embodiment, the packed-bed basket is connected to a harvesting system as described in detail in WO 2012 / 140519 by the same applicant, which is incorporated herein by reference in its entirety, and then gentle vibration of the scaffold basket is performed during the draining step to release trapped suspended immune cells from the formed niches in the packed-bed scaffold without physical damage to the cells. Furthermore, in all embodiments of the harvesting step, cycles of refilling and draining the medium may be performed to collect all cells from the system. With each cycle, extracted cells are drained into a sterile collection element for further processing.

[0070] In one further embodiment of the present invention, at the end of the harvesting step (328, 340), the cells or a portion of the harvested cells are further processed downstream (330, 342) for final product manufacturing. This includes concentration and washing steps, which can be performed using different systems such as continuous flow centrifuges, filters, acoustic filtration devices, etc. The concentration and washing steps are followed by specific cell collection steps using various separation methods and / or final formulation of the product, followed by filling of the final product into final packaging (vials or cryobegs).

[0071] Furthermore, in another embodiment of the present invention, after the harvesting step, the harvested immune cells or a portion of the harvested cells are used for further expansion (332). Further expansion can be performed by performing step 316 in the existing bioreactor or by transferring the cells to a larger bioreactor, e.g., a 1.5 L bioreactor with a 30 g scaffold, a 3.5 L bioreactor with a 100 g scaffold, or a 10 L bioreactor with a 375 g scaffold. Furthermore, the purpose of the new bioreactor can be solely to expand using the ECM-coated scaffold (step 334) or to perform another activation step to reactivate the cells. Reactivation can be performed, for example, using the same activation agent in a new bioreactor (step 336) or a new activation agent in a new bioreactor (step 338). In either embodiment, the new bioreactor is pre-prepared according to steps 310-314 described above.

[0072] In another optional embodiment, reactivation of target cells can be performed in the initial bioreactor or in a new bioreactor by adding soluble activating agents such as transfectants or anti-CD3 and anti-CD28 antibodies, or by adding soluble antigens that can be presented by APCs if they are already located in the bioreactor.

[0073] 3A-3D are schematic partial front views of a packed-bed bioreactor at different stages of the immune cell culture process. Figure 3A shows the setup stage of the system, where the packed-bed basket 116 of the bioreactor 100 contains only uncoated porous scaffolds 10.

[0074] More specifically, the upper and lower boundaries of the packed bed chamber 116 are comprised of perforated disks 132, each having a plurality of holes 1321 of a predetermined diameter. The perforated disks 132 are also referred to below as the "disk," "grid," "upper grid," "lower grid," and "middle grid," all of which are used interchangeably and can refer to the upper, middle, or lower wall of the basket 416. As described in detail above with reference to Figures 2(1) and 2(2), in some optional embodiments, a porous scaffold may be inserted into the basket 116, occupying a portion or most of the basket's volume. At this stage, the bioreactor 100 may not contain any liquid. The basket 116 is preferably connected to one or more vibration rods 136 configured to allow vertical movement of the basket 116. Vibration of the basket 116 may be used to harvest target cells during or at the end of the expansion process, as described in Figures 2(1) and 2(2). The basket 116 has a wall 1161 that is independent of the wall 40 of the bioreactor 100, allowing upward and downward movement of the basket while maintaining the flow direction of the target cell-containing medium solely through the upper and lower disks 132. The basket 116 is located above the bottom of the bioreactor 100, so that an upper section 122 and a lower section 126 separate the middle section 124 containing the packed-bed basket 116.

[0075] Figure 3B shows a packed-bed basket 116 containing the coated scaffold 10' and liquid medium 50. To create an environment suitable for mimicking the immune cell population's natural environment, the porous scaffold must first be coated with an ECM coating, as described in detail in Figures 2(1) and 2(2) above and in the Examples section of the present invention. After the ECM coating is applied, the porous scaffold 10' may be further coated with or coupled to at least one antibody 1022 or antibody-presenting cells (APCs) 1033, as shown in Figure 3C. After the porous scaffold is prepared, immune cells 1044 can be seeded into the bioreactor, as shown in Figure 3D. As shown in Figure 3D, immune cells are seeded in all sections of the bioreactor with flowing medium and can be found in the upper section 122, lower section 126, and middle section 124 of the basket 116. The porous-coated scaffold creates a low-shear niche that mimics the immune cell's natural environment, allowing for optimal proliferation of the immune cells 1044. Furthermore, upon exposure to antigen and / or APC, immune cells 1044 are activated as previously described.

[0076] In certain embodiments, immune cells 1044 are incubated in the bioreactor 100 on an APC-mimicking scaffold (APC-MS). In further embodiments, immune cells are harvested from the porous coated scaffold and then incorporated into a pharmaceutical composition.

[0077] In further optional embodiments, immune cells are seeded with gentle agitation to promote even distribution, for example, in the case of charged beds or solid-state scaffolds. In the case of microcarriers, APC-MS and T cells are gently suspended and gently mixed. In either case, perfusion and agitation are stopped for a period of time after seeding to allow interaction between T cells and APC-MS and promote subsequent activation.

[0078] Figures 4(1) to 4(4) show flow cytometry images of T cell activation and proliferation after 7 days of growth in a packed-bed bioreactor. Cell activation and proliferation were measured on days 0, 5, and 7. The distribution of cell populations was measured using a CytoFLEX flow cytometer with three lasers (405 nm, 488 nm, and 638 nm) and 13 fluorescence detection channels. TM was analyzed by.

[0079] PMBC cells were seeded into a packed-bed bioreactor containing Fibra-Cel® carriers bearing immobilized antibodies (anti-CD3 and anti-CD28 antibodies) that provided T cell activation signals. T cell activation was assessed on days 0, 5, and 7 by measuring the expression of CD69 (an inducible cell surface marker expressed upon activation via the TCR) and CD25 (the α chain of the IL-2 receptor), activation markers commonly associated with T cell activation. Results indicated successful stimulation of T lymphocyte activation and proliferation, with an increase in CD3 markers from -44% on day 0 to -91% on day 7. These results indicated that T lymphocytes, in particular, expanded over the 7-day period. CD69 and CD25 were upregulated from -4% and -8% on day 0 to -42% and 90% on day 7, indicating T cell activation. Furthermore, total cell samples collected after the harvest step on day 7 showed no changes in cell markers, indicating the absence of other cell populations within the bioreactor. Furthermore, no changes in the levels of activation markers were observed, suggesting that cell harvesting did not affect the state of the cells.

[0080] CD69 is an inducible cell surface marker expressed upon activation via the TCR or IL-2 receptor (CD25), which plays a role in the proliferation and survival of activated T lymphocytes.

[0081] Figures 5A(1) to 5A(4) show flow cytometry images of activation and proliferation of MAIT cells grown in a packed-bed bioreactor for 10 days. Cell activation and proliferation were measured on days 0, 5, 7, and 10. The distribution of cell populations was measured using a CytoFLEX flow cytometer. TM was analyzed by.

[0082] Mononuclear cells from the IVB were seeded into a packed-bed bioreactor containing ECM-coated Fibra-Cel® carriers, which mimic the natural environment and promote APC adhesion. MAIT cell activation was induced by 5-OP-RU antigen and IL-15. MAIT cell activation and proliferation were assessed at several time points: days 0, 5, 7, and 10. The MAIT cell population was detected by expression of CD3, Vα7.2, and CD161 markers. Results showed an increase in the proportion of MAIT cells, starting from 22.6% on day 0 and reaching a maximum of 96.26% on day 10. Furthermore, expression of activation markers CD69 and CD25 increased from day 0 to day 7 and decreased by day 10.

[0083] Figures 5B(1)–5B(4) show flow cytometry images of MAIT cell growth after transfer from the first bioreactor to the second bioreactor on day 10 and subsequent growth for an additional 7 days in the second bioreactor. Cells reaching maximum growth capacity in the first bioreactor on day 10 were harvested, and approximately 30% of these cells were then seeded into a second bioreactor similar in design to the first. Cells were grown for an additional 7 days. Flow cytometry results showed that the percentage of MAIT cells remained similar over most of the time period, but decreased from over 90% on day 14 to 82% on day 17. Expression of the CD69 marker was upregulated from 30% to 87% on day 17, indicating that MAIT cells maintained their activation signal, while CD25 expression gradually decreased as expected.

[0084] Figure 6 is a graph showing the cell distribution within and outside the packed bed of the bioreactor for three immune cell types (Jurkat, PBMC, and MAIT cells) on two different growth days, shown as a percentage.

[0085] The properties of the Fibra-Cel® disc and packed-bed structure allow for the formation of a niche within the bioreactor system, mimicking the cells' natural environment, where cells can reside for a period of time under low shear stress. Figure 6 shows the distribution of cells within and outside the bioreactor packed-bed. These values ​​were calculated based on cell counts before and after harvesting the packed-bed, which was performed with several washing steps and bed shaking. The results show that at different bed harvest times during the growth period, at least 40% of viable cells remained within the packed-bed for all three designated immune cell types.

[0086] Since a significant decrease in cell concentration was observed a few hours after cell seeding, it can be assumed that the following cell distribution model is maintained throughout the cell growth period.

[0087] Figure 7 is a schematic diagram of different sized packed-bed bioreactors, illustrating the high scalability of the system of the present invention. According to any embodiment of the present invention, initial seeding of immune cell populations occurs in a mini packed-bed bioreactor 400 with a mini-basket 416, the total maximum volume of which is defined by its vessel dimension 490. After cell expansion in bioreactor 400, the cells or a portion of the cells may be reseeded into a larger-sized bioreactor 500 with a packed-bed basket 516 larger than basket 416, allowing for more porous scaffolding and greater cell expansion compared to bioreactor 400, the total volume of which is larger and determined by its vessel dimension 590. The same process is performed for a larger bioreactor 600 with a basket 616 and vessel 690, and then continues until the largest bioreactor 700 is reached, with the largest basket 716 and vessel dimension 790, allowing for multiplicative expansion of immune cells. According to the methods and examples described above, it can be understood that the conditions for expansion and activation of immune cell populations in each bioreactor can be similar or different from previous expansion sessions in smaller bioreactors, resulting in a large expansion of immune cells activated by similar or different activating agents at the end of the process. [Example]

[0088] Reference is now made to the following examples which, together with the above description, illustrate specific embodiments in a non-limiting fashion. [Example]

[0089] Growth of Jurkat cells in packed-bed bioreactors.

[0090] A 0.5 L packed-bed MiniBio reactor containing 2.5 grams of Fibra-Cel® discs was assembled and then autoclaved by steam sterilization at 122.5°C and 1 bar above atmospheric pressure for 30 minutes. The MiniBio reactor was then connected to the Applikon MiniBio control station.

[0091] Fibra-Cel® discs were preincubated in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) and 0.1% 50 mg / ml gentamicin at 37°C for approximately 24 hours. During incubation, serum proteins electrostatically interacted with the hydrophilic end groups of the Fibra-Cel® discs, creating an extracellular matrix (ECM) coating on the Fibra-Cel® that mimicked the cells' natural environment.

[0092] 81.6×10 6 10 cells were thawed into RPMI-1640 medium supplemented with 10% HI-FBS and 0.1% 50 mg / ml gentamicin, and the thawed cells were seeded at a target concentration of 0.24 × 10 cells. 6 The prepared cell suspension was inoculated into a bioreactor system set at the following conditions: temperature 37°C, dissolved oxygen (DO) 80%, pH 7.4, and agitation speed 150 rpm, until the total volume in the bioreactor reached 340 ml.

[0093] On the third day of culture, the medium was renewed by 25% (i.e., 25% new medium was added). On the fourth day of culture, cells were harvested from the packed-bed bioreactor due to the large-scale expansion in a very short period of time. The total cell number was 830 × 10 6 The cells reached 10.4-fold proliferation (results are detailed in Table 1). The distribution of cells inside and outside the packed bed is shown in FIG. [Example]

[0094] Activation, expansion, and harvest of peripheral blood mononuclear cells (PBMCs) in a packed-bed bioreactor.

[0095] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and subjected to filtration and density gradient media Lymphoprep. TM The cells were separated using Ficoll. Red blood cells (erythrocytes) were removed using RBC X1 lysis buffer. The isolated population was cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) cryopreservation medium.

[0096] A 0.5 L packed-bed MiniBio reactor containing 2.5 grams of Fibra-Cel® discs was assembled and then autoclaved by steam sterilization at 122.5°C and 1 bar above atmospheric pressure for 30 minutes. The MiniBio reactor was then connected to the Applikon MiniBio control station.

[0097] Fibra-Cel® discs were coated with monoclonal anti-CD3 (OKT3) and anti-CD28 (CD28.2) antibodies, both of which provide costimulatory signals that engage the T cell receptor. The amount of each activator was 0.21 μg / cm diluted in PBS. 2 The final solution was incubated at room temperature (RT) for 3 hours and at 37°C for 1.5 hours at 100 rpm.

[0098] After incubation with activator, the Fibra-Cel® discs were blocked and further incubated in 1% BSA solution for 1 hour at room temperature and 100 rpm, after which the Fibra-Cel® discs were drained and washed with PBS for 10 minutes at 150 rpm.

[0099] The packed-bed bioreactor was then prepared for culture and equilibrated for approximately 24 hours at 37°C in growth medium: RPMI-1640 medium supplemented with 10% HI-FBS, 1% sodium pyruvate (100 mM), and 0.1% 50 mg / ml gentamicin. During this incubation, serum proteins electrostatically interact with the hydrophilic end groups of the Fibra-Cel® discs, creating an ECM coating on the Fibra-Cel® that mimics the cells' natural environment.

[0100] 262×10 6 Cells were thawed into RPMI-1640 medium supplemented with 10% HI-FBS, 1% sodium pyruvate (100 mM), IL-2 (100 U / L), and 0.1% 50 mg / ml gentamicin, at a target concentration of 0.97 × 10 cells at the time of seeding. 6 The cell suspension was diluted to cells / ml. The prepared cell suspension was inoculated into a bioreactor system set at the following conditions: temperature 37°C, DO 80%, pH 7.4, and agitation speed 100 rpm, until the total volume in the bioreactor reached 270 ml. Upon inoculation, the cells were distributed between the packed bed and the "external" environment within the bioreactor, and a decrease in cell concentration was observed 3 hours after inoculation.

[0101] During the growth period, the growth medium and cell suspension (in the "external" environment) were monitored for pH, cell concentration (by Vi-Cell), metabolic activity of the cells based on nutrient consumption (by Cedex bioanalyzer), and flow cytometer (CytoFLEX). TM ) were sampled daily to determine the distribution of cell populations.

[0102] Medium renewal was performed at 5.5%, 32%, and 20% on days 3, 5, and 6, respectively. On day 7 of culture, cells were harvested from the packed-bed bioreactor as they had reached maximum growth capacity. The total cell number was 1073 × 10 6The number of cells reached 100,000, 91% of which were CD3 positive, demonstrating an 8.5-fold expansion. The proportions of different cell populations within the culture and their variation over time are shown in Figures 4(1)-(4). The distribution of cells within and outside the packed bed is shown in Figure 6. [Example]

[0103] Cultivation of mucosal-associated invariant T cells (MAIT) in packed-bed bioreactors: activation, expansion, harvest, repopulation, and secondary harvest.

[0104] Blood mononuclear cells were isolated from human placental venous blood (IVB) and filtered using density gradient medium Lymphoprep. TM The cells were separated using Ficoll. Red blood cells (erythrocytes) were removed using RBC X1 lysis buffer. The isolated population was cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) cryopreservation medium.

[0105] A 0.5 L packed-bed MiniBio reactor containing 2.5 grams of Fibra-Cel® discs was assembled and then autoclaved by steam sterilization at 122.5°C and 1 bar above atmospheric pressure for 30 minutes. The MiniBio reactor was then connected to the Applikon MiniBio control station.

[0106] Fibra-Cel® discs were incubated in RPMI-1640 medium supplemented with 10% HI-FBS for approximately 24 hours at 37°C. During incubation, serum proteins electrostatically interacted with the hydrophilic end groups of the Fibra-Cel® discs, creating an ECM coating on the Fibra-Cel® that mimicked the cells' natural environment.

[0107] 300±20×10 6 Cells were thawed into 4Cell® Nutri-T GMP medium supplemented with 1% L-glutamine 200 mM and 0.1% 50 mg / ml gentamicin, and the thawed cells were grown to a target concentration of 1 × 10 6The prepared cell suspension was inoculated into a bioreactor system set at the following conditions: temperature 37°C, DO 80%, pH 7.4, and agitation speed 100 rpm to reach a final volume of 300 ml. Upon inoculation, cells were distributed within the bioreactor between the packed bed and the "external" environment, and a decrease in cell concentration was observed 3 hours after inoculation.

[0108] Three hours after plating, MAIT cells were activated via the T cell receptor (TCR), a pathway that requires recognition of microbial-derived riboflavin metabolites presented on the MHC class I-like molecule MR1 and co-stimulatory signals, including CD28, TLR agonists, bacterial products, or cytokines.

[0109] The initial cell population isolated from human placental venous blood (IVB) contains various antigen-presenting cells (APCs), such as dendritic cells, monocytes, and B cells, which can activate MAIT cells via MR1. 5-OP-RU, a microbial-derived riboflavin intermediate, is added to the growth medium at a concentration of 250 nM and presented by APCs on MR1, where it is recognized by the TCR of MAIT cells. Furthermore, IL-15 at a concentration of 50 ng / ml is also added to the growth medium to stimulate MAIT cells (via the IL-15R expressed on MAIT cells) to produce IFN-γ and release granzyme B and perforin.

[0110] During the growth period, the growth medium and cell suspension were monitored for pH, cell concentration (by Vi-Cell), and metabolic activity of the cells based on nutrient consumption (Cedex TM Bioanalyzer), and flow cytometer (CytoFLEX TM ) were sampled daily to determine the distribution of cell populations.

[0111] Cells were cultured in a packed-bed bioreactor for 10 days, with medium renewals of 5%, 5.2%, and 100% on days 3, 5, and 7, respectively. On day 10 of culture, cells reached maximum growth capacity and were harvested from the packed-bed bioreactor. The total cell number was 1089 × 10 6 The total number of cells reached 94% and was 94% MAIT cells (Vα7.2 positive, CD161 high), demonstrating a 43.5-fold proliferation. The proportions of different cell populations within the culture and their variation over time are shown in Figure 5A(1)–Figure 5A(4).

[0112] After harvesting the cells from the bioreactor on day 10 of culture, further expansion of the cells was considered. An additional 0.5 L packed-bed MiniBioReactor was assembled and prepared similarly to the previous one. This was a sterile system containing 2.5 grams of Fibra-Cel® discs, which were pre-incubated at 37°C for approximately 24 hours in RPMI-1640 supplemented with 10% HI-FBS to provide an ECM coating for the Fibra-Cel® discs and create a natural environment for the reseeded cells.

[0113] After ECM coating, the packed-bed bioreactor was pre-equilibrated for culture with growth medium composed of 4Cell® Nutri-T GMP medium supplemented with 1% L-glutamine 200 mM and 0.1% 50 mg / ml gentamicin. Of the harvested cells, 295 x 10 6 cells are seeded into growth medium at a target concentration of approximately 1 x 10 6 The cell suspension was diluted to 100 cells / ml. The prepared cell suspension was seeded into a bioreactor system set at the following conditions: temperature 37°C, DO 80%, pH 7.4, and agitation speed 100 rpm, to reach a final volume of 300 ml. IL-15 was added to the growth medium at a concentration of 50 ng / ml to induce proliferation of MAIT cells.

[0114] During the growth period, the growth medium and cell suspension were monitored for pH, cell concentration (by Vi-Cell), metabolic activity of the cells based on nutrient consumption (by Cedex bioanalyzer), and by flow cytometer (CytoFLEX). TM ) were sampled daily to determine the distribution of cell populations.

[0115] The cells were cultured in the packed-bed bioreactor for an additional 7 days, with 26.5% and 100% medium renewal on days 12 and 14 of culture, respectively. On day 17 of culture, the cells were harvested from the packed-bed bioreactor. The total cell number was 490 × 10 6 The total number of MAIT cells in the cultures reached 1.5, of which 88% were MAIT cells (Vα7.2 positive, CD161 high), demonstrating a 1.5-fold increase in proliferation. The relative proportions of MAIT cells in the cultures and their variation over time are shown in Figure 5B(1)–Figure 5B(4).

[0116] Table 1 below summarizes the initial and final total viable cell numbers, relative population percentages and fold expansions over the growth period for three different immune cell types (Jurkat cells, PBMCs and MAIT cells).

[0117] [Table 1]

[0118] As shown in Table 1, all fold expansions were greater than 1, confirming cell proliferation for all three given immune cell types, as well as for the secondary growth phase of MAIT cells. Furthermore, Table 1 shows a shift in the balance of the cell populations, with the percentage of target cells exceeding 94% at the end of the growth period investigated (T cells after 7 days of growth, MAIT cells after 10 days of growth). The secondary growth period of MAIT cells shows a slight decrease in the percentage of MAIT cells (from 94% to 86%). [Example]

[0119] Expansion, activation, and harvest of peripheral blood mononuclear cells (PBMC)-derived B cells in a packed-bed bioreactor.

[0120] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and subjected to filtration and density gradient media Lymphoprep. TM The cells are separated using Ficoll. Red blood cells (erythrocytes) are removed using RBC X1 lysis buffer. The isolated population is cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) cryopreservation solution.

[0121] A 0.5 L packed-bed MiniBio reactor containing 2.5 grams of Fibra-Cel® discs is assembled and then autoclaved by steam sterilization at 122.5°C and 1 bar above atmospheric pressure for 30 minutes. The MiniBio reactor is then connected to the Applikon MiniBio control system.

[0122] Fibra-Cel® discs are coated with anti-CD40 antibody to provide an activation signal. The amount of activator is 1 μg / cm diluted in PBS. 2 The final solution is incubated for 3 hours at RT and 1.5 hours at 37°C at 100 rpm. After incubation with the activator, the Fibra-Cel® discs are blocked and further incubated in a 1% BSA solution for 1 hour at RT at 100 rpm. The Fibra-Cel® discs are then drained and washed with PBS for 10 minutes at 150 rpm.

[0123] Fibra-Cel® discs are preincubated in RPMI 1640 medium supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin for approximately 24 hours at 37° C. During this incubation, serum proteins electrostatically interact with the hydrophilic end groups of the Fibra-Cel® discs, creating an ECM coating on the Fibra-Cel® that mimics the cells' natural environment.

[0124] 300×106 Cells are thawed in RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol, 100 IU / ml penicillin-streptomycin, human recombinant IL-4 (10 ng / ml), and IL-21 (10 ng / ml). The thawed cells are seeded at a target concentration of 1 x 10 cells. 6 The cell suspension is diluted to cells / ml. The resulting cell suspension is seeded into a bioreactor system set to the following conditions: temperature 37°C, DO 80%, pH 7.4, and agitation speed 100 rpm, so that the total volume in the bioreactor reaches 300 ml. During seeding, the cells are distributed within the bioreactor between the packed bed and the "external" environment surrounding the packed bed basket. They are sampled daily to measure pH, cell concentration (using Vi-Cell), and metabolic activity of the cells.

[0125] During the growth period, the growth medium and cell suspension (in the "external" environment) were analyzed to measure nutrient consumption (by a Cedex bioanalyzer) and cell viability (by a flow cytometer (CytoFLEX TM ) based on the distribution of cell populations.

[0126] Medium renewal is performed 50% on days 4 and 6. On day 8 of culture, cells are harvested from the packed-bed bioreactor. [Example]

[0127] Activation, expansion, and harvest of peripheral blood mononuclear cell (PBMC)-derived iNKT cells in a packed-bed bioreactor.

[0128] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and subjected to filtration and density gradient media Lymphoprep. TM The cells are separated using Ficoll. Red blood cells (erythrocytes) are removed using RBC X1 lysis buffer. The isolated population is cryopreserved in HI-FBS and DMSO cryopreservation solution.

[0129] A 0.5 L packed-bed MiniBio reactor containing 2.5 grams of Fibra-Cel® discs is assembled and then autoclaved by steam sterilization at 122.5°C and 1 bar above atmospheric pressure for 30 minutes. The MiniBio reactor is then connected to the Applikon MiniBio control system.

[0130] Fibra-Cel® discs are preincubated in RPMI 1640 medium supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin at 37°C for approximately 24 hours. During incubation, serum proteins electrostatically interact with the hydrophilic end groups of the Fibra-Cel® discs, creating an ECM coating on the Fibra-Cel® that mimics the cells' natural environment.

[0131] 600×10 6 Cells are thawed in RPMI 1640 medium supplemented with 10% HI-FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, 0.1 mM MEM non-essential amino acids, 5.5 μM β-mercaptoethanol, 100 IU / ml penicillin-streptomycin, and 100 IU / ml human recombinant IL-2. The thawed cells are seeded at a target concentration of 2 x 10 6 The cell suspension is diluted to cells / ml. The prepared cell suspension is seeded into a bioreactor system set to the following conditions: temperature 37°C, DO 80%, pH 7.4, and agitation speed 100 rpm, so that the total volume in the bioreactor reaches 300 ml. During seeding, the cells are distributed within the bioreactor between the packed bed and the "external" environment. Three hours after seeding, the activation step begins, and 100 ng / ml of α-galactosylceramide is added to the bioreactor medium to allow presentation on CD1d located on antigen-presenting cells (derived from the PBMC population) attached to the Fibra-Cel® disc.

[0132] During the growth period, the growth medium and cell suspension (in the "external" environment) were monitored for pH, cell concentration (by Vi-Cell), metabolic activity of the cells based on nutrient consumption (by Cedex bioanalyzer), and flow cytometer (CytoFLEX). TM ) are sampled daily to measure the distribution of cell populations by chromosome number.

[0133] Medium renewal is performed at 70% on days 3 and 6. On day 7 of culture, cells are harvested from the packed-bed bioreactor. [Example]

[0134] Activation, expansion, and harvest of peripheral blood mononuclear cells (PBMC)-derived γδ T cells in a packed-bed bioreactor.

[0135] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and subjected to filtration and density gradient media Lymphoprep. TM The cells are separated using Ficoll. Red blood cells (erythrocytes) are removed using RBC X1 lysis buffer. The isolated population is cryopreserved in HI-FBS and DMSO cryopreservation solution.

[0136] A 0.5 L packed-bed MiniBio reactor containing 2.5 grams of Fibra-Cel® discs is assembled and then autoclaved by steam sterilization at 122.5°C and 1 bar above atmospheric pressure for 30 minutes, after which the MiniBio reactor is connected to the Applikon MiniBio control station.

[0137] Fibra-Cel® discs are preincubated in RPMI 1640 medium supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin at 37°C for approximately 24 hours. During incubation, serum proteins electrostatically interact with the hydrophilic end groups of the Fibra-Cel® discs, creating an ECM coating on the Fibra-Cel® that mimics the cells' natural environment.

[0138] 300×10 6 Cells are thawed in RPMI 1640 medium supplemented with 10% HI-FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, 0.1 mM MEM non-essential amino acids, 50 μM β-mercaptoethanol, 100 IU / ml penicillin-streptomycin, and 300 IU / ml IL-2. The thawed cells are seeded at a target concentration of 1 x 10 6 The cell suspension is diluted to 100 cells / ml. The resulting cell suspension is seeded into a bioreactor system set at the following conditions: temperature 37°C, DO 80%, pH 7.4, and agitation speed 100 rpm, so that the total volume in the bioreactor reaches 300 ml. During seeding, the cells are distributed within the bioreactor between the packed bed and the "external" environment. Three hours after seeding, the activation step begins, and 5 μM zoledronic acid is added to the bioreactor medium.

[0139] During the growth period, the growth medium and cell suspension (in the "external" environment) were monitored for pH, cell concentration (by Vi-Cell), metabolic activity of the cells based on nutrient consumption (by Cedex bioanalyzer), and flow cytometer (CytoFLEX). TM ) are sampled daily to measure the distribution of cell populations by chromosome number.

[0140] Medium renewal is performed at 50% on days 4, 7, 10 and 13. On day 14 of culture, cells are harvested from the packed-bed bioreactor. [Example]

[0141] Activation, expansion, and harvest of peripheral blood mononuclear cell (PBMC)-derived natural killer (NK) cells in a packed-bed bioreactor.

[0142] Natural killer (NK) cells were isolated from human peripheral blood mononuclear cells (PBMCs) using RosetteSep (STEMCELL Technologies; typically >95% CD56+CD3−) and filtered using the density gradient medium Lymphoprep. TM The cells are separated using Ficoll. Red blood cells (erythrocytes) are removed using RBC X1 lysis buffer. The isolated population is cryopreserved in HI-FBS and DMSO cryopreservation solution.

[0143] A 0.5 L packed-bed MiniBio reactor containing 2.5 grams of Fibra-Cel® discs is assembled and then autoclaved by steam sterilization at 122.5°C and 1 bar above atmospheric pressure for 30 minutes, after which the MiniBio reactor is connected to the Applikon MiniBio control station.

[0144] Fibra-Cel® discs are preincubated in RPMI 1640 medium supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin at 37°C for approximately 24 hours. During incubation, serum proteins electrostatically interact with the hydrophilic end groups of the Fibra-Cel® discs, creating an extracellular matrix (ECM) coating on the Fibra-Cel® that mimics the cells' natural environment.

[0145] 900×10 6 NK cells are thawed in RPMI 1640 medium supplemented with 10% HI-FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, 0.1 mM MEM non-essential amino acids, and 100 IU / ml penicillin-streptomycin. The thawed cells are seeded at a target concentration of 3 x 10 6The cell suspension is diluted to 100 cells / mL. The resulting cell suspension is seeded into a bioreactor system set at the following conditions: temperature 37°C, DO 80%, pH 7.4, and agitation speed 100 rpm, so that the total volume within the bioreactor reaches 300 mL. During seeding, the cells are distributed within the bioreactor between the packed bed and the "external" environment. For activation pretreatment, the medium is supplemented with human recombinant IL-12 (10 ng / mL), IL-18 (50 ng / mL), and IL-15 (50 ng / mL) for 16 ± 2 hours, followed by a wash step and subsequent incubation in growth medium supplemented with human recombinant IL-15 (1 ng / mL).

[0146] During the growth period, the growth medium and cell suspension (in the "external" environment) were monitored for pH, cell concentration (by Vi-Cell), metabolic activity of the cells based on nutrient consumption (by Cedex bioanalyzer), and flow cytometer (CytoFLEX). TM ) are sampled daily to measure the distribution of cell populations by chromosome number.

[0147] Medium renewal is performed at 30% on days 4 and 7. On day 8 of culture, cells are harvested from the packed-bed bioreactor.

[0148] It will be appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Similarly, various features of the invention, which are, for clarity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0149] While the present invention has been described in connection with specific embodiments thereof, it is recognized that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the claims and description. All publications, patents, and patent applications, and GenBank accession numbers mentioned herein are incorporated by reference in their entirety, as if each individual publication, patent, or patent application, or GenBank accession number were specifically and individually indicated to be incorporated by reference herein. Furthermore, citation or identification of any document in this application shall not be construed as an admission that such document is available as prior art to the present invention.

Claims

1. 1. A three-dimensional (3D) bioreactor for large-scale immune cell expansion, comprising: at least one packed bed chamber; at least one porous scaffold coated with one or more extracellular matrix proteins (ECM) surrounded by the at least one packed bed chamber; at least one vessel enclosing said at least one packed bed chamber; a flow medium contained in the at least one container and having at least one immune cell population suspended therein; causing a flow medium having at least one suspended immune cell population to flow through the packed bed chamber and the at least one porous scaffold coated with one or more ECMs; The at least one porous scaffold coated with one or more ECMs comprises: configured to form a retention niche with low shear forces that mimics the natural growth environment of the immune cells; having porosity and allowing for large-scale expansion of said immune cell population through at least one porous scaffold coated with said one or more ECMs; The immune cells are any of Jurkat cells, T2 cells, K562 cells, Raji, U937, THP-1, HL-60, peripheral blood mononuclear cells (PBMC), polymorphonuclear cells (PMN), conventional and non-conventional T cells, B cells, B cell hybridomas, CAR-B, NKT cells, CAR-NKT, gamma delta T (gdT) cells, CAR-gdT, NK cells, CAR-NK, or combinations thereof.

2. 2. The 3D bioreactor of claim 1, wherein the PBMC immune cells are unmodified or modified myeloid cells, myeloid cells with phagocytic capacity, monocytes, CAR monocytes, dendritic cells, CAR dendritic cells, or a combination thereof.

3. 2. The 3D bioreactor of claim 1, wherein the PMN immune cells are unmodified or modified neutrophils, basophils, eosinophils, or a combination thereof.

4. 2. The 3D bioreactor of claim 1, wherein the conventional or non-conventional T cells are unmodified or modified T cells and consist of the following group: CAR-T, CAR-MAIT, modified T cell receptor (TCR)-T, modified TCR-MAIT, tumor infiltrating lymphocytes (TILs), and combinations thereof.

5. 10. The 3D bioreactor of claim 1, wherein at least one porous scaffold coated with said one or more ECMs is further coated or linked with at least one immune cell activator.

6. 6. The 3D bioreactor of claim 5, wherein the immune cell activator is either antigen presenting cells (APCs) loaded or unloaded with antigens, or antigens directly bound to the at least one porous scaffold coated with the one or more ECMs.

7. 2. The 3D bioreactor of claim 1, wherein the expanded immune cells are further activated in said at least one packed bed chamber by exposing said immune cell population to at least one porous scaffold coated with said one or more ECMs that are coated or linked with at least one immune cell activator.

8. 2. The 3D bioreactor of claim 1, wherein the expanded immune cells are further activated in the at least one packed bed chamber by exposure to a suspended soluble immune cell activator and further expanded by the at least one porous scaffold coated with the one or more ECMs.

9. 2. The 3D bioreactor of claim 1, wherein the at least one immune cell population is harvested or reactivated by exposing antigen presenting cells (APCs) attached to the at least one porous scaffold coated with the one or more ECMs to an antigen, thereby generating an additional activation signal for the immune cell population.

10. 10. The 3D bioreactor of claim 1, wherein the at least one immune cell population is harvested or reactivated by transferring the expanded immune cells to another bioreactor comprising at least one porous scaffold coated with a different or similar immune cell activating agent.

11. 2. The 3D bioreactor of claim 1, wherein the at least one porous scaffold coated with one or more ECMs is either a single porous scaffold matrix expanded within the interior space of the at least one packed bed chamber or a plurality of mini- or micro-porous scaffolds filling the at least one packed bed chamber.

12. 10. The 3D bioreactor of claim 1, wherein the fluid medium further comprises one or more genetic modifiers that genetically modify the immune cell population suspended in the fluid medium.

13. 1. A method for large-scale immune cell expansion in a three-dimensional (3D) bioreactor, comprising: a. inserting at least one porous scaffold into at least one packed bed chamber; b. coating the at least one porous scaffold with one or more extracellular matrix proteins (ECM); c) circulating a flow medium having at least one suspended immune cell population contained in at least one container surrounding the at least one packed bed chamber, wherein the flow medium having the at least one suspended immune cell population is configured to flow through the at least one packed bed chamber and the at least one porous scaffold coated with the one or more ECMs; The at least one porous scaffold coated with the one or more ECMs comprises: configured to form a retention niche with low shear forces that mimics the natural growth environment of the immune cells; the at least one porous scaffold is porous and configured to allow for extensive expansion of the immune cell population through the at least one porous scaffold coated with the one or more ECMs; The method, wherein the immune cells are any of Jurkat cells, T2 cells, K562 cells, Raji, U937, THP-1, HL-60, peripheral blood mononuclear cells (PBMC), polymorphonuclear cells (PMN), conventional and non-conventional T cells, B cells, B cell hybridomas, CAR-B, NKT cells, CAR-NKT, gamma delta T (gdT) cells, CAR-gdT, NK cells, CAR-NK, and combinations thereof.

14. 14. The method of claim 13, wherein the PBMC immune cells are unmodified or modified myeloid cells, myeloid cells with phagocytic capacity, monocytes, CAR monocytes, dendritic cells, CAR dendritic cells, or combinations thereof.

15. 14. The method of claim 13, wherein the conventional or non-conventional T cells are unmodified or modified T cells and consist of the group of: CAR-T, CAR-MAIT, modified T cell receptor (TCR)-T, modified TCR-MAIT, tumor infiltrating lymphocytes (TILs), and combinations thereof.

16. coating the at least one porous scaffold coated with one or more ECMs with at least one immune cell activator before or after step b) to expand and activate the immune cell population within the at least one packed bed chamber; 14. The method of claim 13, further comprising the step of exposing said immune cell population to said at least one activating agent after said step.

17. 17. The method of Claim 16, further comprising genetically modifying the immune cell population in the 3D bioreactor by adding into the flowable medium one or more genetic modifying agents that genetically modify the immune cell population suspended in the flowable medium.

18. harvesting the immune cell population or a portion of the immune cell population; 14. The method of claim 13, further comprising expanding and reactivating the harvested immune cell population in the same at least one 3D bioreactor or a different bioreactor.

19. After harvesting the immune cell population, genetically modifying the harvested immune cell population outside the at least one 3D bioreactor; 20. The method of claim 18, further comprising the step of reseeding said genetically modified immune cell population into the same at least one 3D bioreactor or a different bioreactor.

20. harvesting the immune cell population or a portion of the immune cell population; 17. The method of claim 16, further comprising expanding and reactivating the harvested immune cell population in the same at least one 3D bioreactor or a different bioreactor.

21. 1. A three-dimensional (3D) bioreactor for large-scale expansion and activation of immune cell populations, comprising: at least one packed bed chamber; at least one porous antigen-presenting cell-mimetic scaffold (APC-MS) coated with one or more extracellular matrix proteins (ECM) surrounded by the at least one packed-bed chamber; at least one vessel enclosing said at least one packed bed chamber; a flow medium contained in the at least one container and having at least one immune cell population suspended therein; flowing the flow medium having the at least one suspended immune cell population through at least one porous APC-MS coated with the one or more ECMs; The at least one porous APC-MS coated with one or more ECMs comprises: configured to form a retention niche with low shear forces that mimics the natural growth environment of the immune cell population; having porosity and allowing for large-scale expansion of said at least one immune cell population through at least one porous APC-MS coated with said one or more ECMs; The immune cell population is any of Jurkat cells, T2 cells, K562 cells, Raji, U937, THP-1, HL-60, peripheral blood mononuclear cells (PBMC), polymorphonuclear cells (PMN), conventional and non-conventional T cells, B cells, B cell hybridomas, CAR-B, NKT cells, CAR-NKT, gamma delta T (gdT) cells, CAR-gdT, NK cells, CAR-NK, and combinations thereof.

22. 22. The 3D bioreactor of claim 21, wherein said PBMC immune cells are unmodified or modified myeloid cells, myeloid cells with phagocytic capacity, monocytes, CAR monocytes, dendritic cells, CAR dendritic cells, or combinations thereof.

23. 22. The 3D bioreactor of claim 21 , wherein said conventional or non-conventional T cells are unmodified or modified T cells and consist of the following group: CAR-T, CAR-MAIT, modified T cell receptor (TCR)-T, modified TCR-MAIT, tumor infiltrating lymphocytes (TILs), and combinations thereof.

24. 22. The 3D bioreactor of claim 21, wherein the at least one porous APC-MS coated with the one or more ECMs is a single unit extended within the interior space of the at least one packed-bed chamber or a plurality of mini / micro porous APC-MS filling the at least one packed-bed chamber.

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