Systems and methods for large-scale expansion and activation of immune cells

Abstract

This invention discloses a three-dimensional (3D) bioreactor for the large-scale expansion of immune cells and a method of using the same. The 3D bioreactor includes: at least one filled bed chamber comprising at least one porous scaffold; at least one porous scaffold coated with one or more extracellular matrix proteins (ECM); at least one container containing a fluid culture medium configured to flow through the filled bed chamber having at least one porous coated scaffold; and at least one population of immune cells suspended in the fluid culture medium, wherein the at least one porous scaffold coated with the ECM creates a low-shear-force fixation niche that mimics the natural growth environment of immune cells and allows for the large-scale expansion of the immune cell population flowing through the coated porous scaffold.

Description

Technical Field

[0001] This invention relates to systems and methods for large-scale culture and / or activation of immune cells. More specifically, this invention relates to the large-scale culture and / or activation of immune cells in a packed-bed bioreactor. Background Technology

[0002] The culture of mammalian cells is inherently complex due to their high sensitivity, relatively slow proliferation, complex differentiation processes, and the fundamental requirement of absolute sterility. Large-scale cell culture remains a challenging process. Even small-scale cell growth requires attention and specific knowledge because serious problems are frequently encountered. For example, cell cultures are subjected to stress or damage during the preparation or testing process, and any analysis based on the cultured cells may show results that are at least partially a consequence of such damage. Furthermore, applying conclusions drawn from results obtained on damaged cells to in vivo conditions or using cells for immunotherapy can have fatal consequences. Moreover, the damage or stress conditions are not reproducible between individual cell cultures and can affect cell growth. These challenges become even more critical when performing large-scale cell culture. Various attempts have been made in recent decades. U.S. Patent Application No. 2006 / 0194320 describes an apparatus and method for culturing cells and / or tissues that mimic the cellular structure and immune function of immune-active tissues; however, the system and method are limited to a volume of 4 ml. Other patents and patent applications related to the field of this invention are US 8911995 and US Patent No. 10472612.

[0003] Furthermore, the use of bioreactors for cell and tissue culture is well-known. https: / / www.minerva-kg.de / libraryonline / upload / files / file_6400.pdf The document provides a detailed overview of bioreactor design, prototyping, and process control for reproducible three-dimensional tissue culture.

[0004] Cultured immune cells in vitro presents a further challenge. In recent years, the human immune system has been used as the basis for therapeutic techniques capable of recognizing and killing tumor cells and has been a core target of anticancer immunotherapy. Therefore, there is increasing interest in improving the effectiveness and accessibility of this technology for its widespread application in adoptive cell therapy (ACT) such as chimeric antigen receptor T (CAR-T) cells, tumor-infiltrating lymphocytes (TILs), dendritic cells (DCs), natural killer (NK) cells, and so on. However, currently available systems for culturing immune cells are insufficient for implementing this technology because they either damage cells, are inefficient, have limited scalability, and some require very high costs (see: 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 This article reviews the main culture methods known to date for immune cell culture, along with their advantages and disadvantages. More detailed comparisons are made between stirred flasks, G-Rex flasks, rocking motion bioreactors, stirred tank bioreactors, hollow fiber bioreactors, and CliniMACS Prodigy. Therefore, there is an urgent need for scalable, cost-effective, and GMP-compliant bioreactors for culturing immune cells.

[0005] The present invention aims to provide systems and methods for large-scale culture and / or activation of immune cell populations. Summary of the Invention

[0006] In one key aspect, the present invention relates to systems and methods for large-scale culture and / or activation of immune cells. The challenge of effectively growing immune cells on a large scale is to avoid or minimize high shear stresses that could damage cells to obtain a high-scale homogeneous system, and to create conditions that allow for interaction between cells and activators and transfection agents, thereby creating physical niches and conditions that allow for high levels of intercellular interaction.

[0007] In one aspect, this invention discloses a method and system for growing immune cells, comprising a porous stationary phase located within a flowing medium. The stationary phase may be located within a bioreactor or in a separate chamber functionally connected to the bioreactor. The porous elements, as will be described in detail below, are located within baskets of a packed bed bioreactor and are immovable, not moved by the liquid flow surrounding and through them. The stationary phase can be implemented in various forms and is designed to create an environment with low flow rates and low shear forces.

[0008] As used herein, the terms “porous stationary phase,” “porous scaffold,” “porous element,” and “porous coated scaffold” all refer to the same meaning and may be used interchangeably in the following description. In some embodiments, 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 in tissues and lymph nodes.

[0009] As used herein, the terms "activator" and "immune cell activator" have the same meaning and may be used interchangeably in the following description. In some embodiments, "activator" and "immune cell activator" are antigen-presenting cells loaded with an antigen presented on their cell surface. In another embodiment, the activator is an antibody targeting an activating receptor on the surface of an immune cell. In yet another embodiment, the activator is an antigen conjugated to an activating receptor that can be presented to the surface of an immune cell.

[0010] As used herein, the term "niche" refers to a stationary phase with pores, such as, but not limited to, scaffolds, beads, and carriers that allow liquids and particles to flow through. The particles can be cells, or other synthetic or natural components.

[0011] The created niche mimics the microenvironment of lymph nodes / tissues in terms of the natural growth environment of immune cells, achieving optimal cell growth. Furthermore, because the created niche imitates the natural environment of cells, it allows for cell activation, ensuring that only certain cells respond to activation, thus enabling selective cell selection.

[0012] Furthermore, the created niche allows for the large-scale growth of immune cells while maintaining relatively low shear forces to minimize cell damage.

[0013] The terms “media” and “medium” are intended to be synonymous and can be used interchangeably below.

[0014] The terms “filled bed chamber”, “filled bed basket”, “basket”, and “growth basket” are intended to be synonymous and can be used interchangeably in the following text.

[0015] The term "growth" of cells or cell populations in this article is intended to be synonymous with the expansion of activated cell populations, whether or not they contain cells, and the culture of cell populations.

[0016] As used herein, the terms “immune cells,” “immune cell populations,” and “lymphocytes” all have the same meaning and can be used interchangeably in the following description.

[0017] In some embodiments, lymphocytes expand without substantial differentiation. In various embodiments, the expansion described is on a 2D substrate, on a 3D substrate, or on a 2D substrate followed by a 3D substrate.

[0018] In some implementations, lymphocytes are incubated in a bioreactor, non-limiting examples of which include suspension culture and culture on a 3D carrier. The term "bioreactor culture" refers to culture in a typically sterile apparatus (bioreactor) where cells are maintained under controlled conditions, as will be referenced below. Figure 1 As described.

[0019] The term “activation” of immune cells or immune cell populations in this article is intended to be synonymous with the exposure of immune cells to antigens that cause changes in cell morphology and trigger an immune response detected through the rapid proliferation and secretion of a wide variety of cytokines and chemokines.

[0020] Therefore, in one key aspect, the present invention relates to a three-dimensional (3D) bioreactor for the large-scale expansion of immune cells, comprising: a) at least one filled bed chamber including at least one porous scaffold; b) at least one porous scaffold coated with one or more extracellular matrix proteins (ECM); c) at least one container containing a fluid culture medium configured to flow through the filled bed chamber having at least one porous coated scaffold; and d) at least one population of immune cells suspended in the fluid culture medium; wherein the at least one porous scaffold coated with the ECM is configured to create a low-shear-force fixation niche that mimics the natural growth environment of immune cells and allows for the large-scale expansion of the population of immune cells flowing through the coated porous scaffold.

[0021] The at least one porous scaffold may be further coated with or attached to at least one immune cell activator. In some optional embodiments, the immune cell activator is one of antigen-presenting cells (APCs) loaded or unloaded with antigen, or antigen directly conjugated to the coated porous scaffold. In the case where the APC is unloaded with antigen, the antigen may be presented at a later stage as needed to activate immune cells.

[0022] In a further option, the expanded immune cells can be further activated within a bed-filled chamber after the immune cell population has been exposed to at least one porous coated scaffold conjugated with an immune cell activator.

[0023] In another option, after exposure to a suspended soluble immune cell activator, the expanded immune cells can be further activated in a bed-filled chamber and further expanded with at least one porous ECM-coated scaffold.

[0024] In some embodiments of the invention, the expanded and / or activated immune cell population is harvested or reactivated after the APC attached to at least one coated porous scaffold is exposed to the antigen to generate additional activation signals to the immune cell population.

[0025] Furthermore, in some further embodiments, the immune cell population is harvested or reactivated by transferring the expanded immune cells to different bioreactors comprising at least one porous scaffold coated with different or similar immune cell activators.

[0026] Porous scaffolds can be a single porous scaffold matrix that extends within the internal space of a filled bed chamber, or they can be multiple mini or micro porous scaffolds that fill the filled bed chamber.

[0027] In some further optional embodiments of the invention, immune cell populations are genetically modified by using a gene-modifying agent incorporated into the culture medium of a bioreactor.

[0028] The present invention further relates to a method for large-scale expansion of immune cells in a three-dimensional (3D) bioreactor, comprising: a) inserting at least one porous scaffold into at least one filled bed chamber; b) coating the at least one porous scaffold with one or more extracellular matrix proteins (ECM); c) circulating a fluid culture medium from at least one container configured to flow through the filled bed chamber comprising the at least one porous coated scaffold; and d) suspending at least one population of immune cells in the circulating fluid culture medium; wherein the at least one porous scaffold coated with the ECM is configured to create a low-shear fixation niche that mimics the natural growth environment of immune cells and allows for large-scale expansion of the population of immune cells flowing through the at least one porous scaffold.

[0029] The method may further include a step of coating at least one porous scaffold with at least one immune cell activator after the step of coating the scaffold with ECM, and a step of exposing the immune cell population to at least one activator after suspending at least one immune cell population in a circulating fluid culture medium, in order to expand and activate the immune cell population in the packed bed chamber.

[0030] The method may further include the step of genetically modifying immune cells within a 3D bioreactor using a gene-modifying agent incorporated into a fluid culture medium.

[0031] In some optional embodiments, the method further includes the steps of harvesting immune cells or a portion of cells and further expanding and reactivating the immune cell population in the same bioreactor or in a different bioreactor.

[0032] Alternatively or additionally, the above method may further include the step of harvesting immune cells, followed by genetic modification outside the system, and then re-inoculating the genetically modified immune cells into the same or different bioreactors.

[0033] Furthermore, in a further aspect, the present invention aims to provide a three-dimensional (3D) bioreactor for the large-scale expansion and activation of immune cell populations, comprising: a) at least one filled bed chamber including at least one porous antigen-presenting cell mimetic scaffold (APC-MS); b) at least one APC-MS coated with one or more extracellular matrix proteins (ECM); c) at least one container containing a fluid culture medium configured to flow through the coated porous APC-MS; and d) at least one immune cell population suspended in the fluid culture medium; wherein the at least one APC-MS creates a low-shear-force immobilized microenvironment that mimics the natural growth environment of the immune cell population and allows for the large-scale expansion and / or activation of the immune cell population flowing through it.

[0034] After exposing coated porous APC-MS to antigens, immune cell populations can be reactivated to generate additional activation signals to the immune cell populations.

[0035] In some optional embodiments, immune cell populations are reactivated by transferring cells to different bioreactors comprising at least one coated porous APC-MS with different or similar antigens.

[0036] According to an embodiment of the present invention, the at least one porous APC-MS can be made from a single unit amplified within the internal space of a filled bed chamber, or it can be multiple microporous APC-MS / microporous APC-MS filling a filled bed chamber.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While methods and materials similar to or equivalent to those described and used herein may be used in the practice or testing of this invention, suitable methods and materials are described below. In case of conflict, the patent specification including the definitions shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting. Attached Figure Description

[0038] The invention is described herein by way of example only with reference to the accompanying drawings. In the context of now referring specifically to the drawings, it is emphasized that details are shown by way of example and are presented solely for the purpose of illustrative discussion of embodiments of the invention, and to provide a useful and easily understood description of the principles and concepts of the invention. In this respect, no attempt is made to show the structural details of the invention in more detail than necessary for a basic understanding of the invention, and the description taken in conjunction with the drawings makes it apparent to those skilled in the art how various forms of the invention can be practiced.

[0039] In the attached diagram:

[0040] Figure 1 This is a schematic diagram of an optional packed-bed bioreactor for growing, activating, and harvesting immune cell populations according to an embodiment of the present invention.

[0041] Figure 2 This is a flowchart that describes at a high level the sequence of key steps in the preparation and use of a packed-bed bioreactor to activate, expand, and harvest immune cells.

[0042] Figures 3A-3D yes Figure 1 A schematic partial front view of a packed bed bioreactor 100 at different stages of the growth process of immune cell populations, wherein... Figure 3A The initial stage in which the filled bed basket contains an uncoated support is shown; Figure 3B A packed bed basket containing a coated scaffold and filled with ECM protein solution is shown; Figure 3C It shows that Figure 3B The coated scaffold is further connected to a bed-filled basket containing at least one type of immune cell activator; and Figure 3D A packed bed bioreactor is shown in which a coated scaffold is connected to one or more activators and immune cells flow in a culture medium.

[0043] Figure 4 This is a flow cytometry plot of T cell activation and proliferation after 7 days of growth in a packed bed bioreactor, where cell activation and proliferation were measured on days 0, 5, and 7.

[0044] Figure 5A This is a flow cytometry plot of the activation and proliferation of MAIT cells grown in a packed bed bioreactor for 10 days, where cell activation and proliferation were measured on days 0, 5, 7, and 10.

[0045] Figure 5B This is a flow cytometry graph of MAIT cell growth after the cells were transferred from the first bioreactor to the second bioreactor on day 10 and grown for an additional 7 days in the second bioreactor.

[0046] Figure 6 This is a diagram illustrating the cell distribution inside and outside the bioreactor packed bed for three different immune cell types: Jurkat, PBMC, and MAIT cells.

[0047] Figure 7 These are schematic diagrams of packed bed bioreactors of different sizes, illustrating the high scalability of the system of the present invention. Detailed Implementation

[0048] Before explaining at least one embodiment of the invention in detail, it should be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the embodiments. The invention can be implemented or practiced in various ways and with other embodiments. Furthermore, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered limiting.

[0049] The present invention aims to provide systems and methods for the large-scale expansion and activation of various immune cell populations.

[0050] In one key aspect, the present invention provides a 3D bioreactor for the large-scale expansion of immune cells, comprising: a) at least one filled bed chamber including at least one porous scaffold; b) at least one porous scaffold coated with one or more ECM proteins; c) at least one container containing a fluid culture medium configured to flow through the filled bed chamber having at least one porous coated scaffold; and d) at least one population of immune cells suspended in the fluid culture medium; wherein the at least one porous scaffold coated with the ECM is configured to create a low-shear-force fixation niche that mimics the natural growth environment of immune cells and allows for the large-scale expansion of the population of immune cells flowing through the coated porous scaffold.

[0051] In a further aspect, the present invention provides a method for large-scale expansion of immune cells in a three-dimensional (3D) bioreactor, comprising: a) inserting at least one porous scaffold into at least one filled bed chamber; b) coating the at least one porous scaffold with one or more extracellular matrix proteins (ECM); c) circulating a fluid culture medium from at least one container configured to flow through the filled bed chamber comprising the at least one porous coated scaffold; and d) suspending at least one population of immune cells in the circulating fluid culture medium; wherein the at least one porous scaffold coated with the ECM is configured to create a low-shear fixation niche that mimics the natural growth environment of immune cells and allows for large-scale expansion of the population of immune cells flowing through the at least one porous scaffold.

[0052] Furthermore, in a further aspect, the present invention provides a 3D bioreactor for the large-scale expansion and activation of immune cell populations, comprising: a) at least one packed bed chamber including at least one porous APC-MS; b) at least one APC-MS coated with one or more ECM proteins; c) at least one container containing a fluid culture medium configured to flow through the coated porous APC-MS; and d) at least one immune cell population suspended in the fluid culture medium; wherein the at least one APC-MS creates a low-shear-force immobilized microenvironment that mimics the natural growth environment of the immune cell population and allows for the large-scale expansion and / or activation of the immune cell population flowing through it.

[0053] The main aspects of the invention and optional practices of the invention will be better understood from the following detailed description of the various exemplary, non-limiting drawings and embodiments described below. Reference now is made to the drawings:

[0054] Figure 1 This is a schematic cross-sectional view of an optional packed bed bioreactor 100 for the growth, activation, and harvesting of immune cell populations according to an embodiment of the present invention.

[0055] In the depicted embodiment, the growth and vibration chamber 116 (hereinafter referred to as a "basket") is loaded with at least one porous support 10. The terms "carrier" and "support" are used interchangeably and both refer to a configuration as described in the references. Figure 2The porous element, as described in the detailed description, is coated and creates fixed niches within the basket to reduce shear forces and mimic the natural environment of immune cell populations. The basket wall 1161 is preferably separate from the inner wall 40 of the bioreactor and is movable vertically. The bioreactor 100 is supplied with liquid culture medium via an inlet pipe 120, which is configured and operable to deliver various culture media into the bioreactor, and then optionally autoclaved. In other embodiments, after sterilization, the liquid is replaced with growth medium filling the basket 116 and its contents. Basket 116 effectively divides the fluid within bioreactor 100 into three main sections: an upper section 122, which primarily contains fresh culture medium inserted via inlet pipe 120; a middle section 124, which contains culture medium within basket 116 surrounding the coating scaffold; and a lower section 126, which primarily contains culture medium flowing through the basket and, compared to the fresh culture medium at the upper section 122, is generally lacking in nutrients consumed by immune cells at the middle section (where their flow rate decreases as they flow through at least one porous coating scaffold, interact with other cells, and / or are activated), and is rich in compounds and debris secreted by immune cells. In the embodiment described herein, the culture medium at the upper section 122 is agitated by impeller 119, which generates fluid movement as indicated by arrow 109. In a further embodiment, various parameters such as temperature, pH, dissolved oxygen concentration, etc., are set at the beginning of the process as part of the system setup procedure (see below). Figure 2 The process is as follows, and is continuously adjusted to suit the suspension conditions as needed. In a further embodiment, a slow initial stirring rate of the culture medium is used to promote cell adhesion to the coating scaffold, and then the stirring rate can be increased. If desired, cells can be harvested from the culture medium for final product manufacturing or for further expansion of immune cells, as described in detail below ( Figure 2In some embodiments, the rotation of impeller 119 generates a negative pressure in guide tube 150, which draws the culture medium containing cells from the lower part 126 through guide tube 150 and then through the impeller 119 port into the upper part 122, thereby causing the culture medium and immune cells to circulate uniformly in a continuous loop in the direction indicated by arrow 109. In a further embodiment of the invention, the culture medium can be adjusted by monitoring various parameters via electrode 106 to control various parameters of the culture medium. In some optional embodiments, a ring sparger (not visible) is located inside impeller aeration chamber 11 for supplying oxygen to the culture medium flowing through the impeller 119 port via gas added from external port 103 and sparger line 7, external port 103 may be retained inside housing 5. In some other optional embodiments, gas can be added through inlet 120. Optionally, the sparged gas can be confined in a distal chamber and absorbed by the nutrient culture medium of the flushing system. In some optional embodiments, a water jacket 117 covers the culture medium area within the bioreactor 100 and has ports for removing water from the water jacket 13 and 14. A removal tube 110 is disposed along the bioreactor and has an opening within the lower culture medium section 126 to allow, if desired, harvesting of immune cells from the culture medium below basket 116. The removal tube 110 can also be used to remove debris and to refresh the culture medium after partial removal of used culture medium and addition of fresh culture medium.

[0056] In some embodiments, a continuous stirred tank bioreactor may be used, wherein the culture medium is continuously fed into the bioreactor and the product is continuously withdrawn to maintain a time-constant steady state within the bioreactor. Stirred tank bioreactors with fiber bed baskets are available, for example, from New Brunswick Scientific Co., Edison, NJ. Other bioreactors that may be used include, but are not limited to, fixed-bed bioreactors, perfusion bioreactors with multi-active foams, radial-flow perfusion bioreactors comprising tubular poly-L-lactic acid (PLLA) porous scaffolds, and any other bioreactor known in the art suitable for the purposes of this invention. A “fixed-bed bioreactor” refers to a bioreactor in which the cell growth substrate typically does not rise from the bottom of the incubation vessel in the presence of a growth medium. For example, the substrate may have sufficient density to prevent it from rising and / or may be filled with mechanical pressure to prevent it from rising. The substrate may be a single body or multiple bodies. Typically, the substrate remains substantially in situ during the standard stirring rate of the bioreactor. In some embodiments, multiple carriers are loosely packed, for example, forming a loosely packed bed immersed in a nutrient medium.

[0057] Furthermore, in some embodiments, a perfusion bioreactor is used, wherein the perfusion chamber contains a 3D substrate. In some embodiments, the 3D substrate is in the form of a porous scaffold 10. The porous scaffold can be made from a single large unit surrounding the entire or most of the volume of the filled bed chamber. Optionally, the porous scaffold used can be multiple microparticles. In some further optional embodiments, the porous scaffold can 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 (… GE Healthcare), based on collagen ( Percell Biolytica and polystyrene-based microcarriers (SoloHill Engineering).

[0058] In some embodiments, T cells are incubated in a bioreactor equipped with an “APC-MS”, whereby the term “APC-MS” as used herein refers to a scaffold to which a lymphocyte activation portion is attached or associated (in more specific embodiments, it may be any scaffold mentioned herein), and more specifically, a fibrous carrier or mesoporous silica microrod attached to or coated with the activation portion.

[0059] Unless otherwise specified, the term "filled bed bioreactor" refers to a bioreactor in which the cell growth substrate typically does not rise from the bottom of the incubation vessel in the presence of growth medium. For example, the substrate may have sufficient density to prevent it from rising and / or may be filled with mechanical pressure to prevent it from rising. The substrate may be a single body or multiple bodies. Typically, the substrate remains substantially in situ during perfusion at the standard perfusion rate of the bioreactor. In some embodiments, this definition does not preclude the possibility that the substrate may rise at unusually fast perfusion rates (e.g., greater than 200 rpm).

[0060] In other embodiments, the biocontainer is used to expand cells; in further embodiments, it is suitable for suspension culture. In various embodiments, the biocontainer is used for and / or suitable for batch culture, fed-batch culture, or continuous culture.

[0061] Figure 2 This is a flowchart describing at a high level the sequence of main steps in the preparation and use of a packed-bed bioreactor system for amplifying, activating, and harvesting immune cell populations according to an embodiment of the present invention.

[0062] System setup step 310 includes assembling the system, filling the bioreactor basket with the required amount of porous support, connecting the necessary electrodes for monitoring and controlling culture parameters (e.g., pH, dissolved oxygen, temperature) during system use, connecting the necessary piping for fresh nutrient supply and byproduct removal, sealing and performing integrity testing, 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.

[0063] Porous scaffolds, also referred to as "carriers" below, can be made of natural or synthetic materials and can be of various sizes. Some non-limiting examples of commercially available carriers include those based on alginate (GEM, Global Cell Solutions) and dextran (…). GE Healthcare), based on collagen ( Percell Biolytica and polystyrene-based (SoloHill Engineering) carriers. Optionally or additionally, the scaffold may include fibrous materials, optionally adhesive fibrous materials, which may be, for example, a woven fiber matrix, a nonwoven fiber matrix, or both. A non-limiting example of a fiber scaffold is a carrier comprising a polyester web, such as New Brunswick Scientific Co. TM The disk is available from Eppendorf. TM Commercially available from AG, Germany, and includes a polypropylene support; and microporous carriers, such as BioNOC. TM The carrier II is commercially available from CESCO BioProducts (Atlanta, GA) and is made of PET (polyethylene terephthalate). In some embodiments, the mentioned fibrous matrix includes polyester, polypropylene, polyalkylene, polyvinyl fluoride, polyvinyl chloride, polystyrene, or polysulfone. In more specific embodiments, the fibrous matrix is ​​selected from polyester and polypropylene.

[0064] To create an environment that mimics the natural environment of immune cells, an ECM coating step 312 is performed. In this step, by using... The hydrophilic end groups on the disc allow for electrostatic interactions by simply immersing the scaffold in a solution containing ECM proteins, making it possible to coat the scaffold with different materials. A detailed description of the role of the extracellular matrix in immune cells is provided in Sutherland TE et al., “The extracellular matrix and the immune system: A mutually dependent relationship”, Science. 2023 Feb. 17; 379(6633); PMID:36795835. It should be clear that other scaffolds can be used and described The disk is a non-limiting example. Additionally, the scaffold can be coated with either natural or synthetic ECM components.

[0065] In this way, various proteins, such as but not limited to albumin, fibronectin, fibrin, fibrinogen, collagen, hyaluronic acid, elastin, laminin, selectins, etc., can be used to coat the scaffold and create an environment similar to the natural environment. For example, to mimic the structure of a lymph node, the scaffold can be coated with type III collagen, a major building block of the reticular fibers that are a major component of the lymph node ECM. Furthermore, different organs have different combinations of ECM proteins, and therefore different combinations or concentrations can be used for coating. In various embodiments, adhesion molecules can be conjugated to the scaffold or the proteins coating the scaffold to promote stronger 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 be promoters of interactions between leukocytes and the ECM and to help direct leukocytes to inflamed tissue. Based on this, coating the scaffold with these molecules can increase the interaction between leukocytes and the ECM-coated scaffold. Optionally, ECM coating (step 312) occurs after system setup (step 310), followed by scaffold activation coating (step 314). In different embodiments, the porous scaffold activation coating (step 314) occurs after ECM coating (step 312), depending on the product and its activating ligand.

[0066] Step 314 describes the portion of coating the scaffold with ligands for immune cell activation. Part 314 can be performed before or after 312. Immune cells need to be activated by antigen presentation, which typically occurs in lymph nodes or infected tissues. By coating the scaffold with ECM components and providing antigen presentation in a low-shear environment, the created niche mimics the natural environment in which activation occurs. In one embodiment, adherent or semi-adherent cells can attach to the fibers constituting the scaffold after coating the ECM with serum ECM proteins. Because some immune cells require interaction with antigens presented by antigen-presenting cells (APCs) to be activated, antigen-loaded APCs are seeded during step 314 after coating step 312. APCs adhere to the scaffold and can present antigens to various immune cells, such as T cells, B cells, and all their subsets. In different embodiments, APCs can be seeded into a bioreactor and adhere to the scaffold in a non-antigen-presenting form, and then, after cell adhesion, the antigen can be incorporated into the culture medium filling the bioreactor for presentation to immune cells such as T cells and B cells. A more specific example of this implementation is the use of monocytes to activate mucosal-associated invariant T cells (MAIT). In this example, PBMCs are seeded into a bioreactor after a scaffold is coated with fetal bovine serum albumin (FBO), with monocytes adhering to the scaffold and the remaining cells suspended in the culture medium. Following initial cell seeding, 5-OP-RU (5-(2-oxopropylamino)-6-D-ribitolaminouracil) (an antigen for MAIT activation) is incorporated into the culture medium to be presented by monocytes and specifically activate MAIT cells. In a different embodiment of the invention, activation of immune cells can be modulated by antigens conjugated to a scaffold without APCs. The antigen can be an antibody targeting a specific activation receptor on the immune cell, or a protein recognized by an activation receptor such as the TCR. For example, the scaffold is coated with monoclonal anti-CD3 (OKT3) and anti-CD28 (CD28.2) antibodies to provide a co-stimulatory signal that binds to T cell receptors. After an incubation period, a blocking step is performed, followed by a washing step. Following the washing step, the scaffold was coated with fetal bovine serum albumin (PBMC). When the culture medium was changed and PBMCs were seeded into the bioreactor, the cells were activated through interaction with the ECM and the antibody-coated scaffold.

[0067] Another example is the use of MR1 monomers, tetramers, and other forms loaded or unloaded with 5-OP-RU to activate MAIT cells. When using MR1 unloaded with 5-OP-RU, the incorporation of 5-OP-RU occurs after the blocking and washing steps.

[0068] Step 316 describes the inoculation of target cells for activation and expansion. In this step, immune cells from various sources can be used to inoculate the target cells, namely PBMCs collected from blood apheresis, PBMCs isolated from specific organs or tumors. The immune cells can be fresh or frozen. In this step, the immune cells are inoculated into the bioreactor. Using different environmental parameters, such as, but not limited to, agitation speed, pH, dissolved oxygen, temperature, etc., the cells can adhere to or remain suspended in the bioreactor. Adherent cells can enter the packed bed chamber, interact with ECM proteins on the coated scaffold, and create a cellular organized microstructure within the packed bed. According to this embodiment, APCs can be inoculated into the bioreactor and adhere to the scaffold in a non-antigen-presenting mode, creating a lymph node-mimicking environment until activation occurs. A more specific example of this embodiment is the use of monocytes to activate mucosa-associated inertial T cells (MAIT). In such an example, after the scaffold is coated with fetal bovine serum albumin (FBO), PBMCs are seeded in a bioreactor. Monocytes adhere to the scaffold, while the remaining cells are suspended in the culture medium. Non-adherent cells can remain suspended in the bioreactor (within the mobile phase) and migrate into or out of the packed bed chamber to generate cell-to-cell interactions with the adherent cells.

[0069] After inoculating the immune cells in step 316, it is necessary to decide whether to perform gene editing on the expanded cell population (step 318). If gene editing is not performed, proceed to step 326.

[0070] Step 326 describes the target cell expansion phase, where environmental parameters such as stirring speed, pH, dissolved oxygen, and temperature are controlled. In this step, the bioreactor is continuously heated, and a pre-defined gas mixture is supplied to the system to maintain the desired conditions within predetermined ranges. Once cells are seeded and activation occurs, whether via APCs with loaded ligands or via scaffolds coated with activators (antibodies), an infection state is simulated in the bioreactor, triggering a series of mechanisms that lead to the clonal expansion of antigen-specific immune cells. During this period, antigen-specific immune cells proliferate significantly, and up to ~90% of the total immune cells may become antigen-specific. To support the rapid expansion of immune cells, adequate nutrient supply and removal of inhibitory metabolites are provided without disturbing the local microenvironment. In some embodiments, the bioreactor may be operated in batch, fed-batch, and / or perfusion modes. In another embodiment of the invention, a perfusion system (TFF, ATF, BioSep, etc.) may be connected to the bioreactor. Using a perfusion system, the culture medium from the bioreactor can be replaced without extracting cells from the system. The perfusion system allows for the removal of conditioned medium from the system, separating suspended cells from the conditioned medium, and simultaneously supplying fresh medium to the bioreactor via commands from a level electrode, by weight, at a preset flow rate, or manually. In this example, the conditioned medium is sampled daily to measure the number of suspended cells and the concentration of the required substrate, and the amount of fresh medium to be supplied is calculated based on the cell count and nutrient concentration.

[0071] In step 328, the culture medium, along with the immune cells, is removed from the system used to harvest the target cells, and the washing step preferably continues outside the bioreactor system. In this embodiment, the culture medium replacement step can be performed using an intermittent centrifuge or a continuous flow centrifuge (such as kSep and unifuge). After the culture medium replacement, in step 332, the cells or a portion of the cells can be returned to the bioreactor for further expansion with fresh growth medium. Optionally, in step 330, the harvested cells can be transferred downstream of the immune cells for processing to manufacture the final product.

[0072] If gene editing is performed on target cells, then in step 322, immune cells are genetically modified to add or delete specific traits. Immunological cells are transformed using modifications such as adding new target receptors or activating receptors, deleting specific receptors for self-recognition, or deleting unwanted target receptors to better treat different indications. In this step (322), the gene-modifying agent can be inserted into the bioreactor via non-viral agents such as liposomes (lipofectamine, etc.), polymers (PEI, etc.), or electroporation processes as described below, or via viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or other insertions. These gene-modifying agents can be loaded with RNA or DNA constructs that can be used, for example, CRISPR / Cas9, transposons (Sleeping Beauty, PiggyBac), and DNA-binding domains (zinc finger domains, etc.) to add or delete new data into the genome. In these embodiments, a gene-modifying agent, such as a viral vector, is incorporated into the bioreactor culture medium after scaffold coating (312, 314), cell seeding (316) activation, and during cell expansion (326). During this stage, agitation and other environmental parameters, such as pH and temperature, can be altered to prepare the cells for better penetration by the gene-modifying agent. Following incorporation, an incubation period is performed under appropriate conditions to allow the gene-modifying agent to penetrate into the cells.

[0073] Furthermore, in some optional embodiments, activated immune cells can be harvested from the bioreactor according to step (328) and aseptically transferred to an electroporation or other 2D flask device to allow the gene-modifying agent to penetrate into the cells in step 322 via electroporation or viral and non-viral methods. In this embodiment, after the incubation period, the immune cells can be re-inoculated into the bioreactor for a washing step or into a 2D flask for a washing step.

[0074] After the incubation period, washing step 324 begins. During washing step (324), the culture medium in the bioreactor is replaced several times to extract the gene modifier from the system. In one embodiment of the invention, a perfusion system (TFF, ATF, and BioSep, etc.) is connected to the bioreactor. Using a perfusion system, the culture medium from the bioreactor can be replaced without extracting cells from the system. The perfusion system removes the old culture medium and simultaneously injects new culture medium into the bioreactor via instructions from the level electrode, by weight, or manually. This process can be performed in several chamber volumes until no gene modifier is found in the extracted culture medium. Optionally, after cell expansion (step 326), the culture medium containing cells and gene modifier is drained or harvested from the system in step 328, and the washing step continues outside the bioreactor. In this embodiment, the culture medium replacement step can be accomplished using an intermittent centrifuge or a continuous flow centrifuge (e.g., kSep, Unifuge). After the culture medium replacement, the cells can be re-seeded into the starting bioreactor or a new bioreactor (332) for further expansion.

[0075] A cell harvesting step is performed after the amplification duration is reached, or the desired final product is achieved, or the required concentration of immune cells is reached for seeding cells into a larger-scale bioreactor (328, 340). In one embodiment, the harvesting step is performed by simply draining the culture medium from the system with or without stirring. In different embodiments, a filling bed basket is connected to a harvesting system as described in detail in WO 2012 / 140519, which is incorporated herein by reference in its entirety, and then the basket is slowly vibrated during the draining step to release the captured suspended immune cells from the created filling bed scaffold niche without causing physical damage to the cells. Furthermore, in all embodiments of the harvesting step, cycles of refilling and draining of the culture medium can be performed to collect all cells from the system. In each cycle, the extracted cells are drained into a sterile collection element for further processing.

[0076] In a further embodiment of the invention, at the end of the harvesting steps (328, 340), the cells or a portion of the harvested cells undergo further downstream processing (330, 342) to produce a final product. This step includes concentration and washing steps performed using various systems such as continuous flow centrifuges, filters, acoustic filtration devices, etc. Following the concentration and washing steps are specific cell collection steps using a variety of separation methods and / or the final formulation of the product, which is then packaged into its final packaging (vials or frozen bags).

[0077] Furthermore, in different embodiments of the invention, after the harvesting step, the harvested immune cells or a portion of the harvested cells are used for further expansion (332). Further expansion is accomplished by performing step 316 in an existing bioreactor or by transferring the cells to a larger-scale bioreactor, such as a 1.5L bioreactor with a 30g scaffold, a 3.5L bioreactor with a 100g scaffold, or a 10L bioreactor with a 375g scaffold. Alternatively, the new bioreactor may be intended solely for expansion using an ECM-coated scaffold (step 334), or for performing another activation step to reactivate the cells. Reactivation may be performed, for example, in the new bioreactor using the same activator (step 336), or it may be performed in the new bioreactor using a new activator (step 338). In both optional embodiments, the new bioreactor is prepared in advance according to steps 310 to 314 described above.

[0078] In various alternative implementations, reactivation of target cells can be carried out in a first bioreactor or in a new bioreactor by adding soluble activators such as transact or anti-CD3 and anti-CD28 antibodies or by adding soluble antigens that can be presented by APCs (if they are already in the bioreactor).

[0079] Figures 3A-3D This is a schematic partial front view of a packed bed bioreactor at different stages of the immune cell culture process. Figure 3A The diagram illustrates the setup phase of a system in which the filled bed basket 116 of the bioreactor 100 contains only the uncoated porous scaffold 10.

[0080] More specifically, the top and bottom boundaries of the filling chamber 116 are formed by perforated discs 132, each having a plurality of holes 1321 of a predetermined diameter. The perforated discs 132 are also referred to below as "disc," "grid," "upper grid," "lower grid," and "middle grid," which are used interchangeably and refer to the upper, middle, or lower wall of the basket 416. In some optional embodiments, as referenced above... Figure 2 As described in detail, a porous support is inserted into basket 116 and can occupy part or most of the basket's volume. At this stage, bioreactor 100 may be without liquid. Basket 116 is preferably connected to one or more vibrating rods 136 configured to allow vertical movement of basket 116. Vibration of basket 116 can be used for, for example... Figure 2The target cells are harvested during or at the end of the amplification process. Basket 116 has a wall 1161 separate from the wall 40 of bioreactor 100 to allow vertical movement of the basket while maintaining the flow direction of the culture medium containing the target cells only through the upper and lower plates 132. Basket 116 is located above the bottom of the bioreactor within bioreactor 100, such that upper portion 122 and lower portion 126 define a middle portion 124 comprising the filled bed basket 116.

[0081] Figure 3B A packed bed basket 116 is shown, comprising a coated scaffold 10' and filled with liquid culture medium 50. This is to create an environment suitable for simulating the natural environment of an immune cell population, as described above. Figure 2 As described in detail in the embodiments of the present invention, the porous scaffold should first be coated with an ECM coating. After ECM coating, the porous scaffold 10' can be further coated or connected to at least one type of antibody 1022 or antibody-presenting cells (APCs) 1033, such as Figure 3C As shown. After the porous scaffold is prepared, immune cells 1044 can be seeded into the bioreactor, such as... Figure 3D As shown. Figure 3D As shown, immune cells are inoculated with the culture medium flow throughout all parts of the bioreactor and can be found in the upper 122, lower 126, and middle 124 within basket 116. This is because the porous coated scaffold creates low-shear niches that mimic the natural environment of immune cells, thereby allowing for optimal expansion of immune cells 1044. Furthermore, upon exposure to antigens and / or APCs, immune cells 1044 are activated as previously described.

[0082] In some embodiments, immune cells 1044 are incubated on an APC-mimetic scaffold (APC-MS) in bioreactor 100. In a further embodiment, the immune cells are subsequently harvested from the porous coated scaffold for incorporation into a pharmaceutical composition.

[0083] In further optional embodiments, such as in the case of a packed bed or solid scaffold, immune cells are seeded with gentle agitation to promote uniform distribution. In the case of microcarriers, APC-MS and T cells are gently suspended and gently mixed. In either case, after seeding, perfusion and agitation are stopped for at least a period of time to allow for interaction between T cells and APC-MS and subsequent activation.

[0084] Figure 4 This is a flow cytometry graph of T cell activation and proliferation after 7 days of growth in a packed bed bioreactor, where cell activation and proliferation were measured on days 0, 5, and 7. The flow cytometer used was a CytoFLEX, which includes three lasers (405 nm, 488 nm, and 638 nm) and 13 channels for fluorescence detection.TM Analyze cell population distribution.

[0085] PBMC cells were seeded with fixation antibodies (both anti-CD3 and anti-CD28 antibodies) that provide signals for T cell activation. The bioreactor was filled with a carrier. T cell activation was assessed on days 0, 5, and 7 by measuring the expression of CD69 (an inducible cell surface marker expressed via TCR activation) and CD25 (the α-chain of the IL-2 receptor), activation markers commonly associated with T cell activation. The results demonstrated successful stimulation of T lymphocyte activation and proliferation, indicated by an increase in CD3 markers from ~44% on day 0 to ~91% on day 7. These results specifically demonstrate T lymphocyte proliferation over 7 days. Upregulation of CD69 and CD25 from ~4% and ~8% on day 0 to ~42% and 90% on day 7 indicates T cell activation. Furthermore, no changes in cell markers were detected in any of the collected cell samples after the day 7 harvest step, indicating the absence of other cell populations within the bioreactor. Additionally, no changes in activation marker levels were detected, suggesting that cell harvesting did not affect cell state.

[0086] CD69 is an inducible cell surface marker expressed upon activation via the TCR or IL-2 receptor (CD25). It plays a role in the proliferation and survival of activated T lymphocytes.

[0087] Figure 5A This is a flow cytometry plot of activation and proliferation of MAIT cells grown in a packed-bed bioreactor for 10 days, where cell activation and proliferation were measured on days 0, 5, 7, and 10. Flow cytometer CytoFLEX. TM Analyze cell population distribution.

[0088] IVB mononuclear cells were seeded into an ECM coating containing a simulated natural environment that promotes APC attachment. In a packed-bed bioreactor containing the carrier, 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 expression of the MAIT cell population was detected by the expression of CD3, Vα7.2, and CD161 markers. The results showed an increasing proportion of MAIT cells, starting at 22.6% on day 0 and reaching as high as 96.26% on day 10. Furthermore, the expression of the activation markers CD69 and CD25 increased from day 0 to day 7 and decreased by day 10.

[0089] Figure 5BThis is a flow cytometry graph of MAIT cell growth after cells were transferred from the first bioreactor to the second bioreactor on day 10 and grown for an additional 7 days in the second bioreactor. Cells that reached maximum growth capacity by day 10 in the first bioreactor were harvested, and approximately 30% of these cells were then seeded into a second bioreactor designed similarly to the first bioreactor. Cells were allowed to grow for an additional 7 days. Flow cytometry results showed that the percentage of MAIT cells was similar for most of the time, but exhibited a decrease from >90% on day 14 to 82% on day 17. CD69 marker expression was upregulated from 30% to 87% on day 17, indicating that MAIT cells maintained their activation signals, while CD25 gradually decreased as expected.

[0090] Figure 6 This is a diagram illustrating the cell distribution inside and outside the bioreactor packed bed for three different immune cell types: Jurkat, PBMC, and MAIT cells, on two different growth days. The distribution is shown as a percentage.

[0091] The disc-like properties and packed bed structure allow for the creation of niches within the bioreactor system that mimic the natural environment of cells, where cells can remain for a period of time with low internal shear stress. Figure 6 Cell distribution inside and outside the bioreactor packed bed is shown. These values ​​were calculated based on cell counts before and after packed bed harvesting, performed through several washing steps accompanied by packed bed vibration. The results show that at different packed bed harvesting time points throughout the growth period, for all three given immune cell types, at least 40% of all live cells were inside the packed bed.

[0092] Since a significant decrease in cell concentration was observed a few hours after cell seeding, it can be inferred that the following cell distribution pattern is maintained throughout the cell expansion period.

[0093] Figure 7The diagrams illustrate packed-bed bioreactors of different sizes, demonstrating the high scalability of the system of the present invention. According to an optional embodiment of the invention, the initial inoculation of immune cell populations can be performed in a micro-packed-bed bioreactor 400 having micro-baskets 416, wherein the total maximum volume of the micro-bioreactor 400 is defined by its container size 490. After cell expansion in bioreactor 400, cells or a portion of cells can be re-inoculated in a larger-sized bioreactor 500 having a packed-bed basket 516 larger than the basket 416 and allowing a greater number of porous scaffolds and higher cell expansion relative to bioreactor 400, because the total volume of bioreactor 500 is higher and determined by its container size 590. The same process can be performed on a larger bioreactor 600 having baskets 616 and containers 690, and so on until a largest bioreactor 700 with a container size 790 allowing for multiple growth of immune cells and having the largest basket 716 is reached. Based on the methods and examples provided above, it can be understood that the conditions for the growth and activation of immune cell populations in each bioreactor can be similar to or different from the previous growth stage in the smaller bioreactor. At the end of the process, large-scale growth of immune cells activated by similar or different activators is achieved.

[0094] Example

[0095] The following embodiments, together with the above description, illustrate certain implementations in a non-limiting manner.

[0096] Example 1

[0097] Expansion of Jurkat cells in a packed-bed bioreactor.

[0098] Assemble a 0.5L packed bed MiniBio reactor containing 2.5 grams The trays are then sterilized by steam at 122.5°C and 1 bar above atmospheric pressure for 30 minutes in an autoclave. Afterward, the MiniBio reactor is connected to the Applikon MiniBio control station.

[0099] Will The plates were pre-incubated at 37°C for approximately 24 hours with RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) and 0.1% 50 mg / ml gentamicin. During incubation, serum proteins and... The hydrophilic end groups on the disk interact electrostatically and An extracellular matrix (ECM) coating that mimics the natural cellular environment is produced.

[0100] 81.6×10 6Thaw the cells into RPMI-1640 supplemented with 10% HI-FBS and 0.1% 50 mg / ml gentamicin. Dilute the thawed cells to 0.24 × 10⁻⁶ at inoculation. 6 The target concentration was [number of cells / ml]. The prepared cell suspension was inoculated into a bioreactor system set to the following conditions: temperature 37°C, 80% dissolved oxygen (DO), pH 7.4, and stirring at 150 rpm, so that the total volume inside the bioreactor reached 340 ml.

[0101] On day 3 of culture, the culture medium was replaced by 25% (meaning 25% fresh medium was added). On day 4 of culture, due to the rapid proliferation in a very short time, the cells were harvested from the packed bed bioreactor. The total cell count reached 830 × 10⁶ cells / year. 6 Each cell represents a 10.4-fold expansion (see Table 1 for details). Cell distribution inside and outside the packed bed is as follows: Figure 6 As shown.

[0102] Example 2

[0103] Activation, expansion and harvesting of peripheral blood mononuclear cells (PBMCs) in a packed bed bioreactor.

[0104] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and subjected to filtration and density gradient culture medium Lymphoprep. TM (Ficoll) separation. Red blood cells (erythrocytes) were depleted using RBC X1 lysis buffer. The separated population was then cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) freezing solution.

[0105] Assemble a 0.5L packed bed MiniBio reactor containing 2.5 grams The trays are then sterilized by steam at 122.5°C and 1 bar above atmospheric pressure for 30 minutes in an autoclave. Afterward, the MiniBio reactor is connected to the Applikon MiniBio control station.

[0106] Coated with monoclonal anti-CD3 (OKT3) antibody and anti-CD28 (CD28.2) antibody Both discs provide co-stimulatory signals that bind to T cell receptors. Based on a dilution of 0.21 μg / cm in PBS. 2 Calculate the amount of each activator. Incubate the final solution at 100 rpm at room temperature (RT) for 3 hours and then at 37°C for 1.5 hours.

[0107] After incubation with the activator, a blocking procedure was performed, followed by incubation with 1% BSA solution at RT and 100 rpm for 1 hour. Then, the solution was drained and washed with PBS at 150 rpm for 10 minutes.

[0108] The packed bed bioreactor was then prepared and equilibrated at 37°C for approximately 24 hours using growth medium consisting of RPMI-1640 supplemented with 10% HI-FBS, 1% sodium pyruvate (100 mM), and 0.1% 50 mg / ml gentamicin. During this incubation period, serum proteins and... The hydrophilic end groups on the disk interact electrostatically and An ECM coating that mimics the natural environment of cells is produced on the surface.

[0109] 262×10 6 The cells were thawed into RPMI-1640 buffer supplemented with 10% HI-FBS, 1% sodium pyruvate (100 mM), IL-2 (100 U / L), and 0.1% 50 mg / ml gentamicin. At inoculation, the thawed cells were diluted to a concentration of 0.97 × 10⁻⁶ cells / mL. 6 The target concentration was [number of cells / ml]. The prepared cell suspension was seeded into a bioreactor system set to the following conditions: temperature 37°C, 80% DO, pH 7.4, and stirring at 100 rpm, resulting in a total internal volume of 270 ml. After seeding, the cells were distributed within the bioreactor between the packed bed and the “external” environment, and a decrease in cell concentration was observed 3 hours post-seeding.

[0110] During the growth phase, samples of the growth medium and cell suspension (in the “external” environment) were taken daily to measure pH, cell concentration (via Vi-Cell), cell metabolic activity based on nutrient consumption (via Cedex Bioanalyzer), and flow cytometry (CytoFLEX). TM )Measure cell population distribution.

[0111] The culture medium was replenished at 5.5%, 32%, and 20% on days 3, 5, and 6, respectively. Cells were harvested from the packed-bed bioreactor on day 7, as maximum growth capacity had been reached. The total cell count reached 1073 × 10⁻⁶. 6 91% of the cells were CD3 positive, indicating an 8.5-fold amplification. The proportions of different cell populations within the culture and their changes over time are shown below. Figure 4 As shown. The cell distribution inside and outside the packed bed is as follows. Figure 6 As shown.

[0112] Example 3

[0113] Culture of mucosa-associated inert T cells (MAIT) in a packed-bed bioreactor: activation, expansion, harvesting, re-expansion, and second harvest.

[0114] Blood mononuclear cells were isolated from human placental venous blood (IVB) and separated by filtration and density gradient culture medium Lymphoprep. TM (Ficoll) separation. Red blood cells (erythrocytes) were depleted using RBC X1 lysis buffer. The separated population was then cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) freezing solution.

[0115] Assemble a 0.5L packed bed MiniBio reactor containing 2.5 grams The trays are then sterilized by steam at 122.5°C and 1 bar above atmospheric pressure for 30 minutes in an autoclave. Afterward, the MiniBio reactor is connected to the Applikon MiniBio control station.

[0116] Will The plate was incubated with RPMI-1640 supplemented with 10% HI-FBS at 37°C for approximately 24 hours. During incubation, serum proteins and... The hydrophilic end groups on the disk interact electrostatically, and An ECM coating that mimics the natural environment of cells is produced on the surface.

[0117] 300±20×10 6 The cells were thawed and supplemented with 1% L-glutamine 200mM and 0.1% gentamicin 50mg / ml. In Nutri-T GMP medium, thawed cells were diluted to 1×10⁻⁶. 6 The target concentration was [number of cells / ml]. The prepared cell suspension was seeded into a bioreactor system set to the following conditions: temperature 37°C, 80% DO, pH 7.4, and stirring at 100 rpm, until a final volume of 300 ml was reached. After seeding, the cells were distributed within the bioreactor between the packed bed and the “external” environment, and a decrease in cell concentration was observed 3 hours post-seeding.

[0118] Three hours after inoculation, MAIT cells are activated via their T-cell receptors (TCRs). Activation via the TCR requires co-stimulatory signals, including recognition of microbial riboflavin metabolites presented on MHC Class I-like molecules MR1, and co-stimulation by CD28, TLR agonists, bacterial products, or cytokines.

[0119] The initial cell population isolated from human placental IVB included various antigen-presenting cells (APCs) capable of activating MAIT cells via MR1, such as dendritic cells, monocytes, and B cells. 5-OP-RU, a riboflavin intermediate derived from microorganisms, was added to the growth medium at a concentration of 250 nM to present to the MAIT cell TCR by APCs on MR1. IL-15 at a concentration of 50 ng / ml was also added to the growth medium to stimulate MAIT cells (via IL-15R expressed on MAIT cells) to produce IFN-γ and release granzyme B and perforin.

[0120] During the growth phase, samples of the growth medium and cell suspension were taken daily to measure pH, cell concentration (via Vi-Cell), and cell metabolic activity based on nutrient consumption (via Cedex). TM Bioanalyzer) and flow cytometer (CytoFLEX) TM )Measure cell population distribution.

[0121] Cells were grown in a packed-bed bioreactor for 10 days, with culture medium refreshed at 5%, 5.2%, and 100% on days 3, 5, and 7, respectively. Cells were harvested from the packed-bed bioreactor on day 10, as they had reached maximum growth capacity. The total cell count reached 1089 × 10⁶ cells / day. 6 The culture showed 94% MAIT cells (Vα7.2 positive, high CD161), indicating a 43.5-fold expansion. The proportions of different cell populations within the culture and their changes over time are shown below. Figure 5A As shown.

[0122] After harvesting cells from the bioreactor on day 10 of culture, further cell expansion was examined. An additional 0.5L packed-bed MiniBio reactor was assembled, and cells containing 2.5g of [cells / units] were prepared as before. The aseptic system of the trays was pre-incubated at 37°C for approximately 24 hours with RPMI-1640 supplemented with 10% HI-FBS to ensure that... The tray provides an ECM coating and creates a natural environment for re-seeded cells.

[0123] After ECM coating, the packed bed bioreactor was filled with a solution supplemented with 1% L-glutamine 200mM and 0.1% 50mg / ml gentamicin. Pre-equilibrate the growth medium consisting of Nutri-T GMP medium. Introduce 295 × 10⁻⁶ g / cm³ of the medium. 6 The harvested cells were inoculated into growth medium and diluted to ~1×10⁻⁶. 6The target concentration was [number of cells / ml]. The prepared cell suspension was seeded into a bioreactor system set to the following conditions: temperature 37°C, 80% DO, pH 7.4, and stirring at 100 rpm, until a final volume of 300 ml was reached. IL-15 at a concentration of 50 ng / ml was added to the growth medium to induce MAIT cell expansion.

[0124] During the growth phase, samples of the growth medium and cell suspension were taken daily to measure pH, cell concentration (via Vi-Cell), cell metabolic activity based on nutrient consumption (via Cedex Bioanalyzer), and flow cytometry (CytoFLEX). TM )Measure cell population distribution.

[0125] Cells were allowed to grow for an additional 7 days in a packed-bed bioreactor, with 26.5% and 100% of the culture medium replaced on days 12 and 14, respectively. Cells were harvested from the packed-bed bioreactor on day 17. The total cell count reached 490 × 10⁶ cells / day. 6 88% of the cells were MAIT cells (Vα7.2 positive, high CD161), indicating a 1.5-fold expansion. The relative percentage of MAIT cells in the culture and its change over time are shown in the figure. Figure 5B As shown.

[0126] Table 1 below summarizes the initial and final total viable cells, their relative population shares, and expansion folds during the growth phase for three different immune cell types: Jurkat, PBMC, and MAIT.

[0127] Table 1

[0128]

[0129] As shown in the table, all expansion folds were greater than 1, indicating cellular expansion in all three given immune cell types and in the second growth phase of MAIT cells. Additionally, Table 1 shows the shift in cell population balance, which reached over 94% of the target cells (T cells after 7 days of growth and MAIT cells after 10 days of growth) at the end of the growth phase examined. The second growth phase of MAIT showed a slight decrease in the percentage of MAIT cells (from 94% to 86%).

[0130] Example 4

[0131] Expansion, activation, and harvesting of B cells derived from peripheral blood mononuclear cells (PBMCs) in a packed bed bioreactor.

[0132] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and subjected to filtration and density gradient culture medium Lymphoprep. TM(Ficoll) separation. Red blood cells (erythrocytes) were depleted using RBC X1 lysis buffer. The separated population was then cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) freezing solution.

[0133] Assemble a 0.5L packed bed MiniBio reactor containing 2.5 grams The tray is then sterilized by steam at 122.5°C and 1 bar above atmospheric pressure for 30 minutes in an autoclave. Afterward, the MiniBio reactor is connected to the Applikon MiniBio control system.

[0134] Coated with anti-CD40 antibody The activator is used to provide an activation signal. The amount of activator is based on a dilution of 1 μg / cm³ in PBS. 2 The final solution was incubated at 100 rpm at room temperature for 3 hours and then at 37°C for 1.5 hours. Following incubation with the activator was the blocking procedure, with an additional incubation of 1% BSA solution at room temperature and 100 rpm for 1 hour. The solution was then drained and washed with PBS at 150 rpm for 10 minutes.

[0135] Will The plate was pre-incubated with RPMI 1640 supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin at 37°C for approximately 24 hours. During incubation, serum proteins and... The hydrophilic end groups on the disk interact electrostatically and An ECM coating that mimics the natural environment of cells is produced on the surface.

[0136] 300×10 6 Thaw the cells to RPMI 1640 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, recombinant human IL-4 (10 ng / ml), and IL-21 (10 ng / ml). Dilute the thawed cells to 1 × 10⁻⁶ at seed. 6 The target concentration was [number of cells / ml]. The prepared cell suspension was seeded into a bioreactor system set to the following conditions: temperature 37°C, 80% DO, pH 7.4, and stirring at 100 rpm, so that the total volume inside the bioreactor reached 300 ml. After seeding, the cells were distributed within the bioreactor between the packed bed and the “external” environment surrounding the packed bed basket. Samples were taken daily to measure pH, cell concentration (via Vi-Cell), and cell metabolic activity.

[0137] During the growth phase, the growth medium and cell suspension (in the “external” environment) are measured based on nutrient consumption (via Cedex Bioanalyzer) and flow cytometry (CytoFLEX). TM ) cell population distribution.

[0138] The culture medium was replaced by 50% on days 4 and 6. Cells were harvested from the packed bed bioreactor on day 8 of culture.

[0139] Example 5

[0140] Activation, expansion, and harvesting of iNKT cells derived from peripheral blood mononuclear cells (PBMCs) in a packed bed bioreactor.

[0141] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and subjected to filtration and density gradient culture medium Lymphoprep. TM (Ficoll) separation. Red blood cells (erythrocytes) were depleted using RBC X1 lysis buffer. The separated population was then cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) freezing solution.

[0142] Assemble a 0.5L packed bed MiniBio reactor containing 2.5 grams The tray is then sterilized by steam at 122.5°C and 1 bar above atmospheric pressure for 30 minutes in an autoclave. Afterward, the MiniBio reactor is connected to the Applikon MiniBio control system.

[0143] Will The plates were pre-incubated at 37°C for approximately 24 hours with RPMI 1640 supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin. During incubation, serum proteins and... The hydrophilic end groups on the disk interact electrostatically, and An ECM coating that mimics the natural environment of cells is produced on the surface.

[0144] 600×10 6 Thaw the cells to RPMI 1640 buffer 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 recombinant human IL-2. Dilute the thawed cells to 2 × 10⁶ cells / ml at seed. 6The target concentration was [number of cells / ml]. The prepared cell suspension was seeded into a bioreactor system set to the following conditions: temperature 37°C, 80% DO, pH 7.4, and stirring at 100 rpm, so that the total volume inside the bioreactor reached 300 ml. After seeding, the cells were distributed within the bioreactor between the packed bed and the “external” environment. Three hours after seeding, 100 ng / ml α-galactosylceramide was added to the bioreactor culture medium to promote cell adhesion. The activation step begins with the presentation of CD1d on antigen-presenting cells (from the PBMC population) on the disc.

[0145] During the growth phase, samples of the growth medium and cell suspension (in the “external” environment) were taken daily to measure pH, cell concentration (via Vi-Cell), cell metabolic activity based on nutrient consumption (via Cedex Bioanalyzer), and flow cytometry (CytoFLEX). TM )Measure cell population distribution.

[0146] 70% of the culture medium was replaced on days 3 and 6. Cells were harvested from the packed bed bioreactor on day 7 of culture.

[0147] Example 6

[0148] Activation, expansion, and harvesting of γδT cells derived from peripheral blood mononuclear cells (PBMCs) in a packed bed bioreactor.

[0149] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and subjected to filtration and density gradient culture medium Lymphoprep. TM (Ficoll) separation. Red blood cells (erythrocytes) were depleted using RBC X1 lysis buffer. The separated population was then cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) freezing solution.

[0150] Assemble a 0.5L packed bed MiniBio reactor containing 2.5 grams The trays are then sterilized by steam at 122.5°C and 1 bar above atmospheric pressure for 30 minutes in an autoclave. Afterward, the MiniBio reactor is connected to the Applikon MiniBio control station.

[0151] Will The plates were pre-incubated at 37°C for approximately 24 hours with RPMI 1640 supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin. During incubation, serum proteins and... The hydrophilic end groups on the disk interact electrostatically, and An ECM coating that mimics the natural environment of cells is produced on the surface.

[0152] 300×10 6 Thaw the cells to RPMI 1640 buffer 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. Dilute the thawed cells to 1 × 10⁻⁶ at seed. 6 The target concentration was [number of cells / ml]. The prepared cell suspension was seeded into a bioreactor system set to the following conditions: temperature 37°C, 80% DO, pH 7.4, and stirring at 100 rpm, so that the total volume inside the bioreactor reached 300 ml. After seeding, the cells were distributed within the bioreactor between the packed bed and the “external” environment. The activation step was initiated 3 hours post-seeding by incorporating 5 μM zoledronic acid into the bioreactor culture medium.

[0153] During the growth phase, samples of the growth medium and cell suspension (in the “external” environment) were taken daily to measure pH, cell concentration (via Vi-Cell), cell metabolic activity based on nutrient consumption (via Cedex Bioanalyzer), and flow cytometry (CytoFLEX). TM )Measure the distribution of cell populations.

[0154] The culture medium was replaced by 50% on days 4, 7, 10, and 13. Cells were harvested from the packed bed bioreactor on day 14 of culture.

[0155] Example 7

[0156] Activation, expansion, and harvesting of natural killer (NK) cells derived from peripheral blood mononuclear cells (PBMCs) in a packed bed bioreactor.

[0157] Natural killer (NK) cells were isolated from human peripheral blood mononuclear cells (PBMCs) using RosetteSep (STEMCELL Technologies; conventional ≥95% CD56+CD3-) and isolated via filtration and density gradient Lymphoprep medium. TM (Ficoll) separation. Red blood cells (erythrocytes) were depleted using RBC X1 lysis buffer. The separated population was then cryopreserved in HI-FBS and DMSO cryoprotectant.

[0158] Assemble a 0.5L packed bed MiniBio reactor containing 2.5 grams The trays are then sterilized by steam at 122.5°C and 1 bar above atmospheric pressure for 30 minutes in an autoclave. Afterward, the MiniBio reactor is connected to the Applikon MiniBio control station.

[0159] Will The plates were pre-incubated at 37°C for approximately 24 hours with RPMI 1640 supplemented with 10% HI-FBS and 100 IU / ml penicillin-streptomycin. During incubation, serum proteins and... The hydrophilic end groups on the disk interact electrostatically, and An extracellular matrix (ECM) coating that mimics the natural cellular environment is produced.

[0160] 900×10 6 NK cells were thawed into RPMI 1640 buffer 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. At seeding, the thawed cells were diluted to 3 × 10⁶ cells / mL. 6 The target concentration was [number of cells / ml]. The prepared cell suspension was seeded into a bioreactor system set to the following conditions: temperature 37°C, 80% DO, pH 7.4, and stirring at 100 rpm, so that the total volume inside the bioreactor reached 300 ml. After seeding, the cells were distributed within the bioreactor between the packed bed and the “external” environment. For pre-activation, recombinant human IL-12 (10 ng / mL), IL-18 (50 ng / mL), and IL-15 (50 ng / mL) were supplemented to the culture medium and the cells were cultured for 16 ± 2 hours, followed by a washing step, and then cultured in growth medium supplemented with recombinant human IL-15 (1 ng / mL).

[0161] During the growth phase, samples of the growth medium and cell suspension (in the “external” environment) were taken daily to measure pH, cell concentration (via Vi-Cell), cell metabolic activity based on nutrient consumption (via Cedex Bioanalyzer), and flow cytometry using CytoFLEX. TM Measure cell population distribution.

[0162] The culture medium was replaced by 30% on days 4 and 7. On day 8 of culture, the cells were harvested from the packed bed bioreactor.

[0163] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination.

[0164] Although the invention has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations will be apparent to those skilled in the art. Therefore, the invention is intended to include alternatives, modifications, and variations falling within the spirit and broad scope of the claims and description. All publications, patents, and patent applications, as well as GenBank Registry Numbers mentioned in this specification, are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application or GenBank Registry Number were specifically and individually indicated to be incorporated herein by reference. Furthermore, any reference or designation of any reference in this application should not be construed as an admission that such reference is prior art to the invention.

Claims

1. A three-dimensional bioreactor, or 3D bioreactor, for large-scale expansion of immune cells, the 3D bioreactor comprising: At least one filled bed chamber; At least one porous scaffold surrounded by at least one filled bed chamber, wherein the at least one porous scaffold is coated with one or more extracellular matrix proteins, namely ECM; At least one container surrounding the at least one filled bed chamber; and The fluid culture medium having at least one population of immune cells suspended in a fluid culture medium, the fluid culture medium being contained in the at least one container; The fluid culture medium, wherein the fluid culture medium has at least one population of immune cells suspended in the fluid culture medium, flows through the filled bed chamber and the at least one porous scaffold coated with one or more ECMs; The at least one porous scaffold coated with the ECM is configured to create a low-shear fixation niche that mimics the natural growth environment of immune cells and allows for large-scale expansion of immune cell populations flowing through the coated porous scaffold.

2. The 3D bioreactor according to claim 1, wherein at least one porous scaffold coated with one or more ECMs is further coated or connected with at least one immune cell activator.

3. The 3D bioreactor according to claim 2, wherein the immune cell activator is one of antigen-presenting cells (APCs) loaded with or unloaded with antigens, or an antigen directly conjugated to at least one porous scaffold coated with one or more ECMs.

4. The 3D bioreactor of claim 1, wherein the expanded immune cells are further activated in the at least one packed bed chamber after the immune cell population is exposed to at least one porous scaffold coated with one or more ECMs coated or connected to at least one immune cell activator.

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

6. The 3D bioreactor of claim 1, wherein the at least one immune cell population is harvested or reactivated after the antigen-presenting cells (APCs) attached to at least one porous scaffold coated with one or more ECMs are exposed to antigens to generate additional activation signals for the immune cell population.

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

8. The 3D bioreactor according to claim 1, wherein the at least one porous scaffold coated with one or more ECMs is a single porous scaffold matrix extending within the interior space of the at least one packed bed chamber, or a plurality of microporous scaffolds or microporous scaffolds filling the at least one packed bed chamber.

9. The 3D bioreactor of claim 1, wherein the fluid culture medium further comprises one or more gene modifiers capable of genetically modifying the immune cell population suspended in the fluid culture medium.

10. A method for large-scale expansion of immune cells in a three-dimensional bioreactor, the method comprising the following steps: a. Insert at least one porous support into at least one filled bed chamber; b. Coating the at least one porous scaffold with one or more extracellular matrix proteins, i.e., ECM; c. Circulating the fluid culture medium containing at least one population of immune cells suspended in a fluid culture medium, the fluid culture medium being contained in at least one container surrounding the at least one filled bed chamber, the fluid culture medium containing at least one population of immune cells suspended in the fluid culture medium flowing through the at least one filled bed chamber and the at least one porous scaffold coated with one or more ECMs; and The at least one porous scaffold coated with the ECM is configured to create a low-shear fixation niche that mimics the natural growth environment of immune cells and allows for large-scale expansion of immune cell populations flowing through the at least one porous scaffold.

11. The method of claim 10, further comprising the steps of 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, and the step of exposing the immune cell population to the at least one activator after step d, to expand and activate the immune cell population in the at least one packed bed chamber.

12. The method of claim 11, further comprising the step of genetically modifying the immune cell population inside the 3D bioreactor by adding one or more gene modifiers incorporated into the fluid culture medium and capable of genetically modifying the immune cell population suspended in the fluid culture medium.

13. The method of claim 10, further comprising the steps of harvesting the immune cell population or a portion thereof and further expanding and reactivating the harvested immune cell population in at least one of the same 3D bioreactors or in different bioreactors.

14. The method of claim 13, further comprising the step of harvesting the immune cell population, followed by the steps of genetically modifying the harvested immune cell population outside the at least one 3D bioreactor, and re-inoculating the genetically modified harvested immune cell population into the same at least one 3D bioreactor or into different bioreactors.

15. The method of claim 11, further comprising the steps of harvesting the immune cell population or a portion thereof and further expanding and reactivating the harvested immune cell population in at least one of the same 3D bioreactors or in different bioreactors.

16. The method of claim 12, further comprising the steps of harvesting the immune cell population or a portion thereof and further expanding and reactivating the harvested immune cell population in at least one of the same 3D bioreactors or in different bioreactors.

17. A three-dimensional bioreactor, or 3D bioreactor, for the large-scale expansion and activation of immune cell populations, the 3D bioreactor comprising: At least one filled bed chamber; At least one porous antigen-presenting cell mimic scaffold, namely APC-MS, is surrounded by at least one filled bed chamber, wherein the at least one porous APC-MS is coated with one or more extracellular matrix proteins, namely ECM. At least one container surrounding the at least one filled bed chamber; and The fluid culture medium having at least one population of immune cells suspended in a fluid culture medium, the fluid culture medium being contained in the at least one container; The fluid culture medium having at least one population of immune cells suspended in a fluid culture medium flows through at least one porous APC-MS coated with one or more ECMs; and The at least one porous APC-MS creates a low-shear-force immobilized microenvironment that mimics the natural growth environment of the immune cell population and allows for large-scale expansion and / or activation of the immune cell population flowing through it.

18. The 3D bioreactor of claim 17, wherein the at least one porous APC-MS coated with 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 microporous APC-MS or microporous APC-MS filling the at least one packed bed chamber.

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