Compositions and methods for expanding lymphocytes

Abstract

Disclosed are media, supplements, and methods for expanding mammalian lymphocytes or their progenitors, such as B cells, T cells, or NK cells. The disclosed media, supplements, and methods may be used to expand mammalian lymphocytes or their progenitors plated as single cells or at clonal cell density. In some embodiments, the disclosed media, supplements, and methods may also differentiate / activate the lymphocytes during expansion. The disclosed media, supplements, and methods can be used in serum-free and feeder-free culture workflows.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 310,778, filed February 16, 2022, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to cell culture applications, more particularly to cell culture applications using hematopoietic cells, and even more particularly to cell culture applications with a population(s) of lymphocytes. [Background technology]

[0003] Mammalian blood is composed of various cell types including lymphocytes, platelets, red blood cells, and their direct and indirect precursors. Lymphocytes are sometimes called white blood cells, and they function in the host's immune system. Lymphocytes can be further subdivided into B cells, T cells, NK cells, macrophages, eosinophils, basophils, and neutrophils.

[0004] Lymphocytes, like other blood cells, originate from hematopoietic stem cells (HSCs) that are capable of self-renewal and differentiation into each blood cell lineage. T cells are the central component of adaptive immunity, NK cells play a key role in innate immunity, and B cells are the central component of humoral immunity that secrete antibodies upon antigen binding.

[0005] They are of intense research and therapeutic interest, given the involvement of lymphocytes in sensing antigens in the environment and in triggering immune responses upon stimulation. Specific B-, NK- or T-cell clones may be of particular interest for their ability to detect specific antigens or trigger responses, but isolating such cells from the background of cells present in a blood sample is very difficult. Engineering B-, NK- or T-cells to perform specific functions is a relatively new approach to solving the isolation problem.

[0006] Even if the challenge of obtaining individual specific lymphocyte clones is overcome, another problem arises: expanding such cells to meaningful numbers. Because lymphocytes plated as single cells or at clonal density do not readily expand to significant numbers in vitro or ex vivo, reagents and processes are needed to clonally expand single lymphocytes (e.g., B cells, NK cells, or T cells). Summary of the Invention

[0007] The present disclosure relates to nutritional supplements, media, and methods for expanding mammalian lymphocytes, such as B cells, NK cells, or T cells, or their progenitor cells.

[0008] In one aspect of the present disclosure, a cell culture medium supplement for expanding mammalian lymphocytes (e.g., B cells, NK cells, or T cells) or progenitor cells thereof is provided. The nutritional supplement of the present disclosure may include one or more of a ligand for CD40, a mixture of lipids, one or more cytokines, and a diluent. In one embodiment, the nutritional supplement of the present disclosure may include two or more of a ligand for CD40, a mixture of lipids, and one or more cytokines.

[0009] In one embodiment, the diluent is aqueous, hi one embodiment, the diluent is a buffer.

[0010] In one embodiment, the nutritional supplement of the present disclosure may further comprise a first type of extracellular matrix protein. In one embodiment, the nutritional supplement of the present disclosure may further comprise a second type of extracellular matrix protein. In one embodiment, the nutritional supplement of the present disclosure may further comprise a third type of extracellular matrix protein. In one embodiment, any one or all of the extracellular matrix protein(s) are recombinant. In one embodiment, any one or all of the extracellular matrix protein(s) are solubilized in a diluent.

[0011] In one embodiment, the extracellular matrix protein is selected from the group consisting of collagen, ECM1, E-cadherin, laminin, osteopontin, fibronectin, vitronectin, or SPARC. In embodiments including more than one type of extracellular matrix protein, each type is different.

[0012] In one embodiment, the concentration of the first type of extracellular matrix protein, the second type of extracellular matrix protein (if present), or the third type of extracellular matrix protein (if present) is in the range of about 1 μg / mL to 1 mg / mL.

[0013] In one embodiment, the ligand of CD40 is a ligand of human or mouse CD40. In one embodiment, the ligand of CD40 comprises one or more proteins.

[0014] In one embodiment, the one or more cytokines are selected from IL-2, IL-4, IL-6, IL-10, and IL-21. In one embodiment, each of the foregoing cytokines is included in the nutritional supplement. In one embodiment, IL-2 and IL-10, and optionally IL-21, are included in the nutritional supplement.

[0015] In one embodiment, the lipid mixture is chemically defined. In one embodiment, the lipid mixture is free of animal-derived components. In one embodiment, the lipid mixture is stabilized.

[0016] In one embodiment, the nutritional supplement is mixed with the basal medium.

[0017] In one embodiment, the nutritional supplement is animal-derived free and / or serum-free.

[0018] In one embodiment, lymphocytes (eg, T cells, NK cells or B cells) or their precursor cells are plated as single cells or at clonal density.

[0019] In one embodiment, the dietary supplement is concentrated, hi one embodiment, the dietary supplement is concentrated 10 times or more.

[0020] In another aspect of the disclosure, a medium for expanding mammalian lymphocytes (e.g., T cells, NK cells, or B cells) or progenitors thereof is provided. The medium of the disclosure may include a basal medium and one or more of a ligand for CD40, a mixture of lipids, and one or more cytokines. Thus, in another aspect, a basal medium and one or more nutritional supplements may be included in the kit. In one embodiment, the medium is not in contact with surface-bound or immobilized extracellular matrix proteins.

[0021] In one embodiment, the medium of the present disclosure may further comprise two or more of a ligand for CD40, a mixture of lipids, and one or more cytokines.

[0022] In one embodiment, the medium of the present disclosure may further comprise a first type of extracellular matrix protein. In one embodiment, the medium of the present disclosure may further comprise a second type of extracellular matrix protein. In one embodiment, the medium of the present disclosure may further comprise a third type of extracellular matrix protein. In one embodiment, the extracellular matrix protein(s) is recombinant.

[0023] In one embodiment, the extracellular matrix protein is selected from the group consisting of collagen, ECM1, E-cadherin, laminin, osteopontin, fibronectin, vitronectin, or SPARC. In embodiments including more than one type of extracellular matrix protein, each type is different.

[0024] In one embodiment, the concentration of the first type of extracellular matrix protein, the second type of extracellular matrix protein (if present), or the third type of extracellular matrix protein (if present) is in the range of about 1 μg / mL to 1 mg / mL.

[0025] In one embodiment, the medium of the present disclosure may further comprise one or more cytokines. In one embodiment, the one or more cytokines are selected from IL-2, IL-4, IL-6, IL-10, and IL-21. In one embodiment, each of the foregoing cytokines is included in a nutritional supplement. In one embodiment, IL-2 and IL-10, and optionally IL-21, are included in a nutritional supplement.

[0026] In one embodiment, the ligand of CD40 is a ligand of human or mouse CD40. In one embodiment, the ligand of CD40 comprises one or more proteins.

[0027] In one embodiment, the lipid mixture is chemically defined. In one embodiment, the lipid mixture is free of animal-derived components. In one embodiment, the lipid mixture is stabilized.

[0028] In one embodiment, lymphocytes (eg, T cells, NK cells or B cells) or their precursor cells are plated as single cells or at clonal density.

[0029] In one embodiment, the medium supports feeder-free growth of lymphocytes and their progenitor cells.

[0030] In one embodiment, the medium supports animal-derived component-free growth of lymphocytes and their progenitor cells, hi one embodiment, the medium is serum-free.

[0031] In another aspect of the present disclosure, a method for expanding mammalian lymphocytes (e.g., T cells, NK cells or B cells) or their progenitors is provided.In one embodiment, mammalian lymphocytes or their progenitors can be plated as single cells or at clonal density.In one embodiment, mammalian lymphocytes or their progenitors are not plated as single cells or at clonal density.

[0032] The method may include seeding lymphocytes or progenitor cells in a culture environment that includes one or more of a ligand for CD40, a mixture of lipids, and one or more cytokines, and incubating / culturing the seeded cells or progenitor cells in the culture environment for one or more days. In one embodiment, incubating / culturing the cells may include expanding the cells. In one embodiment, the cells may also be differentiated and / or activated during the culturing / incubation step.

[0033] In one embodiment, the disclosed method may further include a first type of extracellular matrix protein in the culture environment. In one embodiment, the disclosed method may further include a second type of extracellular matrix protein in the culture environment. In one embodiment, the disclosed method may further include a third type of extracellular matrix protein in the culture environment.

[0034] In one embodiment, the first type of extracellular matrix protein and / or the second type of extracellular matrix protein (if present) and / or the third type of extracellular matrix protein (if present) are included in the culture medium.

[0035] In one embodiment, the extracellular matrix protein is selected from the group consisting of collagen, ECM1, E-cadherin, laminin, osteopontin, fibronectin, vitronectin, or SPARC. In embodiments including more than one type of extracellular matrix protein, each type is different.

[0036] In one embodiment, the concentration of the first type of extracellular matrix protein, the second type of extracellular matrix protein (if present), or the third type of extracellular matrix protein (if present) is in the range of about 1 μg / mL to 1 mg / mL.

[0037] In one embodiment, the medium of the present disclosure may further comprise one or more cytokines. In one embodiment, the one or more cytokines are selected from IL-2, IL-4, IL-6, IL-10, and IL-21. In one embodiment, each of the foregoing cytokines is included in a nutritional supplement. In one embodiment, IL-2 and IL-10, and optionally IL-21, are included in a nutritional supplement.

[0038] In one embodiment, the ligand of CD40 is a ligand of human or mouse CD40. In one embodiment, the ligand of CD40 comprises one or more proteins.

[0039] In one embodiment, the lipid mixture is chemically defined. In one embodiment, the lipid mixture is free of animal-derived components. In one embodiment, the lipid mixture is stabilized.

[0040] In one embodiment, the seeding and incubation steps are performed under feeder cell-free conditions. In one embodiment, the seeding and incubation steps are performed under serum-free conditions. In one embodiment, the seeding and incubation steps are performed under animal-derived component-free conditions. In one embodiment, the seeding and incubation steps are performed under each of the aforementioned conditions.

[0041] In one embodiment, the lymphocytes or progenitor cells thereof are human or murine.

[0042] In one embodiment, the lymphocyte is a human or mouse B cell, and the B cell is a memory B cell or a plasmablast.

[0043] In one embodiment, the disclosed method may further comprise differentiating lymphocytes or progenitor cells thereof in the culture environment. In one embodiment, the disclosed method may further comprise activating and / or differentiating lymphocytes or progenitor cells thereof in the culture environment.

[0044] In one embodiment, the incubation step is for at least 3 days.

[0045] Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that this detailed description and the specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, and that various changes and modifications which will become apparent to those skilled in the art from this detailed description are within the spirit and scope of the present invention.

[0046] For a better understanding of the various embodiments described herein, and to more clearly show how they may be effectively put into practice, reference is made to the accompanying drawings, which illustrate at least one exemplary embodiment, and which will now be described, and which are not intended to limit the scope of the teachings described herein. [Brief description of the drawings]

[0047] [Figure 1] Bar graphs of marker expression in cells of leukocyte samples either before (light grey) or after enrichment (dark grey) of B lineage cells are shown. [Diagram 2] Representative images of B lineage cell proliferation over time when plated as single cells are shown. Single B cells from a donor were plated under control conditions without exogenously added extracellular matrix proteins diluted in media, and under various media conditions containing different combinations of exogenously added extracellular matrix proteins. Conditions 1, 2, and 3 contained different pairwise combinations of the three extracellular matrix proteins. Condition 4 contained all three extracellular matrix proteins. Single cells on day 0 are circled. [Diagram 3] Representative images of B lineage cell proliferation when plated as single cells are shown. Single B cells from different donors were grown in conditions containing two of the exogenously added extracellular matrix proteins from Figure 2 diluted individually in culture medium (conditions 1 and 2), as well as a combination (condition 3). Scale bar represents 200 μm. [Figure 4] Representative images of B lineage cell proliferation when plated as single cells are shown. Single B cells from different donors from those in Figures 2 and 3 were expanded in individual exogenously added extracellular matrix proteins diluted in culture medium (condition 1, condition 2, and condition 3), as well as in conditions containing two pairwise combinations of exogenously added extracellular matrix proteins (condition 4 and condition 5). Scale bar represents 200 μm. [Diagram 5] Representative images of B lineage cell proliferation when plated as single cells. Single B cells derived from the same donor in Figure 4 were expanded in the presence of individual exogenously added extracellular matrix proteins diluted in culture medium and in the presence or absence of ligand for CD40. Circled areas highlight selected clusters of proliferating cells. Scale bar represents 200 μm. [Figure 6] Representative images of B lineage cell proliferation when plated as single cells are shown. Single B cells derived from the same donor in Figure 2 were expanded in the presence of individual exogenously added extracellular matrix proteins (condition 1 and condition 2) diluted in culture medium (these are different from those tested in Figures 2-5). Scale bar represents 200 µm. [Figure 7] Representative images of B lineage cell proliferation when plated as single cells are shown. Single B cells derived from the same donor in Figure 2 were expanded in the presence of a single exogenously added extracellular matrix protein diluted in culture medium, but in the presence or absence of additional different lipid complexes, as indicated. Scale bar represents 200 μm. [Figure 8] 1 shows line graphs of cell surface marker expression in B lineage cells expanded from single B cells in the presence of two different individual exogenously added extracellular matrix proteins (conditions 1 and 2) and in combination (condition 3). Bars represent the average of duplicate wells. [Figure 9]Representative images of B lineage cell proliferation when plated as single cells are shown. Single B cells were expanded in culture medium containing a single exogenously added extracellular matrix protein and different combinations of cytokines: IL-2 and IL-10 (condition 1) and IL-2, IL-4, IL-6, IL-10 and IL-21 (condition 2). Control conditions included unsupplemented basal medium (control 1), basal medium supplemented with cytokines but not exogenously added extracellular matrix proteins (control 2), and basal medium supplemented with extracellular matrix proteins but not cytokines (control 3). Scale bar represents 200 μm. [Figure 10] Representative images of B lineage cell proliferation when plated as single cells are shown. Single B cells were expanded in the presence of a single exogenously added extracellular matrix protein diluted in different commercial basal media as indicated. White arrows indicate clusters of cells. Scale bar represents 200 μm. [Figure 11] Representative images of human memory B lineage cell expansion when plated as single cells. Single memory B cells derived from a new donor were expanded in conditions containing a combination of two (condition 1) or a combination of three (condition 2) extracellular matrix proteins diluted in culture medium. Scale bar represents 200 μm. [Figure 12] 1 shows the results of single human memory B cell expansion according to the present disclosure. Survival fold expansion after 12 days in culture is shown as the mean ± standard error of the mean (SEM) of 9 donors (A). IgM and IgG production by expanded cells as determined by ELISA is shown as the mean ± SEM of 7 donors (B). Flow cytometry analysis of cell surface marker expression in expanded cells at day 12 is shown as the mean ± SEM of 9 donors (C). [Figure 13]Figure 1 shows the results of single human plasmablast expansion according to the present disclosure. The viable fold expansion after 12 days in culture is shown as the mean ± standard error of the mean (SEM) of five donors (A). The production of IgM and IgG by expanded cells as determined by ELISA is shown as the mean ± SEM of two donors (B). Flow cytometry analysis of cell surface marker expression in expanded cells at day 12 is shown as the mean ± SEM of four donors (C). [Figure 14] Representative images of mouse pan-B lineage cell proliferation when plated as single cells are shown. Single pan-B cells from C57BL / 6 mice were expanded in the presence of two different combinations of two exogenously added extracellular matrix proteins (condition 1 and condition 2) and in the presence of the indicated cytokine cocktails. Scale bar represents 200 μm. [Figure 15] Figure 1 shows the expansion results of single mouse memory B cells according to the present disclosure. Survival fold expansion after 12 days in culture is shown as the mean ± standard error of the mean (SEM) of five donors (A). Production of IgG by expanded cells, as determined by ELISA, is shown as the mean ± SEM of two donors (B). Flow cytometry analysis of cell surface marker expression in expanded cells at day 12 is shown as the mean ± SEM of two to four donors (C). [Figure 16] Figure 1 shows the expansion results of single mouse plasmablasts according to the present disclosure. Viability fold expansion after 12 days in culture is shown as the mean ± standard error of the mean (SEM) of three donors (A). Production of IgG by expanded cells, as determined by ELISA, is shown as the mean ± SEM of two donors (B). Flow cytometry analysis of cell surface marker expression in expanded cells at day 12 is shown as the mean ± SEM of two to three donors (C). [Figure 17]Proliferation results of T lineage cells plated as single cells. Viability fold expansion of single murine CD3+ T cells after a total of 12 days of culture is shown as the mean ± standard error of the mean (SEM) of two donors (A). Flow cytometry analysis of cell surface marker expression in expanded cells at day 12 in (A) is shown as the mean ± SEM of two to three donors (B). Images of expanded human CD3+ T cells plated as single cells in the presence of a single exogenously added extracellular matrix protein diluted in basal medium. Scale bar represents 200 µm (C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] The present disclosure relates to media compositions (and / or nutritional supplements added to the media) and methods for culturing lymphocytes, such as T cells, NK cells, or B cells. More specifically, the disclosure relates to expanding T cells, NK cells, or B cells, seeded as single cells or at clonal cell density, into the media and nutritional supplements disclosed herein and by practicing the methods disclosed herein.

[0049] As used in this disclosure, the term "lymphocyte" refers to a cell of the lymphocyte lineage of the hematopoietic system of a mammal, such as a T cell, a B cell, and / or a NK cell. Mammalian lymphocytes may be human, mouse, rat, or other lymphocytes. Lymphocytes are hematopoietic stem cells (e.g., CD34 + The lymphocytes may be derived from stem cells such as pluripotent stem cells, or any other stem cells that can be obtained from umbilical cord blood, peripheral blood, or bone marrow. In one embodiment, the lymphocytes may be derived from pluripotent stem cells.

[0050] As used in this disclosure, "B cell(s)" refers to a mammalian B lymphocyte or population of B lymphocytes. B cells may be included in a sample, which may include one or more B cell populations, subpopulations, or subsets, including, but not limited to, naive B cells, memory B cells, activated B cells, B2 cells, B1 cells, germinal center B cells, marginal zone B cells, regulatory B cells, follicular B cells, isolated pan B cells, plasmablasts, plasma cells, and / or other B cell lineages. B cells may be distinguished from non-B cells, such as in a sample, based on biomarkers, such as B cell surface markers (e.g., antigens expressed on the B cell surface and that can be targeted with agents that bind to them). Exemplary B cell surface markers include CD10, CD19, CD20 (MS4A1), CD21, CD22, CD23, CD24, CD25, CD27, CD38, CD40, CD53, CD72, CD73, CD79a, CD79b, CD80, CD86, CD138, and TACI. Exemplary mouse B cell surface markers include CD19, CD20, CD22, CD27, CD28, CD38, CD44, CD80, CD73, CD86, CD93, CD98, CD138, CD45R, B220, and CD273.

[0051] As used in this disclosure, "T cell(s)" refers to a mammalian T lymphocyte or population of T lymphocytes. T cells can be contained in a sample, including, but not limited to, pan-T cells, CD4 + T cells, CD8 +The T cell population may include one or more T cell populations, subpopulations, or subsets, including T cells, memory T cells, regulatory T cells, helper T cells, effector T cells, cytotoxic T cells, and / or naive T cells, CAR-T cells, activated T cells, Th1 cells, Th2 cells, or other T cell lineages. T cells may be distinguished from non-T cells, such as in a sample, based on biomarkers, such as T cell surface markers (e.g., antigens that are expressed on the T cell surface and can be targeted with agents that bind to them). T cells are typically characterized by the absence of NK-specific, B-specific, and erythroblast-specific markers, the expression of CD3, and TCRαβ (or TCRγδ), and CD4 or CD8, and their effector functions. Some subpopulations of T cells may express characteristics of both T cells and NK cells, but may or may not express TCRαβ or TCRγδ, and may or may not express CD4, CD8, CD56, CD16, and NK1.1. T cells may be further characterized by expression of CD8α, CD8β, CD45RA and CD27.

[0052] As used in this disclosure, "NK cell(s)" refers to a mammalian NK lymphocyte or population of NK lymphocytes. NK cells may be included in a sample, which may include one or more NK cell populations, subpopulations, or subsets, including, but not limited to, pan-NK cells, tolerant NK cells, cytotoxic NK cells, regulatory NK cells, CAR-NK cells, or other NK cell lineages. NK cells may be distinguished from non-NK cells, such as in a sample, based on biomarkers, such as NK cell surface markers (e.g., antigens expressed on the NK cell surface and that can be targeted with agents that bind to them). NK cells are typically characterized by the absence of T-cell and B-cell specific markers, expression of CD56 with or without CD16 (low affinity Fc gamma receptor 3A expressed on a subpopulation of NK cells), and their effector functions. NK cells may be further characterized by expression of activating and inhibitory receptors called killer immunoglobulin-like receptors (KIRs).

[0053] As used in this disclosure, the term "extracellular matrix" refers to one or more molecules that provide structural and biochemical support to cells. Extracellular matrix compounds are also widely known to promote cell adhesion, cell-cell communication, and differentiation within a given tissue or aggregate of cells in culture medium. Both natural and synthetic extracellular matrices are contemplated within the scope of this disclosure. Many different extracellular matrix proteins are known, including, but not limited to, laminin, collagen, fibronectin, vitronectin, ECM1, osteopontin, fibronectin, and SPARC. An extracellular matrix may include one or more extracellular matrix proteins. If an extracellular matrix includes only one extracellular matrix protein, it can be said to include one type of extracellular matrix protein. If an extracellular matrix includes two different extracellular matrix proteins, it can be said to include two types of extracellular matrix proteins. If an extracellular matrix includes three different extracellular matrix proteins, it can be said to include three types of extracellular matrix proteins, and so on. The components of the extracellular matrix may include one or more of the following proteins: fibronectin, laminin, vitronectin, tenisin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epirinin, kalinin, synthetic polymer-based hydrogels (e.g., polyethylene glycol, polyvinyl, or any derivative or analog thereof), and vegetable hydrogels (e.g., cellulose, hemicellulose, lignin, starch, and pectin, or any derivative or analog thereof). The extracellular matrix proteins of the present disclosure may be included in a nutritional supplement added to the medium of the present disclosure or may be included in the medium of the present disclosure. In both such embodiments, the extracellular matrix protein(s) may be soluble or solubilized.

[0054] As used in this disclosure, the term "mixture of lipids" refers to a preparation of lipids and / or lipid-like substances. The mixture of lipids may be purchased from a commercial distributor or may be prepared immediately prior to use. Examples of lipids that may be included in the mixture of lipids include propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, henicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melissic acid, henatriacontylic acid, racemic acid, psyllic acid, geminate acid, glyceric ... and / or from the group of unsaturated fatty acids including a-linolenic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, docosatetraenoic acid, palmitoleic acid, vaccenic acid, paulic acid, oleic acid, elaidic acid, gondoic acid, erucic acid, nervonic acid, or mead acid. In one embodiment, the mixture of lipids may be included in a concentrated preparation such as a nutritional supplement added to the culture medium. In one embodiment, the mixture of lipids may already be included in the basal or complete medium of the present disclosure.

[0055] As used in this disclosure, the term "cytokine(s)" refers to soluble proteins produced and released from cells. The function of a cytokine includes stimulating or suppressing the cell of origin or other cells with which it is in direct or indirect contact. As an example, a cytokine may mediate the generation or control of an immune response. Common cytokines include interleukins IL-1 through IL-15; tumor necrosis factors alpha and beta; interferons alpha, beta and gamma; TGF-beta; colony stimulating factors (CSFs); and granulocyte monocyte colony stimulating factor (GM-CSF). One or more cytokines may be included in the media or supplements of the present disclosure or may be used in methods to expand lymphocytes, such as T cells, NK cells, or B cells.

[0056] As used in this disclosure, the term "clonal cell density" or "clonal density" refers to the density at which a cell(s) can form individual colonies or clusters. In certain embodiments, cells are seeded at a density of 1 cell / well. In one embodiment, cells are seeded at a density of at least 1 cell / well. In one embodiment, cells may be seeded at a density of 1000 cells / well in a culture dish, e.g., a 10 cm culture dish. In one embodiment, cells are seeded at a sufficiently low density that when plated in a culture dish / well, effective isolation of a single, unbombarded cell is obtained.

[0057] Media and Nutrient Supplements In another aspect of the present disclosure, a medium for expanding mammalian lymphocytes, such as T cells, NK cells or B cells, is provided. In one aspect of the present disclosure, a cell culture medium supplement is provided that is added to the medium for expanding mammalian lymphocytes, such as B cells, NK cells or T cells, or their progenitors. Both the medium and the supplement may contain factors and other components important for the expansion of lymphocytes, such as T cells, B cells or NK cells, or their progenitors, whether plated as single cells or at clonal cell density. In some embodiments, the medium and the supplement may also contain additional factors and other components that improve the efficiency of the expansion and / or differentiation and / or activation of lymphocytes or progenitors, whether plated as single cells or at clonal cell density.

[0058] In one embodiment, the mammalian B cell is a human B cell. In one embodiment, the human B cell is a memory B cell. In one embodiment, the human B cell is a naive B cell. In one embodiment, the human B cell is a pan-B cell. In one embodiment, the mammalian B cell is a plasmablast. In one embodiment, the mammalian B cell is a plasma cell. In one embodiment, the mammalian B cell is a mouse B cell. However, one of skill in the art will readily appreciate that the cell culture media supplements of the present disclosure may also be applied to other cell types, such as T cells or NK cells.

[0059] The culture medium of the present disclosure includes a basal medium. The basal medium can be any medium that can support the proliferation of mammalian lymphocytes or their precursor cells, such as T cells or B cells, when appropriately supplemented. In one embodiment, the basal medium can be any medium that can support the proliferation of human mammalian lymphocytes or their precursor cells, such as T cells or B cells, when appropriately supplemented. In one embodiment, the basal medium can be any medium that can support the proliferation of rodent (e.g., mouse) mammalian lymphocytes or their precursor cells, such as T cells or B cells, when appropriately supplemented. In one embodiment, the basal medium can be any medium that can support the proliferation of human or mouse memory B cells or plasmablast cells.

[0060] Numerous basal media are known in the art, many of which are commercially available. Examples of basal media may include RPMI, DMEM / F-12, IMDM, StemSpan™, and ImmunoCult™ brand media. Basal media typically include one or more of carbohydrates, amino acids, trace elements, lipids, buffers, salts, proteins including albumin, and the like. In some embodiments, basal media may be free of one or more of the aforementioned component types, which may be correspondingly supplemented when formulating a complete medium.

[0061] The basal medium used to formulate the mammalian lymphocyte growth medium of the present disclosure may also be supplemented with additional components to create a complete medium. In one embodiment, the mammalian lymphocyte growth medium of the present disclosure may be provided as a complete medium (i.e., no additional nutritional supplements are required). In one embodiment, the basal medium may be provided (whether in a kit or not) with one or more nutritional supplements that are added to the basal medium to formulate a complete medium prior to use.

[0062] To formulate a complete mammalian lymphocyte growth medium, it may be beneficial and / or necessary to include (or further supplement) one or more of growth factor(s), extracellular matrix(s) such as cytokine(s), lipid(s), hormone(s), salt(s), vitamin(s), other proteins including albumin(s), small molecule(s), and the like.

[0063] In one embodiment, a culture medium (e.g., a mammalian lymphocyte growth medium) of the present disclosure comprises a basal medium and one or more of a mixture of lipids, a ligand for CD40, and one or more cytokines. In one embodiment, a culture medium (e.g., a mammalian lymphocyte growth medium) of the present disclosure comprises a basal medium and two or more of a mixture of lipids, a ligand for CD40, and one or more cytokines. In one embodiment, a culture medium (e.g., a mammalian lymphocyte growth medium) of the present disclosure comprises a basal medium and each of a mixture of lipids, a ligand for CD40, and one or more cytokines.

[0064] In one embodiment, the mixture of lipids may include a single type of lipid. In one embodiment, the mixture of lipids may include multiple types of lipids. In one embodiment, the mixture of lipids may be included in a basal medium. In one embodiment, the mixture of lipids may be included in a nutritional supplement that is added to the basal medium.

[0065] In one embodiment, the mixture of lipids is chemically defined. In one embodiment, the mixture of lipids may be free of animal-derived components.

[0066] The culture medium of the present disclosure may include one or more cytokines and / or one or more growth factors. In one embodiment, the type(s) of one or more cytokines and / or one or more growth factors included in the cell culture medium of the present disclosure are selected depending on the particular type of cells being cultured, e.g., lymphocytes (e.g., T cells, NK cells, or B cells).

[0067] In one embodiment, the one or more cytokines or growth factors may be any one of IL-2, IL-4, IL-6, IL-10, and IL-21, or a combination thereof. In one embodiment, the mammalian lymphocyte growth medium is supplemented with each of IL-2, IL-4, IL-6, IL-10, and IL-21. In one embodiment, the mammalian lymphocyte growth medium is supplemented with one or more of IL-2, IL-4, IL-6, IL-10, and IL-21. In one embodiment, the mammalian lymphocyte growth medium is supplemented with at least IL-2 and IL-10, and optionally, IL-21.

[0068] In certain embodiments, the growth medium for mammalian B cells or T cells comprises each of IL-2, IL-4, IL-6, IL-10 and IL-21. In other specific embodiments, the growth medium for mammalian B cells or T cells comprises at least IL-2 and IL-10, and optionally, IL-21.

[0069] The one or more cytokines included in the culture medium of the present disclosure may each be present at a concentration of about 10 ng / mL to 200 μg / mL, or about 0.1 to 100 μg / mL, or about 1 to 50 μg / mL, or about 5 to 25 μg / mL. In embodiments in which small molecule analogs of one or more of the aforementioned cytokines are included in the mammalian lymphocyte growth medium, they are typically used at low concentrations.

[0070] The culture medium of the present disclosure may include a ligand for CD40. In one embodiment, the ligand for CD40 binds to the CD40L receptor and promotes cell activation. In one embodiment, the ligand for CD40 is a ligand for human CD40. In one embodiment, the ligand for CD40 includes one or more proteins. In one embodiment, the ligand for CD40 is included in a protein complex. In one embodiment, the concentration of the ligand for CD40L in the culture medium ranges from about 0.1 ng / mL to 500 ng / mL, about 0.5 ng / mL to 100 ng / mL, or about 1 ng / mL to 50 ng / mL.

[0071] The culture medium of the present disclosure may include a first type of extracellular matrix protein. In one embodiment, the culture medium of the present disclosure includes a first type and a second type of extracellular matrix protein. In one embodiment, the culture medium of the present disclosure includes a first type of extracellular matrix protein, a second type of extracellular matrix protein, and a third type of extracellular matrix protein. In some embodiments, the culture medium of the present disclosure includes more than three types of extracellular matrix proteins. In one embodiment, the first type of extracellular matrix protein may be selected from collagen, ECM1, laminin, osteopontin, vitronectin, or SPARC. In embodiments including a second, third, or additional type of extracellular matrix protein, they may be selected from, but are not limited to, collagen, ECM1, E-cadherin, laminin (e.g., laminin-511, fragments of laminin-511 (e.g., laminin 511-E8), laminin-521 (also known as laminin 11), osteopontin, fibronectin, vitronectin, SPARC, or combinations thereof. In preferred embodiments, the extracellular matrix protein(s) included in the culture medium of the present disclosure are one or more of ECM1, osteopontin, vitronectin, collagen, and laminin, and more preferably one or more of ECM1, osteopontin, and vitronectin.

[0072] In one embodiment, the concentration of the extracellular matrix protein (whether first, second, third, or further type) is in the range of about 0.1 μg / mL to 1 mg / mL. In one embodiment, the concentration of the extracellular matrix protein (whether first, second, third, or further type) is in the range of about 1 μg / mL to 100 μg / mL, about 3 μg / mL to 50 μg / mL, about 5 μg / mL to 30 μg / mL, or about 10 μg / mL to 20 μg / mL.

[0073] In one embodiment, the first type of extracellular matrix protein, the second type of extracellular matrix protein, or the third type of extracellular matrix protein are coated or immobilized on the surface of a particle / bead or a culture receptacle / vessel containing lymphocytes (e.g., B or T cell(s)). Thus, in such an embodiment, cells cultured in the vessel are cultured in the presence of a medium of the present disclosure and in the presence of the first type of extracellular matrix protein (and any additional types of extracellular matrix protein) coated or bound to the surface of the particle / bead or cell culture receptacle / vessel.

[0074] In one embodiment, each of the extracellular matrix proteins included in the culture medium (or on the particle or container) is recombinant. In one embodiment, some, but not all, of the extracellular matrix proteins are recombinant. In one embodiment, one or more of the extracellular matrix proteins are recombinant human extracellular matrix proteins. In one embodiment, one or more of the extracellular matrix proteins are recombinant mouse extracellular matrix proteins.

[0075] In one embodiment, one or more of the extracellular matrix proteins (such as may be included in the culture medium of the present disclosure) may be of natural origin and may be purified from human, animal, or plant tissue. In one embodiment, the extracellular matrix protein(s) may be genetically engineered and / or recombinant proteins, or synthetic in nature. In one embodiment, the extracellular matrix protein(s) may be whole proteins or fragments thereof, e.g., peptide fragments.

[0076] In one embodiment, the culture medium of the present disclosure expands mammalian lymphocytes or progenitor cells thereof plated as single cells. Expanding cells plated as single cells can be important when cloning a particular cell, such as a genome-edited cell, is intended.

[0077] In one embodiment, the culture medium of the present disclosure expands mammalian lymphocytes or their progenitors plated at clonal density. Cells may be plated at clonal density in microplates, and in applicable embodiments, it is important to select the cell density so that the cells are well spaced apart. Cells plated at clonal density may be less likely to come into contact with each other. Cells plated at clonal cell density may also not confer paracrine effects on other cells in the vessel. For example, the clonal density may be less than 1 cell / cm. 2 , 10 cells / cm 2 , 100 cells / cm 2 , or 1000 cells / cm 2 This may correspond to a cell density of 100 μg / ml.

[0078] In one embodiment, the culture medium of the present disclosure expands mammalian lymphocytes or their progenitor cells that are not plated at clonal density.

[0079] In one embodiment, lymphocytes or their progenitors expanded using the aforementioned nutritional supplements and / or media formulations may be used in high compliance applications such as cell therapy. In one embodiment, the media of the present disclosure are chemically defined. In one embodiment, the media of the present disclosure may be serum-free and / or animal-derived component-free. Such media may, however, include albumin, such as recombinant albumin. Thus, the media of the present disclosure may support serum- and / or animal-derived component-free growth of lymphocytes or their progenitors.

[0080] In one embodiment, lymphocytes or their progenitors cultured in the medium of the present disclosure are not in contact with feeder cells or undefined cell supports (e.g., matrices) such as Matrigel™. Thus, the medium of the present disclosure can support feeder-free growth of lymphocytes or their progenitors.

[0081] Overall, the media of the present disclosure, whether provided as a complete medium or as a basal medium supplemented as described herein, support the proliferation of mammalian lymphocytes. In embodiments where the lymphocytes are B cells, they may be pan-B cells, memory B cells, naive B cells, plasmablasts, plasma cells, or other B cell lineages. In embodiments where the lymphocytes are T cells, they may be pan-T cells, CD4 + T cells, CD8 + They may be T cells, memory T cells, regulatory T cells, helper T cells, effector T cells, cytotoxic T cells, and / or naive T cells, CAR-T cells, or other T cell lineages. In embodiments where the lymphocytes are NK cells, they may be pan-NK cells, cytotoxic NK cells, regulatory NK cells, tolerant NK cells, CAR-NK cells.

[0082] In one embodiment, the mammalian lymphocyte expansion medium of the present disclosure supports 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, or 60 or more population doublings.

[0083] In one embodiment, mammalian lymphocytes or progenitors thereof expanded using the medium of the present disclosure express markers characteristic of mammalian B, NK or T cell lineages. In one embodiment, more than 30% of the expanded cells are positive for markers characteristic of mammalian B, NK or T cell lineages. In one embodiment, more than 50% of the expanded cells are positive for markers characteristic of mammalian B, NK or T cell lineages. In one embodiment, more than 60% of the expanded cells are positive for markers characteristic of mammalian B, NK or T cell lineages. In one embodiment, more than 70% of the expanded cells are positive for markers characteristic of mammalian B, NK or T cell lineages.

[0084] In one embodiment, the medium of the present disclosure may further support differentiation of lymphocytes or their precursor cells in a culture environment. In one embodiment, the medium supports differentiation / maturation of 10% or more of B cells, NK cells or T cells, or their precursor cells. In one embodiment, the medium supports differentiation / maturation of 20% or more of B cells, NK cells or T cells, or their precursor cells. In one embodiment, the medium supports differentiation / maturation of 30% or more of B cells, NK cells or T cells, or their precursor cells. In one embodiment, the medium supports differentiation / maturation of 40% or more of B cells, NK cells or T cells, or their precursor cells. In one embodiment, the medium supports differentiation / maturation of 50% or more of B cells, NK cells or T cells, or their precursor cells. In one embodiment, the medium supports differentiation / maturation of 60% or more of B cells, NK cells or T cells, or their precursor cells.

[0085] In one embodiment, the medium of the present disclosure may further support activation of lymphocytes or their progenitor cells in a culture environment.

[0086] In another aspect of the disclosure, a cell culture medium supplement for expanding mammalian lymphocytes or progenitor cells thereof is provided. In one embodiment, the mammalian lymphocytes are plated as single cells or at a clonal cell density. In one embodiment, the mammalian lymphocytes are not plated as single cells or at a clonal cell density.

[0087] In one embodiment, the cell culture media supplement may include one or more of a CD40 ligand, a lipid mixture, and one or more cytokines. In one embodiment, the cell culture media supplement may include a diluent and one or more of a CD40 ligand, a lipid mixture, and one or more cytokines. In one embodiment, the cell culture media supplement may include two or more of a CD40 ligand, a lipid mixture, and one or more cytokines. In one embodiment, the cell culture media supplement may include a diluent and two or more of a CD40 ligand, a lipid mixture, and one or more cytokines. In one embodiment, the cell culture media supplement may include each of a CD40 ligand, a lipid mixture, and one or more cytokines. In one embodiment, the cell culture media supplement may include a diluent and each of a CD40 ligand, a lipid mixture, and one or more cytokines.

[0088] In one embodiment, the diluent is physiological, e.g., with respect to pH, salt concentration, osmolality, osmolality, etc. In one embodiment, the diluent is inorganic. As a non-limiting example, the diluent may be aqueous (e.g., water). In one embodiment, the diluent may be a buffer. In one embodiment, the diluent solubilizes one or more of the components therein, e.g., a ligand for CD40, a mixture of lipids, and one or more cytokines. In one embodiment, the diluent solubilizes an extracellular matrix protein(s) that may be included in the nutritional supplement.

[0089] In one embodiment, the nutritional supplement of the present disclosure comprises a mixture of lipids. The above description of the mixture of lipids and its concentration in the medium can be applied to the nutritional supplement of the present disclosure. However, in the embodiment where the nutritional supplement is concentrated (e.g., more than 1x), the concentration range is adjusted appropriately.

[0090] In one embodiment, the nutritional supplement of the present disclosure includes a ligand for CD40. The above description regarding CD40 ligand and its concentration in the medium may be applied to the nutritional supplement of the present disclosure. However, in embodiments in which the nutritional supplement is concentrated (e.g., greater than 1x), the concentration range is adjusted appropriately.

[0091] In one embodiment, the nutritional supplement of the present disclosure comprises one or more cytokines and / or one or more growth factors. The above description of cytokines and their concentrations in the medium can be applied to the nutritional supplement of the present disclosure. However, in embodiments where the nutritional supplement is concentrated (e.g., more than 1x), the concentration range is adjusted appropriately.

[0092] In one embodiment, the nutritional supplement may include at least a first type of extracellular matrix protein and one or more of a ligand for CD40, a mixture of lipids, and one or more cytokines. In one embodiment, the nutritional supplement may include at least a first type of extracellular matrix protein and two or more of a ligand for CD40, a mixture of lipids, and one or more cytokines. In one embodiment, the nutritional supplement may include at least a first type of extracellular matrix protein and all of a ligand for CD40, a mixture of lipids, and one or more cytokines.

[0093] In one embodiment, the nutritional supplement comprises a first type and a second type of extracellular matrix protein. In one embodiment, the nutritional supplement comprises a first type of extracellular matrix protein, a second type of extracellular matrix protein, and a third type of extracellular matrix protein. In some embodiments, the nutritional supplement of the present disclosure comprises more than three types of extracellular matrix proteins. In one embodiment, at least one extracellular matrix protein is contained in the nutritional supplement used to formulate the complete growth medium of the present disclosure.

[0094] In the nutritional supplement embodiments of the present disclosure, the above description of one, two or three or more extracellular matrix proteins and their concentration(s) in the medium may be applied to the nutritional supplement of the present disclosure. However, in embodiments in which the nutritional supplement is concentrated (e.g., greater than 1x), the concentration ranges are adjusted appropriately.

[0095] By way of example, the one, two, three or more types of extracellular matrix proteins included in the nutritional supplement may be selected from collagen, ECM1, laminin, osteopontin, vitronectin, or SPARC. In embodiments including a second, third, or additional type of extracellular matrix protein, they may be selected from, but are not limited to, collagen, ECM1, E-cadherin, laminin (e.g., laminin-511, fragments of laminin-511 (e.g., laminin-511-E8), laminin-521 (also known as laminin-11), osteopontin, fibronectin, vitronectin, SPARC, or combinations thereof. In preferred embodiments, the extracellular matrix protein(s) included in the nutritional supplement of the present disclosure are one or more of ECM1, osteopontin, vitronectin, collagen, and laminin, and more preferably one or more of ECM1, osteopontin, and vitronectin.

[0096] In one embodiment, the nutritional supplements of the present disclosure may be concentrated, hi one embodiment, the nutritional supplements may be about 2x concentrated, about 5x concentrated, about 10x concentrated, about 15x concentrated, about 20x concentrated, about 25x concentrated, about 50x concentrated, about 100x concentrated, about 200x concentrated, about 500x concentrated, or more.

[0097] In one embodiment, the nutritional supplement of the present disclosure (when formulated in a mammalian lymphocyte growth medium) supports at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 population doublings.

[0098] In one embodiment, the nutritional supplement of the present disclosure (when incorporated into a mammalian lymphocyte growth medium) may further support differentiation / maturation of lymphocytes or their precursor cells in a culture environment (e.g., from a more primitive fate to a more specified fate). In one embodiment, differentiation / maturation of 10% or more of B cells, NK cells or T cells, or their precursor cells. In one embodiment, differentiation / maturation of 20% or more of B cells, NK cells or T cells, or their precursor cells. In one embodiment, differentiation / maturation of 30% or more of B cells, NK cells or T cells, or their precursor cells. In one embodiment, differentiation / maturation of 40% or more of B cells, NK cells or T cells, or their precursor cells. In one embodiment, differentiation / maturation of 50% or more of B cells, NK cells or T cells, or their precursor cells.

[0099] In one embodiment, the nutritional supplement of the present disclosure (when incorporated into mammalian lymphocyte growth medium) may further support activation of lymphocytes or their progenitor cells in the culture environment.

[0100] In one embodiment, the nutritional supplements (and media combined with the nutritional supplements) of the present disclosure, and cells cultured in their presence, are not in contact with feeder cells or undefined cell supports (e.g., matrices) such as Matrigel™. Thus, the nutritional supplements (and media combined with the nutritional supplements) of the present disclosure can support feeder-free growth of lymphocytes (e.g., B or T cells) or progenitor cells thereof.

[0101] In one embodiment, the nutritional supplement of the present disclosure (and the medium with which the nutritional supplement is combined) is free of animal-derived components. Thus, the nutritional supplement of the present disclosure (and the medium with which the nutritional supplement is combined) can support the proliferation of lymphocytes (e.g., B or T cells) or progenitor cells thereof free of animal-derived components.

[0102] In one embodiment, the nutritional supplements of the present disclosure (and media in which the nutritional supplements are combined) may be serum-free. The serum-free nutritional supplements of the present disclosure (and media in which the nutritional supplements are combined) may, however, include albumin, such as recombinant albumin. method

[0103] In another aspect of the present disclosure, a method for expanding mammalian lymphocytes, such as T cells, NK cells or B cells, or their precursor cells is provided.In one embodiment, mammalian lymphocytes are plated as single cells or at clonal density.In one embodiment, mammalian lymphocytes are not plated as single cells or at clonal density.

[0104] The lymphocytes seeded / cultured in the methods described herein may be isolated / enriched from a mixed sample, e.g., blood or leukapheresis sample. In embodiments where the lymphocytes are B cells, the method may include seeding pan B cells, memory B cells, naive B cells, plasmablasts, and / or plasma cells. In embodiments where the lymphocytes are T cells, the method may include seeding pan T cells, CD4 + T cells, CD8 + The method may include seeding T cells, memory T cells, regulatory T cells, helper T cells, effector T cells, cytotoxic T cells, naive T cells, CAR-T cells. In embodiments where the lymphocytes are NK cells, the method may include seeding pan-NK cells, cytotoxic NK cells, regulatory NK cells, tolerant NK cells, or CAR-NK cells. Nevertheless, one of skill in the art will readily appreciate that the disclosed media / supplements and methods may be used with other subsets or populations of lymphocytes.

[0105] In one embodiment, the mammalian lymphocyte or progenitor cell thereof is human. In one embodiment, the mammalian lymphocyte or progenitor cell thereof is rodent (e.g., mouse).

[0106] In one embodiment, a method of expanding a mammalian lymphocyte or progenitor cell thereof includes seeding the lymphocytes (e.g., a population of B cells or T cells, or their progenitors) into a culture environment as single cells or at clonal density. In one embodiment, a method of expanding a mammalian lymphocyte or progenitor cell thereof includes seeding the lymphocytes (e.g., a population of B cells, NK or T cells, or their progenitors) into a culture environment in bulk or at clonal density, rather than as single cells. After seeding the cells, the method may include incubating (e.g., culturing) the seeded cells or progenitor cells in the culture environment for a time sufficient to expand the lymphocytes or progenitor cells to a desired amount.

[0107] In one embodiment, mammalian lymphocytes or their progenitors are seeded in a culture flask, dish, vessel, plate, or well thereof. Plating lymphocytes may be important in cloning operations, such as when their genome is being edited, or when the cells have a desired phenotype, or perform a desired function. In an embodiment in which mammalian B cells or their progenitors are plated at clonal density, it is important that the cell density is selected so that the cells are well spaced apart. Cells plated at clonal density may be less likely to come into contact with each other. Desirably, cells plated at clonal cell density do not confer paracrine effects on other cells in the vessel. For example, the clonal density may correspond to a cell density of 1 cell / well in a 6-well, 24-well, 48-well, or 96-well plate. In one embodiment, the cells are plated at up to 10, 100, 1000 cells / well or cm in a culture dish, e.g., a 10 cm culture dish. 2As noted herein above, in some applications, the cells are not plated as single cells or at clonal cell density. For example, the cells may be plated at a density of 1000 cells / well or cm 2 It may be seeded at a greater density.

[0108] In one embodiment of the method disclosed herein, the seeded cells are incubated in a culture environment for any time sufficient to allow the lymphocytes or their progenitors to expand to a desired number. In one embodiment, the incubation step may be at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days, or longer.

[0109] In one embodiment, the incubation step can be divided into stages. For example, the first stage of incubation / growth can be 1-10 days in the first culture environment (e.g., culture medium formulation). In one embodiment, the first stage of incubation / growth is about 1-5 days, or about 3-10 days in the first culture environment (e.g., culture medium formulation). In one embodiment, the second stage of incubation / growth is about 1-10 days in the second culture environment (e.g., culture medium formulation). In one embodiment, the second stage of incubation / growth is about 1-5 days, or about 3-10 days in the second culture environment (e.g., culture medium formulation).

[0110] The culture environment in which the lymphocytes are seeded (and in which the lymphocytes are incubated) may include one or more of the following: culture medium, container, temperature, atmospheric conditions, and the like.

[0111] With respect to the culture medium (and nutritional supplements added to the medium to make a complete medium), the above description applies. For example, the culture environment (e.g., culture medium) may include one or more of a mixture of lipids, a ligand for CD40, and one or more cytokines. In one embodiment, the culture environment (e.g., culture medium) may include two or more of a mixture of lipids, a ligand for CD40, and one or more cytokines. In one embodiment, the culture environment (e.g., culture medium) may include each of a mixture of lipids, a ligand for CD40, and one or more cytokines.

[0112] In one embodiment, the lipid mixture comprises a single type of lipid. In one embodiment, the lipid mixture comprises multiple types of lipid. In one embodiment, the lipid mixture may be stabilized and / or may be free of animal-derived components.

[0113] In one embodiment, the ligand of CD40 is a ligand of human CD40. In one embodiment, the ligand of CD40 is a ligand of mouse or rat CD40. In one embodiment, the ligand of CD40 comprises one or more proteins. In one embodiment, the ligand of CD40 is comprised in a complex of proteins.

[0114] In one embodiment, the ligand for CD40 is included in the culture medium, hi one embodiment, the ligand for CD40 is otherwise included in the culture environment, such as being coated on the surface of a container.

[0115] As discussed above, the type(s) of one or more cytokines and / or one or more growth factors included in the cell culture environment (e.g., cell culture medium) of the present disclosure are selected depending on the particular type of cells being cultured.

[0116] In one embodiment, the one or more cytokines or growth factors may be any one of IL-2, IL-4, IL-6, IL-10, and IL-21, or a combination thereof. In one embodiment, the one or more cytokines or growth factors include each of IL-2, IL-4, IL-6, IL-10, and IL-21. In one embodiment, the one or more cytokines or growth factors include at least IL-2 and IL-10, and optionally, IL-21.

[0117] In one embodiment, the one or more cytokines and / or one or more growth factors are contained in the culture medium, hi one embodiment, the one or more cytokines and / or one or more growth factors are otherwise included in the culture environment, such as coated on a surface of a container.

[0118] In one embodiment, the culture environment comprises a vessel (as described above) containing a culture medium (as described in detail herein). In one embodiment, the culture environment may further comprise a surface coating of the vessel. In one embodiment, the culture environment does not comprise a surface coating of the vessel.

[0119] In one embodiment, the culture environment (e.g., culture medium) can further include a first type of extracellular matrix protein. The first type of extracellular matrix protein can be included in the culture medium (or a nutritional supplement added thereto). In one embodiment, the first type of extracellular matrix protein can be solubilized in the culture medium (or a nutritional supplement added thereto).

[0120] In one embodiment, the first type of extracellular matrix protein may be selected from collagen, ECM1, laminin, osteopontin, vitronectin, or SPARC. In one embodiment, the first type of extracellular matrix protein is ECM1, vitronectin, or osteopontin.

[0121] In one embodiment, the culture environment (e.g., culture medium) can further include a second type of extracellular matrix protein. The second type of extracellular matrix protein can be included in the culture medium (or a nutritional supplement added thereto). In one embodiment, the second type of extracellular matrix protein can be solubilized in the culture medium (or a nutritional supplement added thereto).

[0122] In one embodiment, the second type of extracellular matrix protein is different from the first type of extracellular matrix protein and may be selected from collagen, ECM1, laminin, osteopontin, vitronectin, or SPARC, hi one embodiment, the second type of extracellular matrix protein is ECM1, vitronectin, or osteopontin.

[0123] In one embodiment, the culture environment (e.g., culture medium) can further include a third type of extracellular matrix protein. The third type of extracellular matrix protein can be included in the culture medium (or a nutritional supplement added thereto). In one embodiment, the third type of extracellular matrix protein can be solubilized in the culture medium (or a nutritional supplement added thereto).

[0124] In one embodiment, the third type of extracellular matrix protein is different from the first and second types of extracellular matrix proteins and may be selected from collagen, ECM1, laminin, osteopontin, vitronectin, or SPARC, hi one embodiment, the third type of extracellular matrix protein is ECM1, vitronectin, or osteopontin.

[0125] In one embodiment, the second and third types of extracellular matrix proteins may be selected from collagen, ECM1, E-cadherin, laminin (e.g., laminin-511, fragments of laminin-511 (e.g., laminin-511-E8), laminin-521 (also known as laminin-11), osteopontin, fibronectin, vitronectin, SPARC, or a combination thereof.

[0126] Thus, in embodiments of the method in which the culture environment comprises a culture medium, at least a first type of extracellular matrix protein, as well as one or more of a mixture of lipids, a ligand for CD40, and one or more cytokines, may be included in the culture medium. When additional extracellular matrix proteins (e.g., second, third, and further) are included in the culture environment, such extracellular matrix protein(s) may also be included in the culture medium.

[0127] In one embodiment, the culture environment (e.g., culture medium or nutritional supplements added thereto) comprises at least a first type of extracellular matrix protein, optionally a second type of extracellular matrix protein, and only one of a mixture of lipids, a ligand for CD40, and one or more cytokines. In one embodiment, the culture environment (e.g., culture medium or nutritional supplements added thereto) comprises at least a first type of extracellular matrix protein, optionally a second type of extracellular matrix protein, and two of a mixture of lipids, a ligand for CD40, and one or more cytokines.

[0128] The incubation / culturing step may be performed in different or staged culture environments, including, for example, a first stage or culture environment and a second stage or culture environment. In one embodiment, the first stage of incubation / culturing may be performed in a first culture environment, such first culture environment comprising at least the first type of extracellular matrix protein, and the second stage of incubation / culturing may be performed in a second culture environment, such second culture environment not comprising at least the first type of extracellular matrix protein. In one embodiment, the first stage of incubation / culturing may be performed in a first culture environment, such first culture environment not comprising at least the first type of extracellular matrix protein, and the second stage of incubation / culturing may be performed in a second culture environment, such second culture environment comprising at least the first type of extracellular matrix protein.

[0129] It should be noted that the first and second (or additional) stages or culture environments may differ from each other with respect to any of the culture environment components / conditions disclosed herein. For example, the stages or environments may differ with respect to i) the mixture of lipids, ii) a ligand for CD40, iii) one or more cytokines, iv) a second and / or third type of extracellular matrix protein rather than the first type of extracellular matrix protein, v) a coating of the container, and / or vi) their duration.

[0130] In one embodiment, the culture environment further comprises a coating or functionalization applied to the surface of the vessel. In embodiments where the culture environment comprises both a culture medium and a coating / functionalization applied to the surface of the vessel, the coating on the surface of the vessel may comprise one or more of a first type of extracellular matrix protein, a second type of extracellular matrix protein, and a third type of extracellular matrix protein. In such embodiments, the culture medium may also comprise one or more types of extracellular matrix proteins.

[0131] In one embodiment, lymphocytes (e.g., B, NK or T cells) or progenitor cells cultured using the methods of the present disclosure are not in contact with feeder cells or undefined cell supports (e.g., matrices) such as Matrigel™. Thus, the methods of the present disclosure can support feeder-free growth of lymphocytes or their progenitor cells.

[0132] In one embodiment, lymphocytes (e.g., B, NK or T cells) or their progenitors cultured using the method of the present disclosure are not contacted with animal-derived components. Thus, the method of the present disclosure can support the animal-derived component-free growth of lymphocytes or their progenitors. In one embodiment, the animal-derived component-free workflow can be a serum-free workflow.

[0133] Overall, practice of the disclosed methods (and use of the media) can result in the expansion of a population of mammalian lymphocytes (e.g., B, NK or T cells) or their progenitors. More specifically, practice of the disclosed methods (and use of the media) can result in up to a 1000-fold, or up to a 5000-fold, or up to a 10,000-fold, or up to a 15,000-fold, or up to a 20,000-fold, or up to a 25,000-fold or greater increase in mammalian lymphocytes (e.g., B, NK or T cells) or their progenitors.

[0134] In one embodiment, the methods of the disclosure support 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, or 60 or more population doublings.

[0135] In one embodiment, mammalian lymphocytes (e.g., T, NK or B cells) or progenitor cells thereof expanded using the methods of the present disclosure express markers characteristic of the expanded lymphocyte lineage. In one embodiment, more than 30% of the expanded cells are positive for one or more markers characteristic of mammalian B, NK and / or T cell lineages. In one embodiment, more than 40% of the expanded cells are positive for one or more markers characteristic of mammalian B, NK and / or T cell lineages. In one embodiment, more than 50% of the expanded cells are positive for one or more markers characteristic of mammalian B, NK and / or T cell lineages. In one embodiment, more than 60% of the expanded cells are positive for one or more markers characteristic of mammalian B, NK and / or T cell lineages. In one embodiment, more than 70% of the expanded cells are positive for one or more markers characteristic of mammalian B, NK or T cell lineages.

[0136] In one embodiment, the disclosed method may further support differentiation / maturation of lymphocytes or their precursor cells in a culture environment, such as during expansion (e.g., from a more primitive fate to a more specified fate). In one embodiment, differentiation / maturation of 10% or more of B cells, NK cells or T cells, or their precursor cells. In one embodiment, differentiation / maturation of 20% or more of B cells, NK cells or T cells, or their precursor cells. In one embodiment, differentiation / maturation of 30% or more of B cells, NK cells or T cells, or their precursor cells. In one embodiment, differentiation / maturation of 40% or more of B cells, NK cells or T cells, or their precursor cells. In one embodiment, differentiation / maturation of 50% or more of B cells, NK cells or T cells, or their precursor cells.

[0137] In one embodiment, the method of the present disclosure (which can support differentiation of lymphocytes (e.g., B or T cells) or progenitor cells thereof in a culture environment) includes an incubation step. In one embodiment, the incubation step is for 1 to 15 days. In one embodiment, the incubation step is for more than 1 day. In one embodiment, the incubation step is for at least 3 days.

[0138] In one embodiment, the disclosed method may further support activation of lymphocytes or their progenitor cells in a culture environment, such as during proliferation and / or differentiation (e.g., while incubating in culture medium).

[0139] The following non-limiting examples illustrate the present disclosure. EXAMPLES

[0140] Example 1: Treatment of blood samples to enrich for B-lineage cells Pan-B cells were enriched from human peripheral blood mononuclear cells (PBMCs, i.e., leukapheresis samples) using the EasySep™ Human Pan-B Cell Enrichment Kit (STEMCELL Technologies). Cells from the leukapheresis samples and enriched B-lineage cells therefrom were assessed for viability by flow cytometry analysis after staining with fluorescent DRAQ7 dye (Figure 1). B-lineage cells enriched from the leukapheresis samples were also assessed for purity and activation status by flow cytometry using fluorescently labeled antibodies against the surface markers CD19, CD20, CD86, and CD138 (Figure 1).

[0141] Example 2: Seeding and culture of enriched B-lineage cells Pan-B cells were enriched as described in Example 1, and single pan-B cells were sorted into each well of a 96-well plate (BD FACSAria™ fusion cell sorter). Unless otherwise indicated, each well contained ImmunoCult™-XF B cell basal medium (STEMCELL Technologies) supplemented with a ligand for human CD40 (various suppliers including STEMCELL Technologies), one or more extracellular matrix proteins (STEMCELL Technologies and other commercial sources), a mixture of lipids (STEMCELL Technologies), and one or more cytokines (STEMCELL Technologies). Extracellular matrix proteins shown in this study to grow single cells include one or more of collagen, ECM1, E-cadherin, laminin, osteopontin, fibronectin, vitronectin, or SPARC, preferably one or more of collagen, ECM1, laminin, osteopontin, and vitronectin. Wells were imaged periodically during the growth protocol at 40x magnification using an Olympus Q Color3 microscope.

[0142] Example 3: Single B cells proliferate significantly in the presence of soluble, exogenously added extracellular matrix protein(s) Pan-B cells were enriched and seeded essentially as described in Examples 1 and 2, and plating of a single pan-B cell per well on day 0 (indicated by an open circle) was confirmed by imaging with a Solentim Cell Metric microscope at 40x magnification (Figure 2). On days 9 and 12 of culture, wells were imaged with an Olympus Q Color3 microscope at 40x magnification (Figure 2). Single B cells were seeded and cultured in media formulations containing three exogenously added extracellular matrix proteins (condition 1, condition 2, and condition 3), as well as different pairwise combinations of all three proteins (condition 4). Each of condition 1, condition 2, condition 3, and condition 4 showed significant proliferation, as observed on both days 9 and 12. In contrast, proliferation could not be readily observed on day 9 in wells containing cells seeded only in basal medium. The extracellular matrix proteins used in carrying out this embodiment may be selected from among collagen, ECM1, E-cadherin, laminin, osteopontin, fibronectin, vitronectin, or SPARC, and preferably selected from collagen, ECM1, laminin, osteopontin, and vitronectin.

[0143] Example 4: Single B cells proliferate significantly in the presence of individual exogenously added extracellular matrix proteins Pan-B cells were enriched and seeded essentially as described in Examples 1 and 2 from a different leukapheresis sample than that used in Example 3. On days 5 and 12 of culture, individual wells were imaged at 40x magnification using an Olympus Q Color3 microscope (Figure 3). Single B cells were seeded and cultured in media formulations containing different individual exogenously added extracellular matrix proteins (conditions 1 and 2), as well as a combination of both proteins (condition 3). All conditions tested resulted in proliferation of seeded cells at both time points (Figure 3). The extracellular matrix proteins used to carry out this example may be selected from among collagen, ECM1, E-cadherin, laminin, osteopontin, fibronectin, vitronectin, or SPARC, and may be preferably selected from collagen, ECM1, laminin, osteopontin, and vitronectin.

[0144] Example 5: Synergistic Effects of Combinations of Extracellular Matrix Proteins Pan-B cells were enriched and seeded essentially as described in Examples 1 and 2 from different leukapheresis samples than those used in Examples 3 and 4. On days 5 and 12 of culture, wells were imaged at 40x magnification using an Olympus Q Color3 microscope (Figure 4). Single B cells were seeded and cultured in media formulations containing different individual exogenously added extracellular matrix proteins (Condition 1, Condition 2, and Condition 3), as well as different pairwise combinations of the two proteins (Condition 4 and Condition 5). All conditions tested resulted in proliferation of the seeded cells at both time points, but the combination treatments appeared to result in greater proliferation (Figure 4). The extracellular matrix proteins used to carry out this example may be selected from among collagen, ECM1, E-cadherin, laminin, osteopontin, fibronectin, vitronectin, or SPARC, and preferably may be selected from collagen, ECM1, laminin, osteopontin, and vitronectin.

[0145] Example 6: Enhancement of single B cell proliferation in the presence of a ligand for human CD40 Pan-B cells were enriched and seeded essentially as described in Examples 1 and 2 using cells from the same leukapheresis samples used in Run 5. On days 5 and 12 of culture, wells were imaged at 40x magnification using an Olympus Q Color3 microscope (Figure 5). Single B cells were seeded in culture medium containing individual exogenously added extracellular matrix proteins (selected from the preferred list recited in Example 2), but with or without exogenously added ligand for human CD40. Proliferation of seeded single B cells was observed in both conditions and at both time points, but was enhanced in the presence of ligand for CD40 (Figure 5).

[0146] Example 7: Single B cells proliferate in the presence of other individual soluble extracellular matrix proteins Pan-B cells were enriched and seeded from the same donor as in Example 3, essentially as described in Examples 1 and 2. On day 7 of culture, wells were imaged at 40x magnification using an Olympus Q Color3 microscope (Figure 6). Conditions 1 and 2 contained different individual exogenously added extracellular matrix proteins than those tested in the previous examples herein, but the extracellular matrix proteins tested were nevertheless selected from among collagen, ECM1, E-cadherin, laminin, osteopontin, fibronectin, vitronectin, or SPARC, and preferably selected from collagen, ECM1, laminin, osteopontin, and vitronectin. In both conditions tested, by day 7, seeded cells had proliferated to a significant extent (Figure 6).

[0147] Example 8: Role of lipids in single B cell proliferation Pan-B cells were enriched and seeded from the same donor as in Example 3, essentially as described in Examples 1 and 2. On days 5 and 12 of culture, wells were imaged at 40x magnification using an Olympus Q Color3 microscope (Figure 7). To evaluate the role of lipids in B cell proliferation, single B cells were seeded in culture media formulations containing different lipid supplements (Animal-Derived Component-Free Lipid Mix 1 and Stabilized Lipid Mix 2, both from STEMCELL Technologies), but otherwise containing the same single exogenously added extracellular matrix protein (selected from the preferred list listed in Example 2). On day 12 of culture, single B cells showed proliferation in the presence of lipid, but no measurable proliferation in the absence of lipid (Figure 7).

[0148] Example 9: Differentiation and activation of expanded B cells Pan-B cells were enriched and seeded from the same donor as in Example 3 essentially as described in Examples 1 and 2. Cells were expanded in culture for 13 days in culture media containing different individual exogenously added extracellular matrix proteins (Condition 1 and Condition 2) (FIG. 8). The extracellular matrix proteins used to carry out this example may be selected from among collagen, ECM1, E-cadherin, laminin, osteopontin, fibronectin, vitronectin, or SPARC, preferably selected from collagen, ECM1, laminin, osteopontin, and vitronectin.

[0149] After the 13-day culture protocol, the resulting cells were assessed for activation and differentiation status by flow cytometry using fluorescently labeled antibodies against CD19, CD20, CD86, and CD138 (Figure 8). In all three conditions tested, plated pan-B cells were activated and differentiated from a primitive phenotype to a more mature phenotype. Pan-B cell activation is indicated by increased levels of the B cell activation marker CD86. Pan-B cell differentiation towards B lineage cells is indicated by decreased expression levels of CD19 and CD20 surface markers, as well as increased expression levels of the differentiation marker CD138.

[0150] Example 10: Effect of cytokines on single B cell proliferation Pan-B cells were enriched and seeded essentially as described in Examples 1 and 2. On day 13 of culture, wells were imaged at 40x magnification using an Olympus Q Color3 microscope (Figure 9). The extracellular matrix proteins used in carrying out this example may be selected from among collagen, ECM1, E-cadherin, laminin, osteopontin, fibronectin, vitronectin, or SPARC, and preferably selected from collagen, ECM1, laminin, osteopontin, and vitronectin.

[0151] To evaluate the role of cytokines in the proliferation of single B cells, cells were seeded in culture media containing individual exogenously added extracellular matrix proteins (selected from the preferred list recited in Example 2) but differing in the cytokines contained therein: IL-2 and IL-10 (condition 1); and IL-2, IL-4, IL-6, IL-10, and IL-21 (condition 2). Control conditions included unsupplemented basal medium (control 1), basal medium supplemented with cytokines but not with exogenously added extracellular matrix proteins (control 2), and basal medium supplemented with extracellular matrix proteins but not with cytokines (control 3). After 13 days of culture, single B cells showed significant proliferation in condition 1 and condition 2, but no obvious proliferation in control condition 1 (Figure 9).

[0152] Example 11: Quantification of single B cell proliferation Pan-B cells were enriched and seeded essentially as described in Examples 1 and 2. On day 13 of culture, expanded cells were pelleted to remove the supernatant, washed with cold PBS, and trypsinized. Cells were then harvested using a cell scraper and counted using a Nucleocounter to assess proliferation from single B cells. In the first experiment, three different combinations of exogenously added extracellular matrix proteins were tested, and the approximate number of expanded cells ranged from 2400 to 15,000 cells / input cell (data not shown). In the second experiment, a single extracellular matrix protein and a combination of two extracellular matrix proteins were tested, and the approximate number of expanded cells ranged from 2000 to 7000 cells / input cell (data not shown). In the third experiment, a single extracellular matrix protein and a combination of two extracellular matrix proteins were tested, and the approximate number of expanded cells ranged from 15000 to 46000 cells / input cell (data not shown). Thus, even taking into account the potential difficulty of completely dissociating the expanded cells, a surprising level of expansion could be achieved in a variety of media conditions.

[0153] Example 12: Proliferation of single B cells in different basal media Pan-B cells were enriched and seeded essentially as described in Examples 1 and 2. Single B cells were seeded and cultured in different commercial basal media in the presence of individual exogenously added extracellular matrix proteins (selected from the preferred list listed in Example 2) as shown in FIG. 10. On day 13 of culture, cells were imaged at 40x magnification using an Olympus Q Color3 microscope (FIG. 10). No proliferation was observed in the control condition of basal media not supplemented with extracellular matrix proteins. In contrast, when exogenously added extracellular matrix proteins were diluted in either RPMI or DMEM F-12, substantial proliferation was observed (FIG. 10, white arrows), and when diluted in either IMDM or ImmunoCult™ XF, significant proliferation was observed (FIG. 10).

[0154] Example 13: Single human memory B cells proliferate in the presence of exogenously added extracellular matrix proteins Human memory B cells were enriched and isolated from leukapheresis samples using the easySep™ Human Memory B Cell Isolation Kit (STEMCELL Technologies). Single memory B cells were seeded (as above) in media containing either two different exogenously added extracellular matrix proteins (condition 1) or three different exogenously added extracellular matrix proteins (condition 2). On day 13 of culture, cells in the wells were imaged at 40x magnification using an Olympus Q Color3 microscope (Figure 11). Substantial proliferation of single memory B cells was observed in both condition 1 and condition 2.

[0155] Example 14: Expansion of single human memory B cells Human pan-B cells isolated from PBMCs as described in Example 1 were stained with CD19, CD27, CD38, CD138, IgM, IgG, and DRAQ7 antibodies. + CD27+ CD38 - CD138 - IgM + IgG -- Memory B cells were sorted into individual wells of a 96-well plate (BD FACSAria™ fusion cell sorter) and cultured for up to 4 days in media as encompassed in Example 2, including individual exogenously added extracellular matrix proteins. On day 5, each well of cells was transitioned to a media formulation essentially as encompassed in Example 2, except that it was not supplemented with exogenously added extracellular matrix proteins. From day 5 onwards, complete media changes were performed every 2-3 days. After a total of 12 days of culture, output cells were harvested and counted (essentially as described in Example 11), and the viable fold expansion of the cells was calculated as 25,000±1549 (FIG. 12A). Day 12 expanded human cells were also tested by ELISA, where significant secretion of the antibody markers IgM and IgG was detected (FIG. 12B). Finally, the expression of various cell surface markers on the sorted single cells on day 0 and on the expanded human cells on day 12 was assessed by flow cytometry. The results show that single memory B cells expand into a population of B cells including memory B cells, antibody-secreting cells, and plasmablasts (FIG. 12C).The results also showed that among the expanded cells at day 12, i) increased expression of the human B cell marker CD38, which correlates with an increase in antibody-secreting cells, ii) an increase in the antibody marker IgG with a decrease in IgM, indicative of B cell class switching, iii) an increase in the activation marker CD86 (not shown), and iv) an increase in the differentiation marker CD138, indicative of memory B cell differentiation into plasmablasts.

[0156] Example 15: Expansion of single human plasmablasts Human pan-B cells were isolated and stained with antibodies essentially as described in Example 14. On day 0, single viable CD19 + CD27 - CD138 +Human plasmablasts were sorted into individual wells of a 96-well plate and cultured as described in Example 14. After a total of 12 days of culture, the output cells were harvested and counted (essentially as described in Example 11), and the survival fold expansion of the cells was calculated as 9,000±1645 (FIG. 13A). The expanded human cells at day 12 were also tested by ELISA, and significant secretion of antibody markers IgM and IgG was detected (FIG. 13B). Finally, the expression of various cell surface markers on the sorted single cells at day 0 and on the expanded human cells at day 12 was evaluated by flow cytometry. The results show that single human plasmablasts expand into a population of B cells, including memory B cells, antibody-secreting cells, and plasmablasts (FIG. 13C). The results also showed that among expanded cells on day 12, i) increased expression of human plasmablast markers CD38 and CD27, indicative of an increase in antibody-secreting cells, ii) an increase in the antibody marker IgG with a decrease in IgM, indicative of B cell class switching, and iii) a decrease in expression of the differentiation marker CD138.

[0157] Example 16: Single murine pan-B cells proliferate in the presence of exogenously added extracellular matrix proteins Mouse pan-B cells were enriched from C57BL / 6 mice using the easySep™ Mouse Pan-B Isolation Kit (STEMCELL Technologies). On day 0, single B cells were sorted into individual wells of a 96-well plate (BD FACSAria™ fusion cell sorter) and cultured in media formulations containing one exogenously added extracellular matrix protein (selected from the preferred list recited in Example 2) essentially as described in Example 2. On day 13 of culture, cells in the wells were imaged at 40x magnification using an Olympus Q Color3 microscope (Figure 14). Mouse single B cells appeared to proliferate in both conditions tested.

[0158] Example 17: Expansion of single mouse memory B cells Enriched and isolated mouse pan-B cells from C57BL / 6 mice using the easySep™ Mouse Pan-B Isolation Kit (STEMCELL Technologies) were stained with CD19, CD45R, CD138, and DRAQ7 antibodies. + CD45R + CD138 - Mouse memory B cells were sorted into individual wells of a 96-well plate (BD FACSAria™ fusion cell sorter) and cultured for up to 4 days in media as encompassed in Example 16, including individual exogenously added extracellular matrix proteins. On day 5, each well of cells was transitioned to a media formulation essentially as encompassed in Example 2, except that it was not supplemented with exogenously added extracellular matrix proteins. From day 5 onwards, complete media changes were performed every 2-3 days. After a total of 12 days of culture, output cells were harvested and counted (essentially as described in Example 11), and the survival fold expansion of the cells was calculated as 10,000±1423 (FIG. 15A). Day 12 expanded mouse memory cells were also tested by ELISA, where significant secretion of the antibody marker IgG was detected (FIG. 15B). Finally, the expression of various cell surface markers of the sorted single cells on day 0 and the expanded mouse cells on day 12 was assessed by flow cytometry. The results show that single memory B cells expand into a population of B cells including memory B cells, antibody-secreting cells, and plasmablasts (FIG. 15C). The results also showed i) a decrease in CD45R and an increase in CD138, suggesting memory cell differentiation into plasmablasts, ii) an increase in the antibody marker IgG with a decrease in IgM, indicative of B cell class switching, and iii) an increase in the differentiation marker CD138 among the expanded cells at day 12.

[0159] Example 18: Expansion of single mouse plasmablasts Murine pan-B cells were isolated and stained with antibodies essentially as described in Example 17. On day 0, single viable CD19 + CD138 +Mouse plasmablasts were sorted into individual wells of a 96-well plate (BD FACSAria™ fusion cell sorter) and cultured as described in Example 17. After a total of 12 days of culture, output cells were harvested and counted (essentially as described in Example 11), and the viable fold expansion of the cells was calculated as 13,000±600 (FIG. 16A). Day 12 expanded mouse cells were also tested by ELISA, and significant secretion of the antibody marker IgG was detected (FIG. 16B). Finally, the expression of various cell surface markers on the sorted single cells on day 0 and on the expanded mouse cells on day 12 was evaluated by flow cytometry. The results showed that single memory B cells expanded into a population of B cells, which included memory B cells, antibody-secreting cells, and plasmablasts (FIG. 16C). The results also showed that among expanded cells on day 12, i) increased expression of the plasmablast marker CD38, indicative of an increase in antibody-secreting cells, ii) increased expression of the antibody marker IgG accompanied by a decrease in IgM, indicative of B cell class switching, and iii) decreased expression of the differentiation marker CD138.

[0160] Example 19: Expansion of single T cells Mouse splenocytes were isolated from C57BL / 6 mice and stained with CD3 and DRAQ7 antibodies. On day 0, single viable CD3 +Mouse T cells were sorted into individual wells of a 96-well plate (BD FACSAria™ fusion cell sorter) and cultured for up to 4 days in media as encompassed in Example 16, including individual exogenously added extracellular matrix proteins. On day 5, each well of cells was transferred to a media formulation essentially as encompassed in Example 2, except that it was not supplemented with exogenously added extracellular matrix proteins. Complete media changes were performed every 2-3 days after day 5. Seven days after transfer to the second culture environment (total of 12 days), output cells were harvested and counted (essentially as described in Example 11), and the survival fold expansion of the cells was calculated as >10,000±1423 (FIG. 17A). After a total of 12 days of culture, output cells were also analyzed by flow cytometry for surface expression of CD8a (T cell marker), CD25 (T cell activation marker), and CD69 (T cell activation marker) (FIG. 17B). After staining a sample of PBMCs with CD3 and DRAQ7 antibodies, a similar workflow to that described in this example was performed with sorted human T cells. Figure 17C shows single viable human CD3 T cells sorted into wells of a 96-well plate and cultured for 12 days in medium essentially as described in Example 2. + The scale of T cell proliferation is shown.

Claims

1. 1. A cell culture medium supplement for expanding mammalian lymphocytes or their progenitor cells, comprising: one or more of: a ligand for CD40, a mixture of lipids, and one or more cytokines; A diluent; The nutritional supplement comprising: (a) a first type of extracellular matrix protein; (b) a first type of extracellular matrix protein and a second type of extracellular matrix protein; or (c) a first type of extracellular matrix protein, a second type of extracellular matrix protein, and a third type of extracellular matrix protein; 10. The nutritional supplement of claim 1, further comprising:

3. 3. The nutritional supplement of claim 2, wherein the extracellular matrix protein is selected from the group consisting of collagen, ECM1, E-cadherin, laminin, osteopontin, fibronectin, vitronectin, or SPARC. (a) the CD40 ligand comprises one or more proteins; and / or (b) the one or more cytokines are selected from IL-2, IL-4, IL-6, IL-10, and IL-21; The nutritional supplement of claim 1.

5. 10. The nutritional supplement of claim 1, wherein the nutritional supplement is combined with a basal medium.

6. 1. A culture medium for expanding mammalian lymphocytes or their progenitor cells, comprising: A basal medium; one or more of: a ligand for CD40, a mixture of lipids, and one or more cytokines; The culture medium comprising: (a) a first type of extracellular matrix protein; (b) a first type of extracellular matrix protein and a second type of extracellular matrix protein; or (c) a first type of extracellular matrix protein, a second type of extracellular matrix protein, and a third type of extracellular matrix protein; 7. The culture medium of claim 6, further comprising:

8. 8. The culture medium of claim 7, wherein the extracellular matrix protein is selected from the group consisting of collagen, ECM1, E-cadherin, laminin, osteopontin, fibronectin, vitronectin, or SPARC.

9. (a) the one or more cytokines are selected from IL-2, IL-4, IL-6, IL-10, and IL-21; and / or (b) the ligand for CD40 comprises one or more proteins; The culture medium of claim 6.

10. The culture medium comprises: (a) serum-free; and / or (b) supporting the feeder-free growth of lymphocytes and their progenitor cells; and / or (c) supports the animal-origin-free growth of lymphocytes and their progenitor cells; The culture medium according to any one of claims 6 to 9.

11. 1. A method for expanding mammalian lymphocytes or their progenitor cells, comprising: seeding the lymphocytes or their progenitor cells into a culture environment comprising one or more of: a ligand for CD40, a mixture of lipids, and one or more cytokines; Incubating the seeded lymphocytes or progenitor cells thereof in the culture environment for more than one day; The method comprising:

12. In the culture environment, (a) a first type of extracellular matrix protein; (b) a first type of extracellular matrix protein and a second type of extracellular matrix protein; or (c) a first type of extracellular matrix protein, a second type of extracellular matrix protein, and a third type of extracellular matrix protein; The method of claim 11 further comprising:

13. 13. The method of claim 12, wherein the extracellular matrix protein is one or more of collagen, ECM1, E-cadherin, laminin, osteopontin, fibronectin, vitronectin, or SPARC.

14. The method of claim 11 , wherein the one or more of the CD40 ligand, the lipid mixture, and the one or more cytokines are contained in a culture medium.

15. (a) the CD40 ligand comprises one or more proteins; and / or (b) the one or more cytokines are selected from IL2, IL4, IL6, IL10, and IL21; and / or (c) the mixture of lipids is chemically defined; The method according to any one of claims 11 to 14.

16. The method of claim 11, wherein the seeding and incubation are carried out under feeder-free conditions.

17. 12. The method of claim 11, wherein the lymphocytes are human or murine, and the lymphocytes are T cells, B cells, or NK cells.

18. The method of claim 11, further comprising differentiating and / or activating the lymphocytes or their progenitor cells in the culture environment.

19. 12. The method of claim 11, wherein the incubating step is for at least 3 days.

20. The method of any one of claims 17 to 19, wherein the lymphocytes or their precursor cells are seeded as single cells or at clonal density.

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