Culture medium for culturing immune cells

Abstract

The purpose of the present invention is to provide: a culture medium capable of maintaining a more undifferentiated state in the culture of immune cells, especially T cells; a method for culturing cells using the culture medium; an agent for use in the culture medium or the method; and others. Problems in the conventional T cell therapies, including the exhaustion and depletion of T cells, can be overcome by a culture medium for culturing immune cells or the like according to the present invention, wherein the culture medium contains a fatty acid at a concentration of 0.01-50 mg / L.

Description

Immune cell culture medium

[0001] The present invention relates to cell culture technology, particularly to a culture medium for immune cell culture, a culture method, etc.

[0002] Immune cell-based therapies, such as T cell-based therapies, have been shown to be potent mediators of antitumor responses in both hematological and solid tumors (Non-Patent Documents 1 and 2). However, T cell proliferation and chronic activation can weaken their aggressive capabilities, a phenomenon often referred to as T cell exhaustion or terminal differentiation (Non-Patent Document 3). Immune checkpoint blockade therapy, in vivo expanded tumor-infiltrating lymphocyte (TIL) therapy, chimeric antigen receptor (CAR) T cell therapy, and T cell receptor-engineered (TCR) T cell therapy utilize functionally active T cells isolated from patients and require highly functional T cells for efficacy (Non-Patent Document 4). These T cells are engineered and cultured ex vivo to recognize antigens on target cancer cells. T cell therapy has not been consistently effective in curing solid tumors, partly because T cells lose their proliferation or killing capacity over time (Non-Patent Document 5).

[0003] One approach to overcoming T cell depletion is to selectively administer undifferentiated T cells. For example, stem cell memory T cells (TSCM) exert longer-lasting antitumor effects in patients after administration than more differentiated central memory T cells (TCM) or effector memory T cells (TEM) (Non-Patent Document 6). However, there remains a need in the art for methods to efficiently enrich for less differentiated T cells from a mixed population of isolated T cells.

[0004] Lipids, one of the most important nutrients, are components of cell membranes and signal transducers. In different immune response microenvironments, lipids and lipid metabolic programs can promote or inhibit T cell viability and function (Non-Patent Document 7). Activated effector T (Teff) cells upregulate the assimilation of long-chain fatty acids (LCFA) for proliferation, while memory T (TEM) cells burn LCFA to maintain a relatively quiescent, persistent state (Non-Patent Document 8). Lipids have been particularly well documented in tumor infiltrating lymphocytes (TILs), with CD8+ TILs upregulating fatty acid oxidation (FAO) to maintain tumoricidal activity (Non-Patent Document 9). On the other hand, it has been reported that LCFA accumulated in the tumor microenvironment (TME) can directly promote the exhaustion of CD8+ TILs (Non-Patent Documents 10-12). Therefore, the role of fatty acids in the production of more immature T cells remains unclear.

[0005] Dudley ME, J Cancer. 2011;2:360-2.Rosenberg SA, Nat Rev Cancer. 2008 Apr;8(4):299-308.Blank CU et al., Nat Rev Immunol. 2019 Nov;19(11):665-674.Zhao L et al., Front Immunol. 2019 Oct 11;10:2250.Morotti M et al., Br J Cancer. 2021 May;124(11):1759-1776.Sabatino M et al., Blood. 2016 Jul 28;128(4):519-28.Lim SA et al., Nat Chem Biol. 2022 May;18(5):470-481.Raud B et al., Immunol Rev. 2018 May;283(1):213-231.Kurupati R et al., Cancer Cell. 2017 Sep 11;32(3):377-391.Manzo T et al., J Exp Med. 2020 Aug 3;217(8):e20191920.Ma X et al., Cell Metab. 2021 May 4;33(5):1001-1012.e5.Xu S et al., Immunity. 2021 Jul 13;54(7):1561-1577.

[0006] The present invention aims to provide a culture medium that can maintain immune cells, particularly T cells, in a more undifferentiated state when cultured, a method for culturing cells using the medium, and an agent therefor.

[0007] In view of the above problems, the present inventors focused on the components of the medium used for the expansion and culture of immune cells. Serum-free medium is usually used for the culture of immune cells, but the inventors discovered that by adjusting the amount of fatty acids added in such cases, immune cells can be maintained and expanded in a more undifferentiated state. The inventors succeeded in determining the optimal range of fatty acid amounts, leading to the completion of the present invention. Specifically, the present invention is as follows:

[0008] [1] A medium for culturing immune cells, comprising a fatty acid at a concentration of 0.01 to 50 mg / L. [2] The medium according to [1], wherein the immune cells are T cells. [3] The medium according to [2], wherein the T cells express a stem cell marker. [4] The medium according to [3], wherein the stem cell marker is at least one selected from the group consisting of CD45RA, CD62L, CCR7, CD27, TCF1, and TCF7. [5] The medium according to any of [2] to [4], wherein the T cells do not express or express at a low level an exhaustion marker. [6] The medium according to [5], wherein the exhaustion marker is at least one selected from PD1, TIM3, TIGIT, and LAG3. [7] The medium according to any of [1] to [6], wherein the fatty acid is one or more saturated or unsaturated fatty acids having 12 to 22 carbon atoms. [8] The medium according to any one of [1] to [7], wherein the fatty acid comprises at least one selected from the group consisting of lauric acid, myristic acid, palmitoleic acid, heptadecenoic acid, and stearic acid, and the concentrations of each component in the medium are within the following ranges: lauric acid: 0.01 to 50 mg / L, myristic acid: 0.01 to 50 mg / L, palmitoleic acid: 0.01 to 50 mg / L, heptadecenoic acid: 0.01 to 50 mg / L, stearic acid: 0.01 to 50 mg / L (however, the total concentration of each fatty acid does not exceed 50 mg / L). [8-1] The medium according to any one of [1] to [7], wherein the fatty acid comprises at least one selected from the group consisting of lauric acid, myristic acid, palmitoleic acid, heptadecenoic acid, and stearic acid, and the concentrations of each component in the medium are within the following ranges: lauric acid: 0.01 to 10 mg / L, myristic acid: 0.01 to 0.6 mg / L, palmitoleic acid: 0.01 to 10 mg / L, heptadecenoic acid: 0.01 to 10 mg / L, stearic acid: 0.01 to 10 mg / L.[8-2] The medium according to any one of [1] to [7], wherein the fatty acid comprises at least one selected from the group consisting of lauric acid, myristic acid, palmitoleic acid, heptadecenoic acid, and stearic acid, and the concentrations of each component in the medium are within the following ranges: lauric acid: 0.01 to 10 mg / L, myristic acid: 1.0 to 10 mg / L, palmitoleic acid: 0.01 to 10 mg / L, heptadecenoic acid: 0.01 to 10 mg / L, stearic acid: 0.01 to 10 mg / L. [8-3] The medium according to any one of [1] to [7], wherein the fatty acid comprises at least one selected from the group consisting of lauric acid, myristic acid, palmitoleic acid, heptadecenoic acid, and stearic acid, and the concentrations of each component in the medium are within the following ranges: lauric acid: 0.8 to 3.0 mg / L, myristic acid: 0.01 to 0.6 mg / L, palmitoleic acid: 1.0 to 10 mg / L, heptadecenoic acid: 0.01 to 10 mg / L, and stearic acid: 1.1 to 9.0 mg / L. [9] The medium according to any one of [1] to [8], [8-1], [8-2], and [8-3], wherein the fatty acid comprises at least heptadecenoic acid.

[10] The medium according to any one of [1] to [9], [8-1], [8-2], and [8-3], wherein the immune cells are naive T cells and / or stem cell memory T cells.

[11] A method for producing a medium for immunocyte culture, comprising adding a fatty acid to a basal medium to a concentration of 0.01 to 50 mg / L.

[12] The method according to

[11] , wherein the immunocytes are T cells.

[13] The method according to

[12] , wherein the T cells express a stem cell marker.

[14] The method according to

[13] , wherein the stem cell marker is at least one selected from the group consisting of CD45RA, CD62L, CCR7, CD27, TCF1, and TCF7.

[15] The method according to any of

[12] to

[14] , wherein the T cells do not express or express at a low level an exhaustion marker.

[16] The method according to

[15] , wherein the exhaustion marker is at least one selected from PD1, TIM3, TIGIT, and LAG3.

[17] The method according to any of

[11] to

[16] , wherein the fatty acid is one or more saturated or unsaturated fatty acids having 12 to 22 carbon atoms.

[18] The method according to any one of

[11] to

[17] , wherein the fatty acid comprises at least one selected from the group consisting of lauric acid, myristic acid, palmitoleic acid, heptadecenoic acid, and stearic acid, and the concentrations of each component in the medium are within the following ranges: lauric acid: 0.01 to 50 mg / L, myristic acid: 0.01 to 50 mg / L, palmitoleic acid: 0.01 to 50 mg / L, heptadecenoic acid: 0.01 to 50 mg / L, stearic acid: 0.01 to 50 mg / L (provided that the total concentration of each fatty acid does not exceed 50 mg / L). [18-1] The medium according to any one of

[11] to

[17] , wherein the fatty acid comprises at least one selected from the group consisting of lauric acid, myristic acid, palmitoleic acid, heptadecenoic acid, and stearic acid, and the concentrations of each component in the medium are within the following ranges: lauric acid: 0.01 to 10 mg / L, myristic acid: 0.01 to 0.6 mg / L, palmitoleic acid: 0.01 to 10 mg / L, heptadecenoic acid: 0.01 to 10 mg / L, stearic acid: 0.01 to 10 mg / L. [18-2] The medium according to any one of

[11] to

[17] , wherein the fatty acid comprises at least one selected from the group consisting of lauric acid, myristic acid, palmitoleic acid, heptadecenoic acid, and stearic acid, and the concentrations of each component in the medium are within the following ranges: lauric acid: 0.01 to 10 mg / L, myristic acid: 1.0 to 10 mg / L, palmitoleic acid: 0.01 to 10 mg / L, heptadecenoic acid: 0.01 to 10 mg / L, and stearic acid: 0.01 to 10 mg / L. [18-3] The medium according to any one of

[11] to

[17] , wherein the fatty acids comprise at least one selected from the group consisting of lauric acid, myristic acid, palmitoleic acid, heptadecenoic acid, and stearic acid, and the concentrations of each component in the medium are within the following ranges: Lauric acid: 0.8 to 3.0 mg / L, Myristic acid: 0.01 to 0.6 mg / L, Palmitoleic acid: 1.0 to 10 mg / L, Heptadecenoic acid: 0.01 to 10 mg / L, Stearic acid: 1.1 to 9.0 mg / L.

[19] The method according to any one of

[11] to

[18] , [18-1], [18-2], and [18-3], wherein the fatty acids comprise at least heptadecenoic acid.

[20] The method according to any one of

[11] to

[19] , [18-1], [18-2] and [18-3], wherein the immune cells are naive T cells and / or stem cell memory T cells.

[21] A method for maintaining T cell function, characterized by culturing the immune cells in a medium according to any one of [1] to

[10] , [8-1], [8-2] and [8-3].

[22] The method according to

[21] , wherein the T cells are naive T cells and / or stem cell memory T cells.

[23] The method according to

[21] or

[22] , wherein the functions of the T cells are (1) a high self-renewal ability, (2) a high ability to induce downstream memory T cells and / or effector T cells, and (3) a high immune response ability associated with activation.

[24] A method for suppressing T cell exhaustion, characterized by culturing the immune cells in a medium according to any one of [1] to

[10] , [8-1], [8-2] and [8-3].

[25] The method of

[24] , wherein the T cells are naive T cells and / or stem cell memory T cells.

[26] A method for producing T cells, comprising culturing the T cells in the medium according to any one of [1] to

[10] , [8-1], [8-2] and [8-3].

[27] The method of

[26] , wherein the T cells are naive T cells and / or stem cell memory T cells.

[0009] According to the present invention, immune cells, particularly T cells, can be maintained and proliferated in an undifferentiated state, which makes it possible to produce a large amount of T cells that: 1) have a high self-replication capacity and are long-lived; 2) are highly differentiated T cells that have antitumor effects, etc.; and 3) are quickly activated upon reinfection and can induce an immune response. Therefore, it is possible to overcome the problems associated with previous T cell therapies, such as T cell exhaustion and depletion.

[0010] Fig. 1 is a graph showing the results of examining the total cell number and the proportion of cells expressing stem cell markers (CD62L+, CCR7+) (high stemness T cells) after 6 days of culture of PBMCs in media supplemented with various fatty acids and in media not supplemented with fatty acids. Fig. 2 is a graph showing the results of examining the total cell number and the proportion of cells expressing stem cell markers (CD62L+, CCR7+) (high stemness T cells) after 7 days of culture of PBMCs in media supplemented with various fatty acids and in media not supplemented with fatty acids. Fig. 3 is a graph showing the results of examining the total cell number and the proportion of cells expressing stem cell markers (CD62L+, CCR7+) (high stemness T cells) after 10 days of culture of PBMCs in media supplemented with various fatty acids and in media not supplemented with fatty acids. Figure 4 is a graph showing the results of examining the total cell number and the proportion of stem cell marker-expressing (CD62L+, CCR7+) cells (high stemness T cells) after 6 days of culture of PBMCs in media prepared with varying final concentrations of heptadecenoic acid (C17:1). Figure 5 is a graph showing the results of examining the total cell number and the proportion of stem cell marker-expressing (CD62L+, CCR7+) cells (high stemness T cells) after 6 days of culture of PBMCs in media prepared with the addition of lauric acid (C12:0), myristic acid (C14:0), palmitoleic acid (C16:1), and stearic acid (C18:0).

[0011] The present invention will be described below. Terms used in this specification have the meanings commonly used in the relevant field unless otherwise specified. 1. Cell Culture Medium and Method for Producing the Same The present invention provides a medium for immune cell culture containing a fatty acid (hereinafter also referred to as the cell culture medium of the present invention) and a method for producing the same (hereinafter also referred to as the production method of the present invention). Another embodiment of the present invention contains human serum albumin in addition to the fatty acid. In yet another embodiment of the present invention, the fatty acid is present in the medium in a state supported by human serum albumin.

[0012] (1-1. Fatty Acids) As used herein, "fatty acids" may be either saturated or unsaturated fatty acids. Fatty acids may be short-chain fatty acids (5 carbon atoms or less), medium-chain fatty acids (6 to 12 carbon atoms), long-chain fatty acids (13 to 21 carbon atoms), or very long-chain fatty acids (22 or more carbon atoms). Saturated fatty acids do not have a C═C double bond and include, for example, propionic acid (C3:0), butyric acid (C4:0), valeric acid (C5:0), caproic acid (C6:0), enanthic acid (C7:0), caprylic acid (C8:0), pelargonic acid (C9:0), capric acid (C10:0), undecylic acid (C11:0), lauric acid (C12:0), tridecylic acid (C13:0), myristic acid (C14:0), pentadecylic acid (C15:0), palmitic acid (C16:0), margaric acid (C17:0), stearic acid (C18:0), nonadecylic acid (C19:0), arachidic acid (C20:0), heneicosylic acid (C21:0), behenic acid (C22:0), tricosyl Acids include, but are not limited to, glyceric acid (C23:0), lignoceric acid (C24:0), pentacosylic acid (C25:0), cerotic acid (C26:0), carboselic acid (C27:0), montanic acid (C28:0), nonacosylic acid (C29:0), melissic acid (C30:0), hentriacontylic acid (C31:0), russellic acid (C32:0), psyllic acid (C33:0), gedaic acid (C34:0), ceroplastic acid (C35:0), hexatriacontylic acid (C36:0), heptatriacontylic acid (C37:0), octatriacontylic acid (C38:0), nonatriacontylic acid (C39:0), and tetracontylic acid (C40:0). Preferably, the saturated fatty acid has 8 to 22 carbon atoms, more preferably 12 to 22 carbon atoms (e.g., lauric acid, myristic acid, palmitic acid, stearic acid). The unsaturated fatty acid has a C=C double bond and may be in either a cis or trans configuration.For example, octenoic acid (C8:1), decenoic acid (C10:1), decadienoic acid (C10:2), lauroleic acid (C12:1), lauronolic acid (C12:2), myristovacenic acid (C14:1), myristolinoleic acid (C14:2), myristolinolenic acid (C14:3), farneseic acid (C15:3), palmitrinolenic acid (C16:3), palmitidonic acid (C16:4), α-linolenic acid (C18:3), stearidonic acid (C18:4), dihomo-α-linolenic acid (C20:3), eicosatetraenoic acid (C20:4), and eicosapentaenoic acid (C20:5), clupanodonic acid (C22:5), docosahexaenoic acid (C22:6), 9,12,15,18,21-tetracosapentaenoic acid (C24:5), 6,9,12,15,18,21-tetracosahexaenoic acid (C24:6), myristoleic acid (C14:1), palmitovaccenic acid (C16:1), α-eleostearic acid (C18:3), β-eleostearic acid (trans-C18:3), punicic acid (C18:3), 7,10,13-octadecatrienoic acid (C18:3), 9,12,15-eicosatrienoic acid (C20:3), β- Eicosatetraenoic acid (C20:4), 8-tetradecenoic acid (C14:1), 12-octadecenoic acid (C18:1), linoleic acid (C18:2), linolelaidic acid (trans-C18:2), gamma-linolenic acid (C18:3), calendic acid (C18:3), pinolenic acid (C18:3), dihomo-linoleic acid (C20:2), dihomo-gamma-linolenic acid (C20:3), arachidonic acid (C20:4), adrenic acid (C22:4), osbondic acid (C22:5), palmitoleic acid (C16:1), heptadecenoic acid (17:1), vaccenic acid (C18:1), These include, but are not limited to, rumenic acid (C18:2), paulic acid (C20:1), 7,10,13-eicosatrienoic acid (C20:3), oleic acid (C18:1), elaidic acid (trans-C18:1), gondoic acid (C20:1), erucic acid (C22:1), nervonic acid (C24:1), 8,11-eicosadienoic acid (C20:2), mead acid (C20:3), sapienic acid (C16:1), gadoleic acid (C20:1), 4-hexadecenoic acid (C16:1), petroselinic acid (C18:1), and 8-eicosenoic acid (C20:1).Preferably, unsaturated fatty acids having 8 to 22 carbon atoms are used, and more preferably 12 to 22 carbon atoms (e.g., oleic acid, palmitoleic acid, heptadecenoic acid). The above examples are expressed as "fatty acid name (number of carbon atoms: number of double bonds)."

[0013] The cell culture medium of the present invention is characterized by containing one or more fatty acids in the medium. Examples of fatty acids include those listed above. Preferably, the cell culture medium of the present invention contains multiple types of fatty acids. The concentration of fatty acids in the medium varies depending on the type and combination of fatty acids used, but is characterized by containing fatty acids at a total fatty acid concentration (the concentration of that fatty acid when one type is used, or the total concentration when two or more types are used) of 0.01 mg / L or more, preferably 0.05 mg / L or more, and more preferably 0.1 mg / L or more. Furthermore, the cell culture medium of the present invention can contain fatty acids at a concentration of 50 mg / L or less, preferably 40 mg / L or less, more preferably 30 mg / L or less, even more preferably 20 mg / L or less, and particularly preferably 10 mg / L or less. Furthermore, the cell culture medium of the present invention contains fatty acids at a concentration of 0.01 to 50 mg / L, preferably 0.05 to 50 mg / L, and more preferably 0.1 to 50 mg / L. In another embodiment, the cell culture medium of the present invention contains fatty acids at a concentration of 0.01 to 40 mg / L, preferably 0.05 to 30 mg / L, more preferably 0.1 to 20 mg / L, and particularly preferably 0.1 to 10 mg / L. If the fatty acid concentration in the medium is too high (too much fatty acid), the effect will plateau or will have a negative effect on cell growth, while if the fatty acid concentration is too low (too little fatty acid), sufficient effects will not be obtained, which is undesirable. The cell culture medium of the present invention preferably contains at least one acid selected from the group consisting of lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), heptadecenoic acid (C17:1), stearic acid (C18:0), and oleic acid (C18:1). The cell culture medium of the present invention preferably contains at least one, two, three, four, or more preferably all five fatty acids selected from the group consisting of lauric acid (C12:0), myristic acid (C14:0), palmitoleic acid (C16:1), heptadecenoic acid (C17:1), and stearic acid (C18:0). When two or more fatty acids are contained, the fatty acid concentration refers to the total concentration of the fatty acids. However, preferred concentrations of individual fatty acids include the following ranges.Lauric acid (C12:0): 0.01 to 50 mg / L, preferably 0.01 to 10 mg / L, more preferably 0.8 to 3.0 mg / L Myristic acid (C14:0): 0.01 to 50 mg / L, preferably 0.01 to 10 mg / L, more preferably 0.01 to 0.6 mg / L, 1.0 to 10 mg / L, particularly preferably 0.01 to 0.6 mg / L Palmitoleic acid (C16:1): 0.01 to 50 mg / L, preferably 0.01 to 10 mg / L, more preferably 1.0 to 10 mg / L Heptadecenoic acid (C17:1): 0.01 to 50 mg / L, preferably 0.01 to 10 mg / L Stearic acid (C18:0): 0.01 to 50 mg / L, preferably 0.01 to 10 mg / L, more preferably 1.1 to 9.0 mg / L However, the total concentration of each fatty acid does not exceed 50 mg / L.

[0014] In the cell culture medium of the present invention, fatty acids may be present in a state where they are supported by human serum albumin. The amount of fatty acid supported by human serum albumin is 0.004 to 20 mg, preferably 0.008 to 18 mg, and more preferably 0.04 to 16 mg per gram of albumin. If the amount of fatty acid supported by albumin is too high, the fatty acid concentration in the medium will be too high, adversely affecting cell growth, while if the amount of fatty acid supported by albumin is too low, sufficient effects will not be obtained, which is undesirable. When two or more types of fatty acids are contained, the amounts of each fatty acid supported by albumin may be in the following molar ratios: lauric acid is present in the medium at a molar ratio to albumin ranging from 3.0 to 26.0; myristic acid is present in the medium at a molar ratio to albumin ranging from 5.0 to 10.3; and palmitoleic acid is present in the medium at a molar ratio to albumin ranging from 19.3 to 71.8. Heptadecenoic acid is present in the medium at a molar ratio to albumin ranging from 0.8 to 3.7. Stearic acid is present in the medium at a molar ratio to albumin ranging from 0.6 to 4.2.

[0015] The amount of fatty acids in a culture medium can be measured using a method commonly used in the art or a method based thereon. Fatty acids attached to albumin are measured after isolating the fatty acids from the albumin to free fatty acids. In addition to detection by GC-MS after methyl esterification and quantification by infrared spectroscopy, Duncombe's extraction method and the ACS-ACOD method using acyl-CoA synthetase (ACS) and acyl-CoA oxidase (ACOD) can be used. Commercially available measurement kits can be used for all of these methods.

[0016] (1-2. Albumin) Albumin may be commercially available or prepared in-house. In one embodiment, the albumin is a full-length albumin polypeptide. In another embodiment, the albumin may be a partial peptide or a mutant peptide, as long as it has the desired function, such as functioning as an additive component to a culture medium or carrying fatty acids. Preferably, the albumin is a full-length albumin polypeptide. The albumin may be from any suitable species, and in one embodiment, the albumin is of human origin. In another embodiment, the albumin is of bovine origin. In another embodiment, the albumin is of porcine origin. The albumin may be derived from any source, for example, from blood (e.g., whole blood, plasma, serum). In one embodiment, the albumin is recombinant albumin. Recombinant albumin may be produced in any host cell, including, but not limited to, bacteria, yeast, fungi, plants, mammalian cells, and insect cells. In the present invention, the albumin is preferably human-derived albumin, and more preferably human serum albumin. Human serum albumin may be derived from blood or may be recombinant. Recombinant human serum albumin is more preferred. Albumin may be present in the cell culture medium at any suitable concentration to achieve the desired effect. The albumin concentration (final concentration) in the cell culture medium ranges, for example, from 0.2 mg to 20 g / L, preferably from 0.2 mg to 10 g / L, more preferably from 0.2 mg to 5 g / L, and even more preferably from 0.2 mg to 3 g / L. In another embodiment, the albumin concentration (final concentration) in the cell culture medium ranges, for example, from 0.2 mg to 20 g / L, preferably from 0.5 mg to 10 g / L, more preferably from 0.8 mg to 5 g / L, and even more preferably from 1 mg to 3 g / L. The albumin concentration in the culture medium can be measured using a method commonly used in the art or a method based thereon.

[0017] (1-3. Immune Cells) The type of immune cells to be cultured in the cell culture medium of the present invention is not particularly limited, and examples thereof include antigen-presenting cells, dendritic cells, B cells, and T cells, with T cells being preferred. In the present invention, T cells refer to cells expressing an antigen receptor known as a T cell receptor (TCR) on their surface. Examples of T cells include helper T cells, cytotoxic T cells, regulatory T cells, naive T cells, stem cell memory T cells (TSCM), central memory T cells (TCM), effector memory T cells, and terminal effector T cells, with undifferentiated T cells being preferred. Examples of undifferentiated T cells include, in descending order of degree of undifferentiation, naive T cells, stem cell memory T cells, and central memory T cells. Among these, naive T cells and stem cell memory T cells are particularly preferred from the viewpoint of maintaining the undifferentiated state of the cells and improving their proliferation and biological persistence. Naive T cells refer to T cells in an undifferentiated state that have not yet been presented with an antigen. After naive T cells are activated by antigen presentation, most die, but some become memory T cells in preparation for the next antigen presentation. Stem cell memory T cells are similar to naive T cells, have the longest lifespan, and are thought to retain immunological memory for a long period of time. In the present invention, the term "undifferentiated state" refers to a high expression rate of stem cell markers, and similarly, the term "more undifferentiated" refers to a higher expression rate of stem cell markers. Such a more undifferentiated state with a high expression rate of stem cell markers can be referred to as a high stemness state. For example, T cells that are more undifferentiated and have a high expression rate of stem cell markers are high stemness T cells. A characteristic of naive T cells and stem cell memory T cells is the expression of stem cell markers. Examples of stem cell markers include CD45RA, CD62L, CCR7, CD27, CD28, CD95, IL-2Rβ, BCL-2, CXCR3, TCF1, and TCF7. Preferably, the cells express at least one, two, or three, and preferably all four, stem cell markers selected from the group consisting of CD45RA, CD62L, CCR7, and CD27.In one embodiment of the present invention, the stem cell marker is a combination of CD62L and CCR7. Expression of stem cell markers may mean that 60% or more of a cell population are CCR7+ and CD62L+ (both positive). The immune cells, preferably T cells, more preferably naive T cells and stem cell memory T cells, to be cultured in the cell culture medium of the present invention are highly undifferentiated T cells that express stem cell markers. Highly undifferentiated T cells are characterized by expressing stem cell markers while not expressing or expressing low levels of exhaustion markers. Therefore, the immune cells, preferably T cells, to be cultured in the cell culture medium of the present invention are characterized by not expressing or expressing low levels of exhaustion markers. Examples of exhaustion markers include CD45RO, CD39, CD69, KLRG1, CD122, CD95, PD1, TIM3, TIGIT, and LAG3. Preferably, at least one, two, or three, and preferably all four exhaustion markers selected from the group consisting of PD1, TIM3, TIGIT, and LAG3 are not expressed or are expressed at low levels. Low expression may mean, for example, that less than 30% of the cell population is PD1+ and TIM3+ (both positive) and / or less than 50% is PD1+ and LAG3+ (both positive). Furthermore, because exhausted T cells do not express CD45RO (CD45RO-), T cells expressing CD45RO are also preferred as T cells to be cultured in the cell culture medium of the present invention.

[0018] The presence or absence and intensity of expression of stem cell markers and / or exhaustion markers can be determined by examining the expression of each target factor. By examining the expression status of these markers, immune cells, particularly T cells, preferably stem cell memory T cells, targeted by the present invention can be identified and isolated. Methods for identification and isolation, depending on the expression status of each target factor, include flow cytometry (FACS), column chromatography, magnetic bead panning, Western blotting, radiography, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and hyperdiffusion chromatography, as well as various immunological methods, such as fluid or gel precipitation, immunodiffusion (simple or duplex), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay. FACS is conveniently used.

[0019] As immune cells, T cells, particularly isolated human T cells, are preferably used. Such isolated human T cells can be obtained by treating peripheral blood collected from a blood vessel or by apheresis, preferably peripheral blood collected from a vein, using a conventional method. T cells can be separated from peripheral blood by well-known T cell separation methods, such as discontinuous density gradient centrifugation using sucrose or a commercially available T cell separator. Peripheral blood for T cell isolation can be supplemented with heparin or citrate to prevent blood clotting. The amount of peripheral blood used is not particularly limited and can be appropriately determined based on the burden on the donor, the effort required for blood collection, the T cell separation procedure, and the like.

[0020] As described above, preferred examples of human T cells in the present invention include peripheral blood-derived human T cells (preferably, peripheral blood-derived human T cells that have been cryopreserved and then thawed), and particularly preferred examples of such human T cells include peripheral blood-derived CD3-positive human T cells (preferably, peripheral blood-derived CD3-positive human T cells that have been cryopreserved and then thawed).

[0021] The T cells of the present invention may be T cells derived from pluripotent stem cells. Methods for inducing T cells from pluripotent stem cells can be performed using known methods. Specific examples include the methods described in International Publication No. 2016 / 076415 and International Publication No. 2017 / 221975.

[0022] "Pluripotent stem cells" refer to cells that have the ability to self-replicate and differentiate / proliferate, and have the potential to differentiate into all tissues and cells that constitute a living organism. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), and pluripotent stem cells induced and selected by stress or cell stimulation. Stem cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei are also preferred as pluripotent stem cells. In the present invention, iPS cells are preferred as pluripotent stem cells. Identification of iPS cells can be performed using undifferentiated markers, which are due to the undifferentiated nature of iPS cells. Examples of undifferentiated markers include alkaline phosphatase, Oct3 / 4, Sox2, Nanog, ERas, and Esgl. Methods for detecting these undifferentiated markers include mRNA detection methods (using primers and probes) and immunological detection methods (using antibodies and labels).

[0023] Specifically, the production method of the present invention is carried out by adding a fatty acid to a basal medium. Alternatively, the method may be carried out by adding albumin supported by the fatty acid. Here, the type of fatty acid, the concentration in the medium, and the amount supported on albumin are as described above, and the albumin supported by the fatty acid is added to the basal medium so that these conditions are met.

[0024] As used herein, the term "basal medium" refers to a medium containing carbon sources, nitrogen sources, inorganic salts, and the like that are essential for cell culture. The basal medium that can be used as a component of the cell culture medium of the present invention is not particularly limited and may be appropriately selected depending on the type of cell to be cultured. The basal medium may be prepared by a method known per se, or a commercially available product may be used.

[0025] Usable basal media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium (EMEM), and alpha Modified Eagle's Minimum Essential Medium (alpha Modified Eagle's Minimum Essential Medium). Examples of suitable medium include αMEM, Roswell Park Memorial Institute (RPMI) 1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, and Fischer's Medium.

[0026] Furthermore, examples of basal media that can be used when preparing the cell culture medium of the present invention, particularly for culturing T cells, include IMDM, RPMI1640, and DMEM / F-12.

[0027] Furthermore, in addition to the basal medium, the cell culture medium of the present invention can also contain components that are favorable for cell growth. Such components include, for example, sugars such as glucose, fructose, sucrose, maltose, etc.; amino acids such as asparagine, aspartic acid, glutamine, glutamic acid, etc.; proteins such as albumin, transferrin, etc.; peptides such as glycylglycylglycine, soybean peptide, etc.; serum; vitamins such as choline, vitamin A, B vitamins (thiamine, riboflavin, pyridoxine, cyanocobalamin, biotin, folic acid, pantothenic acid, nicotinamide, etc.), vitamin C, vitamin E, etc.; fatty acids such as oleic acid, arachidonic acid, linoleic acid, etc.; lipids such as cholesterol; inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, sodium dihydrogen phosphate, etc.; trace elements such as zinc, copper, selenium, etc.; N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffering agents such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (BES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and N-[tris(hydroxymethyl)methyl]glycine (Tricine); antibiotics such as amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin; Type I collagen, Type II collagen, and the like; Examples of such components include cell adhesion factors and extracellular matrix components such as collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine; cytokines and growth factors such as interleukin, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), transforming growth factor (TGF)-α, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), and activin A; and hormones such as dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin. Appropriate components can be selected and used depending on the type of cells to be cultured.

[0028] The cell culture medium of the present invention may be provided in a liquid state, or may be prepared in a more concentrated state than the concentration at the time of use, or in a solid state such as a freeze-dried powder, and then diluted with a solvent such as water, or dissolved or dispersed in a solvent such as water, at the time of use.

[0029] 2-1. Cell Culture Method (Maintenance and Proliferation Culture) The present invention also provides a method for culturing immune cells, preferably T cells (hereinafter, sometimes referred to as the "culturing method of the present invention"), which comprises culturing the cells in the cell culture medium of the present invention (described above).

[0030] The culture method of the present invention is not particularly limited as long as it allows immune cells, preferably T cells, to proliferate while maintaining their undifferentiated state. In the case of immune cells, preferably T cells, activation culture may be included as necessary. Activation culture is culture that involves activation with a stimulatory substance. For example, a method of culturing in the cell culture medium of the present invention in the presence of an anti-CD3 antibody is mentioned. Of these, a method of culturing in the cell culture medium of the present invention in the presence of IL-2 (interleukin 2) and an anti-CD3 antibody is preferred. Methods using anti-CD3 antibodies or IL-2 to activate immune cells have been known for some time (e.g., JP-A-3-80076), and these methods can also be used in the culture method of the present invention. For example, a suitable example is a method in which the cell culture medium of the present invention supplemented with IL-2 is placed in a culture vessel on which an anti-CD3 antibody has been immobilized, and cells are cultured by suspending them in the medium.

[0031] The culture period is not particularly limited, but can be, for example, 1 to 20 days, preferably 1 to 15 days, more preferably 1 to 10 days, even more preferably 2 to 8 days, more preferably 3 to 7 days, and even more preferably 4 to 6 days. The frequency of medium replacement during this culture period is not particularly limited, but to prevent deterioration of the medium, it is preferable to replace the medium once every 1 to 7 days, and it is preferable to add new medium in an amount that is about 0.1 to 5 times the volume of the medium before adding the new medium.

[0032] The culture temperature is not particularly limited as long as immune cells, preferably T cells, can grow while maintaining an undifferentiated state. For example, the culture temperature may be 36 to 38°C (preferably 36.5 to 37.5°C), humidity 90 to 98% (preferably 93 to 97%, more preferably 94 to 96%), CO ２ The concentration may be 2 to 13% (preferably 3 to 10%, more preferably 5 to 10%).

[0033] In the above-mentioned expansion culture, there is no particular limitation on the seeding number (seeding concentration) of immune cells used at the start of culture, but if the number of cells at the start of cell culture is too small, there is a disadvantage that it takes a long time for the cell growth curve to rise, and conversely, if the number of cells is too large, there is a disadvantage that the time when cell growth reaches a plateau becomes too early, making it difficult to adopt methods such as adding medium to increase the culture scale, and making it impossible to obtain a sufficient number of cells. Taking these into consideration, the seeding number (seeding concentration) of immune cells used at the start of expansion culture is, for example, 1.0 × 10 ５ ~1.0 x 10 １０ cells / mL, preferably 1.0 x 10 ６ ~5.0 x 10 ９ cells / mL, more preferably 1.0 x 10 ７ ~1.0 x 10 ９ The volume of the culture vessel and the volume of the culture solution can be appropriately selected in consideration of operability, for example, 225 cm ２ Examples include using 50 to 200 mL (preferably 80 to 180 mL, more preferably 100 to 180 mL) of medium in a flask, and using 50 to 1000 mL (preferably 100 to 1000 mL, more preferably 200 to 1000 mL) of medium in a 1000 mL culture bag.

[0034] 2-2. Cell Culture Method (Method for Maintaining T Cell Function, Method for Suppressing T Cell Exhaustion) The culture method of the present invention (described above) allows immune cells, particularly T cells, to proliferate while maintaining their function. T cells are classified based on their degree of differentiation as follows: stem cell memory T cells (TSCM), central memory T cells (TCM), and effector memory T cells (TEM). The culture method of the present invention allows T cells to proliferate while maintaining their undifferentiated state, so seeded T cells can maintain their original function without differentiation after seeding. Furthermore, because the undifferentiated state can be maintained, the culture method of the present invention can also be considered a method for suppressing T cell exhaustion. Stem cell memory T cells (1) have a high self-renewal capacity and are long-lived, (2) can generate other memory T cells and effector T cells, and (3) can be quickly activated upon reinfection and induce an immune response, making them useful as a source of material for T cell therapy, etc. Therefore, stem cell memory T cells are preferred as immune cells to which the "method for maintaining T cell function" and "method for suppressing T cell exhaustion" of the present invention are applied.

[0035] 3. Method for Producing T Cells The present invention also provides a method for producing T cells (hereinafter, sometimes referred to as the "production method of the present invention"), characterized by culturing the cells in the cell culture medium of the present invention (described above). Specifically, the production method of the present invention involves carrying out the culture method of the present invention using the cell culture medium of the present invention, and the obtained T cells maintain an undifferentiated state (suppression of exhaustion) and maintain their function. Therefore, the T cells obtained by the production method of the present invention are preferably naive T cells and / or stem cell memory T cells.

[0036] The present invention will be described in more detail below with reference to examples, but these examples are not intended to limit the scope of the present invention in any way.

[0037] (Fatty Acids) The fatty acids used in this example are as follows: 12:0 Lauric acid (C12:0) 14:0 Myristic acid (C14:0) 16:1 Palmitoleic acid (C16:1) 18:0 Stearic acid (C18:0) 17:1 Heptadecenoic acid (C17:1)

[0038] (Activated Charcoal Purification of Albumin) 0.5 N HCl was added to a human serum albumin solution to adjust the pH to 3.9. Activated charcoal (50 wt% vs. HSA, GW_1) was added and the mixture was stirred at 4°C for 3 hours, followed by centrifugation at 12,000 x g for 60 minutes at 4°C. The precipitated activated charcoal was removed by decantation, and 0.5 N NaOH was added to adjust the pH to 6.9. The mixture was filtered once with a 0.45 μm syringe filter and three times with a 0.1 μm syringe filter.

[0039] (Fatty acid loading of albumin) The method for preparing a fatty acid loaded albumin solution consists of the following steps: 1) Dissolve 1 to 100 mg of each fatty acid in 1 ml of absolute ethanol (preparation of fatty acid-ethanol solution), 2) Dilute the fatty acid-ethanol solution prepared by method 1) above with sterile water to prepare a solution with a final concentration of 0.01 to 100 g / L (preparation of fatty acid solution), 3) Add the fatty acid solution prepared by method 2) above to the albumin solution so that the final target concentration in the medium is reached, and 4) Mix using a rotator at room temperature for 2 hours.

[0040] (Culture Evaluation) The proliferation effect of high-stemness T cells when using each test medium was evaluated. PBMCs purchased from Lonza Co., Ltd. were used. The culture process was as follows: 1) PBS containing anti-CD3 antibody (5 μg / ml, BD Bioscoence) and RetroNectin (25 μg / ml, Takarabio) was placed in a culture plate and incubated at 37°C and 5% CO ２ The plates were incubated in an incubator for 2 hours for coating. 2) The entire coating solution was removed, and the plates were washed three times with 1 mL of PBS. 3) A cell suspension in each test medium was added to the plate, and IL-2 (200 U / ml, Peprotech) was added. 4) The plates were incubated at 37°C, 5% CO ２The cells were cultured in an incubator. 5) On days 4 to 10 of culture, the cells were collected by centrifugation, and the cell count was measured using a nucleocounter, and the cell expression markers CD62L and CCR7 (both stem cell markers) were detected by flow cytometry. 6) The value of "number of viable cells (cells) x stem cell marker positivity rate (CCR7+, CD62L+ (both positive))" on the evaluation day was used as the number of highly stem T cells, and a relative evaluation was performed. When evaluating the exhaustion marker positivity rate, after 4), 5') on days 4 to 10 of culture, the cells were collected by centrifugation, and the cell expression markers CD3 and LAG3 were detected by flow cytometry. 6') The percentage of LAG3+ (exhaustion marker) cells among CD3+ cells on the day of evaluation (CD3+ and LAG3+ (both positive)) was defined as the exhaustion marker positivity rate (%). The medium used was a basal medium IMDM supplemented with ethanolamine, N-acetyl-L-cysteine, sodium selenite, human insulin, and human transferrin (medium A).

[0041] Example 1: Test media were prepared by adding human serum albumin (2.5 g / L) and various fatty acids (Sigma-Aldrich) to Medium A. A control medium was prepared by adding only human serum albumin (2.5 g / L) to Medium A without adding any fatty acids (FA-free). The final concentrations of various fatty acids in each test medium are shown in Table 1. Culture results were compared using each test medium. For (1), HSA from Oriental Yeast Co., Ltd. (OYC) was used, and for (2), HSA from Sigma-Aldrich was used. Activated carbon purification was performed to remove fatty acids (Chen, The Journal of Biological Chemistry Vol. 242, Issue of January 25, pp. 173-181, 1967). 300 μL of coating solution was added per well of a 24-well plate, and 60,000 cells were seeded. The culture period was 6 days. The results are shown in Table 1 and Figure 1. When fatty acids (FA) were not added (FA-free), high-stemness T cells did not proliferate. However, when fatty acids were added, high-stemness T cells proliferated. These results demonstrated that fatty acids contained in the medium positively contribute to the proliferation of high-stemness T cells.

[0042]

[0043] Example 2: Culture results were compared using media prepared by adding C12:0, C14:0, C16:1, C18:0, and C17:1 fatty acids to Medium A at final concentrations as shown in Table 2 (1) to (6). Albumin was prepared using HSA from Oriental Yeast Co., Ltd., and activated carbon purification was performed to remove fatty acids. 300 μL of coating solution was added per well of a 24-well plate, and 60,000 cells were seeded. The culture period was 7 days. The results are shown in Table 2 and Figure 2. Cell proliferation was not observed without the addition of fatty acids. However, the addition of each fatty acid alone resulted in cell proliferation and the production of highly stemmed T cells. These results demonstrated that even a single fatty acid contained in the medium is effective in promoting the proliferation of highly stemmed T cells. Furthermore, the effect was further enhanced by adding a combination of fatty acids to the medium at high concentrations, as in (5) and (6).

[0044]

[0045] Example 3: Culture results were compared using media prepared by adding C12:0, C14:0, C16:1, and C18:0 to Medium A so that the final concentrations of each fatty acid were three points for each fatty acid, as shown in Table 3 (1) to (12). Albumin was HSA from Oriental Yeast Co., Ltd., and purified with activated carbon to remove fatty acids. 300 μL of coating solution was added per well of a 24-well plate, and 100,000 cells were seeded. The culture period was 10 days. The results are shown in Table 3 and Figure 3.

[0046]

[0047] Example 4: Culture results were compared using media prepared by adding C17:1 to Medium A at final concentrations of fatty acids as shown in Table 4 (1) to (4). Oriental Yeast Co., Ltd. (OYC) HSA was used for (1) and (2), and Sigma-Aldrich HSA for (3) and (4). Activated carbon purification was performed to remove fatty acids. 300 μL of coating solution was added per well of a 24-well plate, and 60,000 cells were seeded. The culture period was 6 days. The results are shown in Table 4 and Figure 4. Cell proliferation was not observed without the addition of fatty acids. However, the addition of C17:1 at high concentrations in (2) and (4) resulted in the proliferation of highly stemmed T cells. These results demonstrate that the addition of a single C17:1 medium is effective in promoting the proliferation of highly stemmed T cells.

[0048]

[0049] Example 5: Culture results were compared using media prepared by adding C12:0, C14:0, C16:1, and C18:0 to Medium A so that the final concentrations of each fatty acid were 2 points for each fatty acid, as shown in Tables 5(1) to 5(8). Albumin was HSA from Oriental Yeast Co., Ltd., and activated carbon purification was performed to remove fatty acids. 300 μL of coating solution was added per well of a 24-well plate, and 100,000 cells were seeded. The culture period was 6 days. The results are shown in Table 5 and Figure 5.

[0050]

[0051] Example 6: Culture results were compared using media prepared by adding the final concentrations of C12:0, C14:0, C16:1, C18:0, and C17:1 fatty acids to Medium A as shown in Table 6 (1). Albumin was prepared using HSA from Oriental Yeast Co., Ltd., and activated carbon purification was performed to remove fatty acids. 300 μL of coating solution was added per well of a 24-well plate, and 100,000 cells were seeded. The culture period was 6 days. The results are shown in Table 6. Compared to the group in which fatty acids were completely removed, the addition of fatty acids was shown to reduce the proportion of exhausted cells (CD3+ / LAG3+).

[0052]

[0053] Table 7 shows the results of a comparison of the "number of viable cells (cells) x stemness marker positivity rate (CCR7+, CD62L+ (both positive))" under each condition.

[0054]

[0055] According to the present invention, immune cells, particularly T cells, can be maintained and proliferated in an undifferentiated state, thereby making it possible to produce large quantities of T cells that: 1) have high self-replication capacity and longevity; 2) produce highly differentiated T cells with antitumor effects, etc.; and 3) can be rapidly activated upon reinfection and induce an immune response. This overcomes the problems associated with previous T cell therapies, such as T cell exhaustion and depletion. This application is based on Patent Application No. 2023-166529 filed in Japan (filing date: September 27, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A medium for culturing immune cells, comprising a fatty acid at a concentration of 0.01 to 50 mg / L.

2. The medium of claim 1, wherein the immune cells are T cells.

3. The medium according to claim 2, wherein the T cells express a stem cell marker.

4. The medium according to claim 3, wherein the stemness marker is at least one selected from the group consisting of CD45RA, CD62L, CCR7, CD27, TCF1 and TCF7.

5. The medium according to claim 2, characterized in that the T cells do not express or express low levels of exhaustion markers.

6. The medium according to claim 5, wherein the exhaustion marker is at least one selected from the group consisting of PD1, TIM3, TIGIT and LAG3.

7. The medium according to claim 1, wherein the fatty acid is one or more saturated or unsaturated fatty acids having 12 to 22 carbon atoms.

8. The medium according to claim 7, wherein the fatty acid comprises at least one selected from the group consisting of lauric acid, myristic acid, palmitoleic acid, heptadecenoic acid and stearic acid, and the concentrations of each component in the medium are within the following ranges: lauric acid: 0.01-50 mg / L, myristic acid: 0.01-50 mg / L, palmitoleic acid: 0.01-50 mg / L, heptadecenoic acid: 0.01-50 mg / L, stearic acid: 0.01-50 mg / L (however, the total concentration of each fatty acid does not exceed 50 mg / L).

9. The medium according to claim 7, wherein the fatty acids include at least heptadecenoic acid.

10. The medium according to any one of claims 1 to 9, wherein the immune cells are naive T cells and / or stem cell memory T cells.

11. A method for producing a medium for culturing immune cells, comprising adding a fatty acid to a basal medium to a concentration of 0.01 to 50 mg / L.

12. The method of claim 11, wherein the immune cell is a T cell.

13. The method according to claim 12, characterized in that the T cells express stem cell markers.

14. The method according to claim 13, wherein the stemness marker is at least one selected from the group consisting of CD45RA, CD62L, CCR7, CD27, TCF1 and TCF7.

15. The method according to claim 12, characterized in that the T cells do not express or express low levels of exhaustion markers.

16. The method of claim 15, wherein the exhaustion marker is at least one selected from the group consisting of PD1, TIM3, TIGIT and LAG3.

17. The method according to claim 11, wherein the fatty acid is one or more saturated or unsaturated fatty acids having 12 to 22 carbon atoms.

18. The method according to claim 17, wherein the fatty acid comprises at least one selected from the group consisting of lauric acid, myristic acid, palmitoleic acid, heptadecenoic acid, and stearic acid, and the concentrations of each component in the medium are within the following ranges: lauric acid: 0.01-50 mg / L, myristic acid: 0.01-50 mg / L, palmitoleic acid: 0.01-50 mg / L, heptadecenoic acid: 0.01-50 mg / L, stearic acid: 0.01-50 mg / L (however, the total concentration of each fatty acid does not exceed 50 mg / L).

19. The method of claim 18, wherein the fatty acid comprises at least heptadecenoic acid.

20. The method according to any one of claims 11 to 19, wherein the immune cells are naive T cells and / or stem cell memory T cells.

21. A method for maintaining the function of T cells, comprising culturing the cells in a medium according to any one of claims 1 to 10.

22. The method of claim 21, wherein the T cells are naive T cells and / or stem cell memory T cells.

23. The method according to claim 21, wherein the functions of the T cells are (1) high self-renewal ability, (2) high ability to induce downstream memory T cells and / or effector T cells, and (3) high immune response ability upon activation.

24. A method for suppressing exhaustion of T cells, comprising culturing the cells in a medium according to any one of claims 1 to 10.

25. The method of claim 24, wherein the T cells are naive T cells and / or stem cell memory T cells.

26. A method for producing T cells, comprising culturing the cells in a medium according to any one of claims 1 to 10.

27. The method of claim 26, wherein the T cells are naive T cells and / or stem cell memory T cells.

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