Liquid culture medium for preparing human lymphocyte population containing human NK cells

Abstract

The present invention addresses the problem of providing: a liquid culture medium capable of specifically increasing the proportion and number of human NK cells in a human lymphocyte population; and others. Provided is a basal liquid culture medium for culturing human cells. The medium contains human albumin, a selenious acid compound, a buffer, and human serum, and is used as a liquid culture medium for preparing a human lymphocyte population containing human NK cells.

Description

Culture medium for preparing human lymphocyte cell populations, including human NK cells.

[0001] The present invention relates to a culture medium for preparing a population of human lymphocytes including human NK (Natural Killer) cells, comprising the essential components and human serum; a method for preparing a population of human lymphocytes including human NK cells, comprising culturing the human lymphocyte population in the culture medium; and a kit for preparing the culture medium.

[0002] Malignant tumors are the leading cause of death in Japan, making countermeasures an urgent necessity. In particular, advanced, refractory malignant tumors are resistant to existing treatments such as surgery, radiation therapy, and chemotherapy, making the development of new treatment methods extremely important. In recent years, immunotherapy, such as immune checkpoint inhibitors and therapy using chimeric antigen receptor (CAR) gene-modified T cells (CAR-T therapy), has attracted attention as a fourth treatment method. However, since most of these use T cells that are activated by recognizing antigens as effectors, there is a fundamental barrier: restriction to specific antigens.

[0003] NK cell therapy, which uses NK cells as a major factor in innate immunity, is attracting attention as a treatment method with relatively few side effects. This therapy involves proliferating NK cells in vitro before administering them to the patient. However, challenges remain, as the number of NK cells obtainable from peripheral blood, etc., is relatively small, and their proliferative capacity in vitro is low. Therefore, techniques for culturing and proliferating NK cells have been investigated. For example, it has been reported that NK cells can be amplified with high purity by removing T cells from a cell population containing NK cells and then culturing them in a culture medium containing a specific concentration of IL-2 (Patent Document 1). Furthermore, a method has been reported for preparing NK cells by amplifying hematopoietic progenitor cells under a single culture condition containing IL-15, SCF, IL-7, and Flt3L, and then inducing differentiation into NK cells under culture conditions containing IL-2 (Patent Document 2). Furthermore, it has been reported that when hematopoietic mononuclear cells from which T cells have been removed are suspended in a serum-free culture medium containing ultra-high concentrations of IL-2 and IL-18 to prepare a cell suspension, and then cultured in a culture vessel coated with anti-human NKp46 antibody, the amplification factor of NK cells is enhanced (Patent Document 3).

[0004] On the other hand, the present inventors have reported a serum-free culture medium (iMedium® in this embodiment) that can increase the proliferation efficiency of human lymphocyte cell populations (Patent Document 4). However, it was previously unknown that adding human serum to iMedium can specifically increase the proportion and number of NK cells in the human lymphocyte cell population.

[0005] Japanese Patent Publication No. 2013-027385, Japanese Patent Publication No. 2014-226079, Japanese Patent Publication No. 2023-103632, International Publication No. 2020 / 044538 (brochure)

[0006] The object of the present invention is to provide a culture medium or the like that can specifically increase the proportion and number of human NK cells in a human lymphocyte cell population.

[0007] The inventors of this invention have been diligently conducting research to solve the above problems. In the process, they happened to add human serum, which is not necessary to be added, to iMedium, a serum-free culture medium, and found that when a population of human lymphocytes was cultured in iMedium containing human serum, the proportion and number of human NK cells in the human lymphocyte population could be specifically increased compared to when the population of human lymphocytes was cultured in iMedium alone. This led to the completion of the present invention.

[0008] In other words, the present invention is as follows: [1] A culture medium for preparing a human lymphocyte cell population including human NK cells, comprising a basic culture medium for human cell culture comprising human albumin, a selenite compound, and a buffer, and human serum. [2] The culture medium for preparation according to [1] above, further comprising one or more selected from the group consisting of human transferrin, human insulin, glutathione, ascorbic acid phosphate or a salt thereof, ethanolamine, pyruvate, L-alanyl-L-glutamine, acetic acid or a salt thereof, and antibiotics. [3] The culture medium for preparation according to [1] or [2] above, wherein the buffer is one or two selected from the group consisting of sodium bicarbonate and HEPES. [4] A method for preparing a human lymphocyte cell population including human NK cells, comprising culturing the human lymphocyte cell population in the presence of the culture medium for preparation according to any one of [1] to [3] above. [5] A kit for preparing a culture medium for preparing a population of human lymphocyte cells including human NK cells, comprising human albumin, a selenite compound, and a buffer, wherein the culture medium for preparation comprises a basic culture medium for human cell culture comprising the human albumin, the selenite compound, and the buffer, and human serum. [6] The kit according to [5], further comprising human serum and a basic culture medium for human cell culture. [7] The kit according to [5] or [6], further comprising one or more selected from the group consisting of human transferrin, human insulin, glutathione, ascorbic acid phosphate or a salt thereof, ethanolamine, pyruvate, L-alanyl-L-glutamine, acetic acid or a salt thereof, and antibiotics. [8] The kit according to any one of [5] to [7], wherein the buffer is one or two selected from the group consisting of sodium bicarbonate and HEPES.

[0009] According to the present invention, the proportion and number of human NK cells in a human lymphocyte population can be specifically increased. Human NK cells not only have the function of directly attacking tumor cells and virus-infected cells, but recent research has also revealed that they play a role in eliminating senescent cells. Therefore, increasing the proportion and number of human NK cells in a human lymphocyte population is useful for immunotherapy for diseases such as cancer, viral infections, and age-related diseases (e.g., Alzheimer's disease).

[0010] This figure shows the results of measuring the proportion of T cells and NK cells in a human lymphocyte population before culture. This figure shows the results of measuring the proportion of T cells in a human lymphocyte population after culture using three types of culture media (iMedium without human serum ["0%HS iMedium" in the figure], iMedium containing 5% human serum ["5%HS iMedium" in the figure], or iMedium containing 10% human serum ["10%HS iMedium" in the figure]). The "*" in the figure indicates a statistically significant difference (p < 0.05) (the same applies below). This figure shows the results of measuring the proportion of NK cells in a human lymphocyte population after culture using the three types of culture media mentioned above. This figure shows the results of measuring the total number of cells in a human lymphocyte population after culture using the three types of culture media mentioned above.

[0011] <Culture medium for NK cell preparation> The culture medium for preparation of the present invention is not particularly limited as long as it is a basic culture medium for human cell culture (hereinafter referred to as "the culture medium for NK cell preparation") that contains human albumin, selenite compounds, and buffers (collectively referred to as "the essential components" in this specification) and human serum, and is specified for the use of "preparing a human lymphocyte cell population including human NK cells". The culture medium for NK cell preparation can be prepared by adding the essential components in solid form (e.g., powder, granules, etc.), a solid basic culture medium for human cell culture, and human serum to water, or by adding the essential components in solid form and human serum to a basic culture medium for human cell culture. When a human lymphocyte cell population is cultured using the culture medium for NK cell preparation, the proportion and number of human NK cells contained in the human lymphocyte cell population can be specifically increased.

[0012] <Human Albumin> In this invention, the human albumin may be naturally derived human albumin (for example, human plasma-derived albumin), or a human-type recombinant albumin (recombinant human albumin [rHSA]), and may be commercially available or prepared in-house. Examples of commercially available rHSAs include genetically modified rice-derived products such as Sigma Aldrich's A9731 (model number), Science Research Laboratory's OsrHSA-10 (model number), Healthgen Biotechnology's HY01E-10g (model number), eEnzyme®'s HSA-1r (model number), and BioVerde's IBK-A1-10 (model number); and genetically modified yeast-derived products such as Sigma Aldrich's A7223 (model number), A6608 (model number), A7736 (model number), and Novozymes' Albucult® (product name), Recombumin alpha® (product name), and AlbIX® (product name). Furthermore, commercially available human plasma-derived albumins include, for example, CSL Behring's "Albumina-20% Injection" (product name) and "Albumina-25% Injection" (product name), Sigma Aldrich's A1887 (model number), A1653 (model number), A9511 (model number), A3782 (model number), A8763 (model number), A4327 (model number), and Biological Industries' Bio-Pure HSA 10% Solution (product name).

[0013] The concentration of human albumin in the culture medium for NK cell preparation is not particularly limited. Examples of lower limits include 0.1 g / L or higher, 0.3 g / L or higher, 0.6 g / L or higher, 0.9 g / L or higher, 1 g / L or higher, etc., and examples of upper limits include 6 g / L or lower, 5 g / L or lower, 4 g / L or lower, 3 g / L or lower, etc. These lower and upper limits can be combined in any way.

[0014] <Selenite Compounds> Examples of selenite compounds in the present invention include selenite or a salt thereof. Examples of such selenite salts include sodium selenite, potassium selenite, magnesium selenite, calcium selenite, and the like.

[0015] The concentration of selenite compound in the culture medium for NK cell preparation is not particularly limited. Examples of lower limits include 0.6 μg / L or higher, 1.0 μg / L or higher, 2.0 μg / L or higher, 3.0 μg / L or higher, 4.0 μg / L or higher, 5.0 μg / L or higher, 6.0 μg / L or higher, etc. Examples of upper limits include 60 μg / L or lower, 30 μg / L or lower, 20 μg / L or lower, 15 μg / L or lower, 10 μg / L or lower, etc. These lower and upper limits can be combined arbitrarily.

[0016] <Buffering Agent> The buffering agent in this invention can be any substance that has the effect of mitigating pH fluctuations caused by the addition of acids or bases. Examples include sodium bicarbonate, PBS (Phosphate buffered saline), TBS (Tris buffered saline), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), etc., with sodium bicarbonate and / or HEPES being preferred. The buffering agent may be a solid or a liquid (buffer solution). If the buffering agent is a solid, a buffer solution can be prepared by adding it to a solvent such as water.

[0017] The concentration of the buffer in the culture medium for NK cell preparation cannot be specified in general terms, as it depends on the type of buffer, etc. However, possible lower limits include, for example, 0.2 g / L or more, 0.4 g / L or more, 0.6 g / L or more, 0.8 g / L or more, 1.0 g / L or more, 2.0 g / L or more, etc., and possible upper limits include, for example, 40 g / L or less, 20 g / L or less, 10 g / L or less, 8 g / L or less, 6 g / L or less, 4 g / L or less, etc. These lower and upper limits can be combined arbitrarily.

[0018] <Human Serum> In the present invention, human serum may be serum prepared from human blood (i.e., blood that has been coagulated and coagulation factors removed), and human serum can be obtained by a blood separation operation using beads coated with blood coagulation-promoting enzymes.

[0019] The concentration of human serum in the culture medium for NK cell preparation is not particularly limited. Examples of lower limits include 0.5% or more, 1.0% or more, 1.5% or more, 2.0% or more, 2.5% or more, 3.0% or more, 3.5% or more, 4.0% or more, etc. Examples of upper limits include 20% or less, 18% or less, 16% or less, 14% or less, 12% or less, etc. These lower and upper limits can be combined arbitrarily.

[0020] <Basic Culture Medium for Human Cell Culture> In this specification, "basic culture medium for human cell culture" can be any culture medium containing nutrients necessary for the proliferation of human cells, for example, DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12), DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's minimal essential medium), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's Minimum Essential Medium), RPMI-1640 (Roswell Park Memorial Institute 1640 Medium), α-MEM (Alpha modification of Eagle's minimal essential medium), Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F-12K, ATCC-CRCM30, BME (Basal Medium Eagle), Fischer's medium, McCoy's 5A medium, Leibovitz's L-15 medium, RITC80-7 medium, MCDB105 medium, MCDB107 medium, MCDB153 medium, MCDB201 medium, NCTC109 medium, NCTC135 medium, Waymouth'sMB 752 / 1 medium, Williams' medium E, and ReproFF2 cell Examples include one culture medium or a mixture of two or more culture media selected from the group consisting of culturemedium (ReproCELL, Inc.). In addition, as a basic culture medium for human cell culture, a culture medium modified for human stem cell culture or a mixture of a basic culture medium for human cell culture and other culture media may be used.

[0021] <Human Lymphocyte Cell Population> In this specification, "human lymphocyte cell population" means a cell population containing human lymphocyte cells. Examples of human lymphocyte cell populations include lymphocyte cell populations collected from humans, lymphocyte cell populations proliferated from lymphocyte cell populations collected from humans, and human lymphocyte cell populations differentiated from stem cells such as iPS cells. Human lymphocyte cell populations include human NK cells, human T cells, human B cells, and human monocytes. Examples of human lymphocyte cell populations include peripheral blood-derived lymphocyte cell populations, umbilical cord blood-derived lymphocyte cell populations, and bone marrow-derived lymphocyte cell populations. Peripheral blood-derived lymphocyte cell populations are preferred due to the ease and availability of collection. Furthermore, human lymphocyte cell populations may also be human lymphocyte cell populations obtained by thawing cryopreserved human lymphocyte cell populations.

[0022] In this specification, "human NK cells" means large granular lymphocytes of human origin that do not express the T cell receptor (TCR), the universal T cell marker CD3, and the membrane immunoglobulin B cell receptor, and are typically CD16-positive and CD56-positive. Whether or not a cell is an NK cell can be easily determined by a person skilled in the art based on the expression pattern of cell surface markers, etc. NK cells have cytotoxic activity, and the presence and degree of this cytotoxic activity can be measured by various known methods.

[0023] In this specification, "human T cell" means a cell that expresses TCR and is usually CD3-positive.

[0024] The culture medium for NK cell preparation may contain optional components in addition to the essential components (for example, stabilizers, isotonic agents, pH adjusters, reducing agents, organic solvents, physiologically active substances, cell growth factors, cytokines [for example, IL-2, IL-15, etc.], solvents, etc.), and preferred optional components include one or more selected from the group consisting of human transferrin, human insulin, glutathione, ascorbic acid phosphate ester or salt thereof, ethanolamine, pyruvate, L-alanyl-L-glutamine, acetic acid or salt thereof, and antibiotics (hereinafter referred to as "the optional components").

[0025] <Human Transferrin> In the present invention, human transferrin may be naturally derived human transferrin, or human-type genetically modified transferrin (recombinant human transferrin), and may be commercially available or prepared in-house. Examples of commercially available recombinant human transferrins include products derived from recombinant rice such as Fujifilm Wako Pure Chemical Industries' "Transferrin, Human, Recombinant" 205-18084 (model number), Sigma Aldrich's T3705 (model number), and PROSPEC's PRO-747 (model number). Examples of commercially available human plasma-derived transferrins include Fujifilm Wako Pure Chemical Industries' "Transferrin (Holo), Human Blood Derived" 208-18971 (model number), Sigma Aldrich's 90190 (model number), etc.

[0026] The concentration of human transferrin in the culture medium for NK cell preparation is not particularly limited. Examples of lower limits include 0.1 mg / L or higher, 0.2 mg / L or higher, 0.3 mg / L or higher, 0.4 mg / L or higher, 0.5 mg / L or higher, 0.6 mg / L or higher, 0.7 mg / L or higher, 0.8 mg / L or higher, etc. Examples of upper limits include 10 mg / L or lower, 9.0 mg / L or lower, 8.0 mg / L or lower, 7.0 mg / L or lower, 6.0 mg / L or lower, 5.0 mg / L or lower, 4.0 mg / L or lower, 3.0 mg / L or lower, 2.0 mg / L or lower, etc. These lower and upper limits can be combined arbitrarily.

[0027] <Human Insulin> In the present invention, human insulin can be, for example, human-type recombinant insulin (recombinant human insulin), which may be commercially available or prepared in-house. Examples of commercially available recombinant human insulin include recombinant yeast-derived products such as Fujifilm Wako Pure Chemical Industries 094-06484 (model number) and Sigma Aldrich I9278 (model number); and recombinant Escherichia coli-derived products such as Cell Prime®'s r Insulin.

[0028] The concentration of human insulin in the culture medium for NK cell preparation is not particularly limited. Examples of lower limits include 0.1 mg / L or higher, 0.2 mg / L or higher, 0.3 mg / L or higher, 0.4 mg / L or higher, 0.5 mg / L or higher, 0.6 mg / L or higher, 0.7 mg / L or higher, 0.8 mg / L or higher, etc. Examples of upper limits include 10 mg / L or less, 9.0 mg / L or less, 8.0 mg / L or less, 7.0 mg / L or less, 6.0 mg / L or less, 5.0 mg / L or less, 4.0 mg / L or less, 3.0 mg / L or less, 2.0 mg / L or less, etc. These lower and upper limits can be combined arbitrarily.

[0029] <Glutathione> The glutathione used in this invention may be commercially available or prepared in-house. Examples of commercially available glutathione include Fujifilm Wako Pure Chemical Industries 073-02013 (model number) and Sigma Aldrich G4251 (model number).

[0030] The concentration of glutathione in the culture medium for NK cell preparation is not particularly limited. Examples of lower limits include 0.3 mg / L or higher, 0.6 mg / L or higher, 1.0 mg / L or higher, 1.3 mg / L or higher, 1.6 mg / L or higher, 2.0 mg / L or higher, 2.3 mg / L or higher, 2.6 mg / L or higher, etc. Examples of upper limits include 30 mg / L or lower, 20 mg / L or lower, 10 mg / L or lower, 8.0 mg / L or lower, 6.0 mg / L or lower, 4.0 mg / L or lower, etc. These lower and upper limits can be combined arbitrarily.

[0031] <Ascorbic acid phosphate ester or salt thereof> Examples of ascorbic acid phosphate ester salts used in the present invention include magnesium salt, sodium salt, calcium salt, potassium salt of ascorbic acid phosphate ester, etc. One or more selected from the group consisting of ascorbic acid phosphate ester and salts thereof may be used as the ascorbic acid phosphate ester or salt thereof. The ascorbic acid phosphate ester or salt thereof may be a commercially available product or one that has been prepared in-house. Examples of commercially available magnesium salts of ascorbic acid phosphate include Fujifilm Wako Pure Chemical Industries 013-19641 (model number) (L-ascorbic acid phosphate magnesium salt n hydrate) and Sigma Aldrich A8960 (model number) (L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate). Examples of commercially available sodium salts of ascorbic acid phosphate include Sigma Aldrich 49752 (model number) (2-phospho-L-ascorbic acid trisodium salt).

[0032] The concentration of ascorbic acid phosphate ester or its salt in the culture medium for NK cell preparation is not particularly limited. Examples of lower limits include 2.7 mg / L or more, 3.0 mg / L or more, 6.0 mg / L or more, 10 mg / L or more, 13 mg / L or more, 16 mg / L or more, 20 mg / L or more, 23 mg / L or more, 26 mg / L or more, etc. Examples of upper limits include 270 mg / L or less, 230 mg / L or less, 200 mg / L or less, 160 mg / L or less, 130 mg / L or less, 100 mg / L or less, 80 mg / L or less, 60 mg / L or less, 40 mg / L or less, 30 mg / L or less, etc. These lower and upper limits can be combined in any way.

[0033] <Ethanolamine> Examples of ethanolamines used in the present invention include monoethanolamine, diethanolamine, and triethanolamine, with monoethanolamine being preferred. Alternatively, one or more ethanolamines selected from the group consisting of monoethanolamine, diethanolamine, and triethanolamine may be used. The ethanolamine may be a commercially available product or one prepared in-house. Examples of commercially available monoethanolamines include Fujifilm Wako Pure Chemical Industries, Ltd. 016-12453 (model number) and Sigma Aldrich E9508 (model number). An example of a commercially available diethanolamine is Tokyo Chemical Industry Co., Ltd. S0376 (model number). An example of a commercially available triethanolamine is Tokyo Chemical Industry Co., Ltd. S0377 (model number).

[0034] The concentration of ethanolamine in the herein culture solution for NK cell preparation is not particularly limited. The lower limit value can be, for example, 1.0 mg / L or more, 3.0 mg / L or more, 6.0 mg / L or more, 8.0 mg / L or more, 10 mg / L or more, 11 mg / L or more, etc., and the upper limit value can be, for example, 123 mg / L or less, 100 mg / L or less, 80 mg / L or less, 60 mg / L or less, 40 mg / L or less, 30 mg / L or less, 20 mg / L or less, 15 mg / L or less, etc. The lower limit values and the upper limit values can be arbitrarily combined, respectively.

[0035] <Pyruvate> Examples of the pyruvate in the present invention include sodium pyruvate, potassium pyruvate, magnesium pyruvate, calcium pyruvate, and the like.

[0036] The concentration of pyruvate in the herein culture solution for NK cell preparation is not particularly limited. The lower limit value can be, for example, 0.010 g / L or more, 0.020 g / L or more, 0.040 g / L or more, 0.060 g / L or more, 0.080 g / L or more, 0.10 g / L or more, etc., and the upper limit value can be, for example, 1.1 g / L or less, 1.0 g / L or less, 0.8 g / L or less, 0.6 g / L or less, 0.4 g / L or less, 0.3 g / L or less, 0.2 g / L or less, etc. The lower limit values and the upper limit values can be arbitrarily combined, respectively.

[0037] <L-alanyl-L-glutamine> The concentration of L-alanyl-L-glutamine in the herein culture solution for NK cell preparation is not particularly limited. The lower limit value can be, for example, 0.050 g / L or more, 0.10 g / L or more, 0.15 g / L or more, 0.20 g / L or more, 0.25 g / L or more, 0.30 g / L or more, 0.35 g / L or more, 0.40 g / L or more, 0.45 g / L or more, etc., and the upper limit value can be, for example, 5.0 g / L or less, 4.5 g / L or less, 3.0 g / L or less, 2.5 g / L or less, 2.0 g / L or less, 1.5 g / L or less, 1.0 g / L or less, etc. The lower limit values and the upper limit values can be arbitrarily combined, respectively.

[0038] <Acetic acid or a salt thereof> Examples of the salt of acetic acid (acetate) in the present invention include sodium acetate, magnesium acetate, calcium acetate, and the like.

[0039] The concentration of acetic acid or a salt thereof in the culture solution for preparing NK cells of the present invention is not particularly limited. Examples of the lower limit thereof include 1.0 µg / L or more, 2.0 µg / L or more, 3.0 µg / L or more, 4.0 µg / L or more, 5.0 µg / L or more, 6.0 µg / L or more, 7.0 µg / L or more, 8.0 µg / L or more, 9.0 µg / L or more, and the like. Examples of the upper limit thereof include 100 µg / L or less, 90 µg / L or less, 80 µg / L or less, 70 µg / L or less, 60 µg / L or less, 50 µg / L or less, 40 µg / L or less, 30 µg / L or less, 20 µg / L or less, and the like. Any combination of these lower limits and upper limits is possible.

[0040] <Antibiotics> Examples of the antibiotics in the present invention include aminoglycoside antibiotics such as kanamycin, streptomycin, gentamicin, tobramycin, and amikacin; β-lactam antibiotics such as penicillin (e.g., penicillin G), ampicillin, and bacampicillin; polyene antibiotics such as amphotericin B; and the like. As the antibiotics, one type may be used alone, or two or more types may be used in combination.

[0041] The concentration of antibiotics in the culture medium for NK cell preparation cannot be specified in general terms, as it depends on the type of antibiotic, etc. However, possible lower limits include, for example, 2.0 mg / L or more, 3.0 mg / L or more, 6.0 mg / L or more, 1.0 mg / L or more, 2.0 mg / L or more, 3.0 mg / L or more, 6.0 mg / L or more, 10 mg / L or more, 20 mg / L or more, 30 mg / L or more, 60 mg / L or more, 70 mg / L or more, etc., and possible upper limits include, for example, 1000 mg / L or less, 800 mg / L or less, 600 mg / L or less, 400 mg / L or less, 200 mg / L or less, 100 mg / L or less, 90 mg / L or less, 80 mg / L or less, 60 mg / L or less, 40 mg / L or less, 30 mg / L or less, etc. These lower and upper limits can be combined arbitrarily.

[0042] <IL-2> In the present invention, IL-2 may be naturally derived IL-2, genetically modified IL-2 (recombinant IL-2 [rIL-2]), and may be commercially available or prepared in-house, but human IL-2 is preferred. Examples of commercially available recombinant IL-2 include those manufactured by GC Lymphotec, PeproTech, and R&D Systems.

[0043] The concentration of IL-2 in the culture medium for NK cell preparation is not particularly limited. Examples of lower limits include 35 U / mL or higher, 60 U / mL or higher, 100 U / mL or higher, 130 U / mL or higher, 160 U / mL or higher, 200 U / mL or higher, 230 U / mL or higher, 260 U / mL or higher, 300 U / mL or higher, 330 U / mL or higher, etc. Examples of upper limits include 1500 U / mL or lower, 1400 U / mL or lower, 1200 U / mL or lower, 1000 U / mL or lower, 900 U / mL or lower, 800 U / mL or lower, etc. These lower and upper limits can be combined arbitrarily.

[0044] <Method for Preparing NK Cells> The method for preparing human lymphocyte cells according to the present invention is not particularly limited as long as it includes culturing a population of human lymphocyte cells in the presence of the culture medium for preparing NK cells according to the present invention (hereinafter referred to as "the method for preparing NK cells according to the present invention"). For example, the population of human lymphocyte cells may be cultured in the culture medium for preparing NK cells according to the present invention containing a human lymphocyte stimulating substance (hereinafter referred to as "activated culture"), or the population of human lymphocyte cells may be cultured in the culture medium for preparing NK cells according to the present invention without a human lymphocyte stimulating substance (hereinafter referred to as "deactivated culture"). The human lymphocyte stimulating substance may be any substance that can stimulate and activate human lymphocyte cells (e.g., human T cells), and examples include mitogens, cytokines, antigens, antibodies, etc. More specifically, examples include IL-2, IL-18, anti-CD3 antibodies, etc. While such anti-CD3 antibodies can be produced in animals or cells using purified CD3 molecules, commercially available OKT-3 antibodies (manufactured by eBioscience, Takara Bio, etc.) that offer superior stability and ease of use can be advantageously used.

[0045] The human lymphocyte cell population in the activated culture described above includes cells that adhere to and proliferate on the bottom of the culture vessel, such as a flask, as well as cells that proliferate while suspended in the culture medium after activation. The human lymphocyte cell population that adheres to and proliferates on the bottom of the culture vessel is a living cell, just like the suspended cells. It is possible to perform activated culture without increasing the volume of the NK cell preparation culture medium from the start of activated culture, or it is possible to add the NK cell preparation culture medium further during activated culture and then continue activated culture for several days (for example, 1 to 2 days) to further proliferate the human lymphocyte cell population. The amount of NK cell preparation culture medium to be added is preferably 0.5 to 2 times the volume of the NK cell preparation culture medium at the start of activated culture, and more preferably the same volume as the volume of the NK cell preparation culture medium at the start of activated culture. If the NK cell preparation culture medium is added during the activation culture process and the activation culture is continued, it is possible to subplant only the human lymphocyte cell population suspended in the culture medium, or to subplant both the suspended human lymphocyte cell population and the human lymphocyte cell population attached to the culture vessel.

[0046] In the activated and deactivated cultures described above, there are no particular restrictions on the number of human lymphocyte cells used at the start of culture. However, if the number of cells at the start of culture is too small, it has the disadvantage of prolonging the time it takes for the cell proliferation curve to rise. On the other hand, if the number is too large, the proliferation of the human lymphocyte cell population reaches a plateau too quickly, making it difficult to adopt the method of increasing the culture scale by adding culture medium, and thus it becomes difficult to obtain a sufficient amount of cells. Considering these factors, for example, 1.0 × 10⁶ human lymphocyte cells should be used at the start of activated culture. ５ ~1.0 x 10 ６ Within the range of cells / mL, preferably 1.5 × 10 ５ ~6.0 x 10 ５ Cells / mL, more preferably 4.0 × 10 ５ ~6.0 x 10 ５ The cell count is cells / mL. Furthermore, the number of human lymphocytes used at the start of inactivation culture is, for example, 1.0 × 10⁶.５ to 1.0×10 １０ cells / mL, preferably 1.0×10 ６ to 5.0×10 ９ cells / mL, more preferably 1.0×10 ７ to 1.0×10 ９ cells / mL. The volume of the culture vessel and the amount of the culture medium can be appropriately selected in consideration of operability. For example, for a 225 cm ２ flask, an example includes using 20 to 200 mL (e.g., 50 to 200 mL, 30 to 100 mL, 30 to 70 mL, 80 to 180 mL, 100 to 180 mL, etc.) of the present NK cell preparation culture medium; for a 25 cm ２ flask, an example includes using 2 to 20 mL (preferably 3 to 10 mL, more preferably 3 to 7 mL, even more preferably 5 mL) of the present NK cell preparation culture medium; and an example for a 1000 mL culture bag includes using 50 to 1000 mL (e.g., 100 to 1000 mL, 200 to 1000 mL, etc.) of the present NK cell preparation culture medium.

[0047] When the present NK cell preparation method comprises the above-mentioned activation culture step, after the activation culture step, the method further comprises: a step of cryopreserving the human lymphocyte cell population; a step of non-activated culture of the human lymphocyte cell population in the presence of the present NK cell preparation culture medium; a step of activation culture of the human lymphocyte cell population in the presence of the present NK cell preparation culture medium; and a step of non-activated culture of the human lymphocyte cell population in the presence of a basal culture medium for human cell culture containing the present essential components (preferably containing the present optional components as optional components). The cryopreservation method in the "step of cryopreserving the human lymphocyte cell population" may be any general method for cell cryopreservation. For example, the human lymphocyte cell population is suspended in a cell cryopreservation solution such as Bambanker (registered trademark) (manufactured by Lymphotec Inc.), CP-1 (manufactured by Kyokuto Pharmaceutical Industrial Co., Ltd.), KM Banker (manufactured by Kojin Bio Co., Ltd.), stored in a cryopreservation container, temporarily stored in a -80°C ultra-low temperature freezer (manufactured by Sanyo Electric Co., Ltd.) as needed, and can be cryopreserved in liquid nitrogen. As a method for thawing the cryopreserved human lymphocyte cell population, a general thawing method for frozen human cells can be used.

[0048] The human lymphocyte cell population after thawing may be used directly for cancer immunotherapy, or it may be used for further activation culture and / or deactivation culture.

[0049] The culture period for the activated and inactivated cultures described above is not particularly limited. For example, the lower limit can be 6 hours or more, 12 hours or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, etc., and the upper limit can be 20 days or less, 18 days or less, 16 days or less, 14 days or less, 12 days or less, 10 days or less, 8 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, etc. These lower and upper limits can be arbitrarily combined. The culture temperature for the activated and inactivated cultures described above is, for example, in the range of 36 to 38°C (preferably 36.5 to 37.5°C), and the humidity for the activated and inactivated cultures described above is, for example, in the range of 90 to 98% (preferably 93 to 97%, more preferably 94 to 96%), and the CO2 of the activated and inactivated cultures described above ２ The concentration is, for example, in the range of 2 to 13% (preferably 3 to 10%, more preferably 5 to 10%).

[0050] <NK Cell Preparation Kit> The kit of the present invention is not particularly limited as long as it contains human albumin, selenite compound, and buffer (which may be referred to as "NK cell preparation kit" in this specification), and is specified for the purpose of "preparing a culture medium for preparing a human lymphocyte cell population including human NK cells (i.e., the NK cell preparation culture medium) comprising human albumin, selenite compound, buffer, and human serum." When preparing the NK cell preparation culture medium using the NK cell preparation kit, human serum and a human cell culture medium (solid or liquid [culture medium]) are prepared separately, and the NK cell preparation culture medium can be prepared by adding human albumin, selenite compound, buffer, solid human cell culture medium, and human serum to water, or by adding human albumin, selenite compound, buffer, and human serum to a human cell culture medium.

[0051] Each component of this NK cell preparation kit may be a liquid or a solid. Furthermore, the liquid or solid state may differ for each component. From the viewpoint of distribution and storage, it is preferable that this NK cell preparation kit be packaged in a container. In this NK cell preparation kit, each component may be in its own separate container, one container may contain two or more components, or all components of this NK cell preparation kit may be in a single container. Such containers can be ordinary containers used for products similar to this NK cell preparation kit.

[0052] The NK cell preparation kit in this case preferably further includes human serum and a basal culture medium for human cells. Here, the basal culture medium for human cells may be a solid or a liquid (i.e., a basal culture solution for human cells), but a basal culture solution for human cells is preferred. Furthermore, the NK cell preparation kit in this case may also include the optional components mentioned above, and the optional components in this case are examples of preferred optional components.

[0053] The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to these examples. The following operations should be performed aseptically, and the human lymphocyte cell population should be cultured at 37.0°C, 95% or higher humidity, and CO2. ２ The experiment was conducted under conditions of a 5.0% concentration. In addition, rIL-2 (manufactured by GC Lymphotec) was added to the culture medium at a concentration of 700 U / mL during activation culture and curing culture, and at a concentration of 350 U / mL during amplification culture.

[0054] 1. Method 1-1 Preparation of Human Lymphocyte Cell Population Suspension 50 mL of peripheral blood was collected from the vein of a healthy person with heparin added, and transferred to a centrifuge tube. 100 mL of washing culture medium (RPMI-1640; manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was gently mixed to dilute it three-fold. The diluted blood was overlaid on a 50 mL centrifuge tube dispensed with 15 mL of Lymphoprep (manufactured by Serumwerk Bernburg), and the tube was centrifuged at 1800 rpm for 20 minutes at room temperature without braking. The mononuclear cell layer of the intermediate layer was collected in a 50 mL centrifuge tube dispensed with the washing culture medium. The lid of the centrifuge tube was closed, mixed by inversion 2-3 times, and then centrifuged at 1800 rpm for 15 minutes at room temperature. After centrifugation, the supernatant fraction was removed, and the precipitate fraction (pellet) was dispersed. Then, the above-mentioned washing culture medium was added to prepare a total volume of 50 mL of human lymphocyte cell population suspension. A portion (5 mL) of this suspension was recovered, and the proportion of T cells and NK cells in the human lymphocyte cell population before culture was measured according to the method described in section "1-4" below.

[0055] 1-2 Human Lymphocyte Cell Population Culture and Subsequent Cryopreservation The prepared human lymphocyte cell population suspension was centrifuged at 1800 rpm for 10 minutes at room temperature. After centrifugation, the supernatant fraction was removed, and the precipitate fraction (pellet) was dispersed. Then, 50 mL of three types of culture media (iMedium without human serum [see Table 1], iMedium containing 5% human serum [GeminiBio], or iMedium containing 10% human serum) were added to prepare human lymphocyte cell population suspensions. 50 mL of the prepared human lymphocyte cell population suspension was transferred to an anti-CD3 antibody-immobilized flask 225 (GC Lymphotek) and activated cultured for 4 days. After that, the human lymphocyte cell population attached to the bottom of the flask was detached by tapping to obtain suspension cells, and all cells were collected. The collected human lymphocyte cell population was divided into groups of 3 × 10⁶ cells. ７The cells were transferred to centrifuge tubes in an individual manner and centrifuged at 1500 rpm for 5 minutes at room temperature. After centrifugation, the supernatant was removed from each centrifuge tube, the resulting pellet was dispersed, and cell cryopreservation solution Banbankar (manufactured by GC Lymphotec) was added to suspend the cells. The cells were then transferred to cryopreservation tubes and temporarily stored in an ultra-low temperature freezer at -80°C for 2 days before being transferred to a liquid nitrogen tank for cryopreservation.

[0056]

[0057] 1-3 Thawing of the human lymphocyte cell population and subsequent culture. The cryopreservation tubes containing the cryopreserved human lymphocyte cell population were heated for 4 minutes in a dry thermo unit (manufactured by Taitec) set to 37°C to thaw the human lymphocyte cell population. The thawed human lymphocyte cell population was transferred to a centrifuge tube while suspended in the three culture media described above, and centrifuged at 1800 rpm for 10 minutes at room temperature. After centrifugation, the supernatant fraction was removed, and the pellet was dispersed. The cell concentration was 6 × 10⁶. ５ The three types of culture media described above were adjusted by adding and suspending them to a concentration of cells / mL, and 2 mL of each was seeded per well in a 24-well plate (Sumitomo Bakelite Co., Ltd.). The cells were then cultured in a non-activating state for two days. Subsequently, all cells of the human lymphocyte population were transferred to a 225-liter suspension cell flask (Sumitomo Bakelite Co., Ltd.) and cultured in a non-activating state for two days in the presence of the three types of culture media described above. After that, all cells of the human lymphocyte population were transferred to a 225-liter anti-CD3 antibody-immobilized flask (GC Lymphotek Co., Ltd.) and cultured in an activated state for one day in the presence of the three types of culture media described above. Subsequently, all cells of the human lymphocyte population were transferred to a culture bag (manufactured by GC Lymphotek) filled with iMedium without human serum, and after amplification culture (deactivation culture) for 9 days, a portion (5 mL) of the human lymphocyte population suspension was collected, and the proportion of T cells and NK cells in the human lymphocyte population after culturing in the three types of culture media described above was measured according to the method described in item "1-4" below, and the total number of cells was measured according to the standard method.

[0058] 1-4 Measurement of the proportion of T cells and NK cells in a human lymphocyte population. The recovered human lymphocyte population suspension was transferred to a 15 mL centrifuge tube and centrifuged at 1800 rpm for 5 minutes at room temperature. The supernatant fraction after centrifugation was removed, and Iso sheath solution (Beckman Coulter) was added to the pellet, and 5.0 × 10⁶ cells were measured. ６ Human lymphocyte population suspensions were prepared at a concentration of 10 cells / mL. Three fluorescently labeled antibodies (APC [Allophycocyanin]-labeled anti-human CD3 antibody, FITC [Fluorescein isothiocyanate]-labeled anti-human CD16 antibody, and PE [Phycoerythrin]-labeled anti-human CD56 antibody; all from BioLegend) were dispensed into 5 mL round tubes (BD Falcon), and 200 μL of the prepared human lymphocyte population suspension was dispensed into each tube. The tubes were incubated in the dark at 4°C for 30 minutes under static conditions. Subsequently, 2 mL of Iso sheath solution was added, and the cells were centrifuged at 1800 rpm for 5 minutes at room temperature. The supernatant fraction was removed after centrifugation, and 500 μL of Iso sheath solution was added to each pellet. After suspension, 10,000 human lymphocyte populations were measured using a flow cytometer (CytoFLEX; Beckman Coulter). After measurement, the proportion of T cells (CD3-positive cells) and NK cells (CD16-positive and CD56-positive cells) was analyzed using analysis software (Kaluza; Beckman Coulter).

[0059] 2. Results: The proportion of T cells and NK cells in the human lymphocyte population before culture was measured, and the proportion of T cells was approximately 61%, while the proportion of NK cells was approximately 18% (see Figure 1).

[0060] On the other hand, when the percentage of T cells in the human lymphocyte population after culturing in three types of culture media (iMedium without human serum, iMedium containing 5% human serum, or iMedium containing 10% human serum) was measured, the percentage was approximately 98% when cultured in iMedium without human serum, an increase compared to before culturing. In contrast, when cultured in iMedium containing 5% or 10% human serum, the percentages were approximately 65% ​​and 60%, respectively, showing almost no change from before culturing (see Figure 2). As a result, when the human lymphocyte population was cultured in iMedium containing 5% or 10% human serum, the percentage of T cells in the human lymphocyte population was significantly reduced compared to when the human lymphocyte population was cultured in iMedium without human serum (see Figure 2).

[0061] Furthermore, when the percentage of NK cells in the human lymphocyte population after culturing in the three types of culture media described above was measured, it was approximately 3% when cultured in iMedium without human serum, a decrease compared to before culturing. In contrast, when cultured in iMedium containing 5% or 10% human serum, the percentage was approximately 36% and 40%, respectively, an increase compared to before culturing (see Figure 3). As a result, culturing the human lymphocyte population in iMedium containing 5% or 10% human serum significantly increased the percentage of NK cells in the human lymphocyte population compared to culturing the human lymphocyte population in iMedium without human serum (see Figure 3).

[0062] Furthermore, the total number of human lymphocyte cells cultured in the three types of culture media described above tended to increase when cultured in iMedium containing 5% or 10% human serum compared to when cultured in iMedium without human serum (see Figure 4).

[0063] These results indicate that culturing human lymphocyte populations in iMedium containing human serum specifically increases the proportion and number of NK cells in the human lymphocyte population compared to culturing them in iMedium without human serum.

[0064] This will be useful for immunotherapy for diseases such as cancer, viral infections, and age-related diseases.

Claims

1. A culture medium for preparing a human lymphocyte cell population, including human NK cells, comprising a basic culture medium for human cell culture containing human albumin, selenite compounds, a buffer, and human serum.

2. The culture medium for preparation according to claim 1, further comprising one or more selected from the group consisting of human transferrin, human insulin, glutathione, ascorbic acid phosphate ester or a salt thereof, ethanolamine, pyruvate, L-alanyl-L-glutamine, acetic acid or a salt thereof, and antibiotics.

3. The culture medium for preparation according to claim 1, wherein the buffer is one or two selected from the group consisting of sodium bicarbonate and HEPES.

4. A method for preparing a human lymphocyte cell population including human NK cells, comprising culturing a human lymphocyte cell population in the presence of a culture medium for preparation described in any one of claims 1 to 3.

5. A kit for preparing a culture medium for preparing a population of human lymphocyte cells including human NK cells, comprising human albumin, a selenite compound, and a buffer, wherein the culture medium for preparation consists of a basic culture medium for human cell culture comprising the human albumin, the selenite compound, and the buffer, and human serum.

6. The kit according to claim 5, further comprising human serum and a basic culture medium for human cell culture.

7. The kit according to claim 5 or 6, further comprising one or more selected from the group consisting of human transferrin, human insulin, glutathione, ascorbic acid phosphate or a salt thereof, ethanolamine, pyruvate, L-alanyl-L-glutamine, acetic acid or a salt thereof, and antibiotics.

8. The kit according to claim 5 or 6, wherein the buffer is one or two selected from the group consisting of sodium bicarbonate and HEPES.

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