Culture method for NK cells

Abstract

The invention relates to the technical field of NK cell culture, in particular to a culture method for NK cells. Comprising the following steps: (1) separating PBMC cells to obtain NK cells; (2) inoculating the NK cells into a culture bottle for preliminary culture; (3) transferring the NK cells obtained in the step (2) into a new culture bottle for amplification culture; (4) transferring the NK cells obtained in the step (3) into a three-dimensional bioreactor for multiplication culture; an amplification culture medium is added into the three-dimensional bioreactor. The culture method of the NK cells can be used for in-vitro long-term culture and amplification of various NK cells, the NK cells subjected to large-scale amplification are stable in performance, efficient amplification of the NK cells is achieved, a stable, safe and large-scale cell source is provided for cell treatment schemes of tumors, infectious diseases and the like, and the culture method has remarkable clinical transformation value.

Description

Technical Field

[0001] The present invention relates to the technical field of NK cell culture, and in particular to a method for culturing NK cells. Background Art

[0002] Immune cells refer to cells that participate in or are related to immune responses. As an important carrier for the induction of immune cell proliferation in vitro, the culture medium is directly related to the induction results in vitro, including the number of proliferation, tumor killing effect, etc. Immune cells have the following characteristics: fast proliferation rate, main effector cell CD3 + CD56 + T (anti-tumor activity) can proliferate in large quantities, and the cell activity is also greatly enhanced; the killing activity is strong, far superior to traditional lymphokine-activated killer cells; the tumor killing spectrum is broad, not restricted by the major histocompatibility complex (MHC), and has a broad-spectrum tumor and virus killing effect.

[0003] In recent years, immune cell therapies (such as CAR-T and CAR-NK therapies) have shown great potential in the clinical application of tumors, infectious diseases, and autoimmune diseases. However, traditional two-dimensional static culture methods have problems such as limited culture scale, easily damaged cell function, and large batch-to-batch variability, making it difficult to meet the cell quantity and quality requirements of clinical-grade cell therapy products.

[0004] A 3D bioreactor is an advanced cell culture system that mimics the in vivo microenvironment. By providing a three-dimensional growth space, dynamic nutrient supply, and precise environmental control, it significantly improves cell expansion efficiency, functional activity, and production standardization, becoming a core tool for cell expansion and culture. The use of 3D bioreactors can significantly increase cell yield per unit volume, addressing the technical challenges of insufficient yield and high costs in cell therapy. Therefore, how to apply 3D bioreactors to the expansion of NK cells is an urgent issue facing those skilled in the art. Summary of the Invention

[0005] The object of the present invention is to provide a method for culturing NK cells.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for culturing NK cells, comprising the following steps:

[0008] (1) separating peripheral blood to obtain PBMC cells; separating the PBMC cells to obtain NK cells;

[0009] (2) inoculating NK cells into a culture flask for preliminary culture; the culture flask is supplemented with activation medium;

[0010] (3) transferring the NK cells obtained in step (2) to a new culture flask for expansion culture; the culture flask is added with expansion medium;

[0011] (4) transferring the NK cells obtained in step (3) to a three-dimensional bioreactor for expansion culture; the three-dimensional bioreactor is added with expansion culture medium;

[0012] Preferably, the activation medium comprises the following components: 3.5-4.5% (v / v) serum replacement, 0.8-1.2% penicillin-streptomycin, 18-22 mg / L transferrin, 25-29 mg / L human recombinant insulin, 12-18 mg / L adenosine triphosphate disodium, 20-30 ng / mL IL-4, 20-30 ng / mL IL-6, 25-35 ng / mL IL-15, and the remainder is basal medium;

[0013] Preferably, the expansion medium comprises the following components: 1.5-2.5% (v / v) serum replacement, 0.8-1.2% penicillin-streptomycin, 18-22 mg / L transferrin, 25-29 mg / L human recombinant insulin, 12-18 mg / L adenosine disodium triphosphate, 60-70 mg / L acetylcysteine, 55-65 nM salvianolic acid B, 40-50 ng / mL IL-4, 30-40 ng / mL IL-6, 45-55 ng / mL IL-15, and the rest is basal medium.

[0014] Preferably, the basal culture medium is RPMI 1640 or DMEM.

[0015] Preferably, the pH of the activation medium and the expansion medium are both 7.2-7.4.

[0016] Preferably, the initial culture time is 1 to 4 days;

[0017] Preferably, the seeding density of the initial culture and the expanded culture is 1-2×10 6 pieces / mL.

[0018] Preferably, the expansion culture time is 3 to 5 days;

[0019] Preferably, the expansion culture time is 5 to 14 days.

[0020] Preferably, the culture flask in step (2) is a T75 culture flask;

[0021] Preferably, the culture flask in step (3) is a G-Rex100 culture flask.

[0022] Preferably, during the initial culture and expanded culture, the temperature is 36.5-37.5° C. and the carbon dioxide concentration is 4.5-5.5%.

[0023] Preferably, during the amplification culture in step (4), the temperature is 36.5-37.5° C. and the dissolved oxygen is 45%-55%.

[0024] Preferably, during the amplification culture in step (4), the seeding density is 1 to 2×10 6 pieces / mL.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides two culture media for culturing NK cells: activation medium and expansion medium. The activation medium promotes the cells to enter a state of proliferation and functional activation. The expansion medium supports the large-scale proliferation of NK cells while maintaining their functional properties and stability.

[0027] Among them, cytokines such as IL-4, IL-6, and IL-15 added to the culture medium effectively promote the activation and function maintenance of NK cells (especially NK cells). Disodium adenosine triphosphate is used to directly supplement ATP, alleviate energy stress in early culture, and reduce cell apoptosis. Transferrin and human recombinant insulin can promote iron ion transport and sugar metabolism to meet the synthetic needs of activated cells. The tansolic acid B added to the expansion culture medium protects cells by inhibiting ROS, and acetylcysteine ​​increases glutathione levels and reduces oxidative damage. The two components are used in combination to protect cells and maintain cell activity, which allows NK cells to adapt to the dynamic environment of the three-dimensional bioreactor and ensure uniformity and reproduction speed during expansion.

[0028] The NK cell culture method provided by the present invention can be used for long-term in vitro culture and expansion of various NK cells. The performance of the NK cells after large-scale expansion is stable, achieving efficient expansion of NK cells, providing a stable, safe and scalable cell source for cell therapy programs for tumors, infectious diseases, etc., and has significant clinical application value. DETAILED DESCRIPTION

[0029] The present invention provides a method for culturing NK cells, comprising the following steps:

[0030] (1) separating peripheral blood to obtain PBMC cells; separating the PBMC cells to obtain NK cells;

[0031] (2) inoculating NK cells into a culture flask for preliminary culture; the culture flask is supplemented with activation medium;

[0032] (3) transferring the NK cells obtained in step (2) to a new culture flask for expansion culture; the culture flask is added with expansion medium;

[0033] (4) transferring the NK cells obtained in step (3) to a three-dimensional bioreactor for expansion culture; the three-dimensional bioreactor is added with expansion culture medium;

[0034] In the present invention, the activation medium comprises the following components: 3.5-4.5% (v / v) serum replacement, 0.8-1.2% penicillin-streptomycin, 18-22 mg / L transferrin, 25-29 mg / L human recombinant insulin, 12-18 mg / L adenosine triphosphate disodium, 20-30 ng / mL IL-4, 20-30 ng / mL IL-6, 25-35 ng / mL IL-15, and the rest is basal medium;

[0035] Preferably, the medium comprises 4% (v / v) serum replacement, 0.9-1.1% penicillin-streptomycin, 19-21 mg / L transferrin, 26-28 mg / L human recombinant insulin, 13-17 mg / L adenosine triphosphate disodium, 22-28 ng / mL IL-4, 22-28 ng / mL IL-6, 27-33 ng / mL IL-15, and the remainder is basal culture medium;

[0036] More preferably, the medium comprises 4% (v / v) serum replacement, 1% penicillin-streptomycin, 20 mg / L transferrin, 27 mg / L human recombinant insulin, 14-16 mg / L adenosine triphosphate disodium, 24-26 ng / mL IL-4, 24-26 ng / mL IL-6, 29-31 ng / mL IL-15, and the remainder is basal medium;

[0037] More preferably, it is 4% (v / v) serum replacement, 1% penicillin-streptomycin, 20 mg / L transferrin, 27 mg / L human recombinant insulin, 15 mg / L adenosine triphosphate disodium, 25 ng / mL IL-4, 25 ng / mL IL-6, 30 ng / mL IL-15, and the rest is basal culture medium.

[0038] In the present invention, the expansion medium comprises the following components: 1.5-2.5% (v / v) serum replacement, 0.8-1.2% penicillin-streptomycin, 18-22 mg / L transferrin, 25-29 mg / L human recombinant insulin, 12-18 mg / L adenosine disodium triphosphate, 60-70 mg / L acetylcysteine, 55-65 nM salvianolic acid B, 40-50 ng / mL IL-4, 30-40 ng / mL IL-6, 45-55 ng / mL IL-15, and the rest is basal medium;

[0039] Preferably, the medium comprises 2% (v / v) serum replacement, 0.9-1.1% penicillin-streptomycin, 19-21 mg / L transferrin, 26-28 mg / L human recombinant insulin, 13-17 mg / L adenosine disodium triphosphate, 62-68 mg / L acetylcysteine, 57-63 nM salvianolic acid B, 42-48 ng / mL IL-4, 32-38 ng / mL IL-6, 47-53 ng / mL IL-15, and the remainder is basal culture medium;

[0040] More preferably, the medium comprises 2% (v / v) serum replacement, 1% penicillin-streptomycin, 20 mg / L transferrin, 27 mg / L human recombinant insulin, 14-16 mg / L adenosine disodium triphosphate, 64-66 mg / L acetylcysteine, 59-61 nM salvianolic acid B, 44-46 ng / mL IL-4, 34-36 ng / mL IL-6, 49-51 ng / mL IL-15, and the remainder is basal medium;

[0041] More preferably, it is 2% (v / v) serum replacement, 1% penicillin-streptomycin, 20 mg / L transferrin, 27 mg / L human recombinant insulin, 15 mg / L adenosine disodium triphosphate, 65 mg / L acetylcysteine, 60 nM salvianolic acid B, 45 ng / mL IL-4, 35 ng / mL IL-6, 50 ng / mL IL-15, and the rest is basal culture medium.

[0042] In the present invention, the basal culture medium is RPMI 1640 or DMEM; preferably RPMI 1640.

[0043] In the present invention, the pH of the activation medium and the expansion medium are both 7.2-7.4, preferably 7.3.

[0044] In the present invention, the initial culture time is 1 to 4 days, preferably 2 to 3 days, and more preferably 2 days.

[0045] In the present invention, the inoculation density of the initial culture and the expanded culture is 1 to 2×10 6 / mL; preferably 1.5×10 6 pieces / mL.

[0046] In the present invention, the time of the expanded culture is 3 to 5 days, preferably 3 days.

[0047] In the present invention, the expansion culture time is 5 to 14 days, preferably 7 to 14 days, and more preferably 10 days.

[0048] In the present invention, the culture bottle in step (2) is a T75 culture bottle;

[0049] In the present invention, the culture bottle in step (3) is a G-Rex100 culture bottle.

[0050] In the present invention, during the initial culture and expanded culture, the temperature is 36.5-37.5° C. and the carbon dioxide concentration is 4.5-5.5%; preferably, the temperature is 37° C. and the carbon dioxide concentration is 5%.

[0051] In the present invention, during the amplification culture in step (4), the temperature is 36.5-37.5°C, and the dissolved oxygen is 45%-55%; preferably, the temperature is 37°C, and the dissolved oxygen is 47%-93%; further preferably, the temperature is 37°C, and the dissolved oxygen is 50%.

[0052] In the present invention, during the amplification culture in step (4), the seeding density is 1 to 2×10 6 / mL. Preferably 1.5×10 6 pieces / mL.

[0053] In the present invention, the three-dimensional bioreactor is derived from the three-dimensional (3D) bioreactor for large-scale expansion of immune cells in Patent 202380041907.X.

[0054] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0055] Example 1

[0056] A method for culturing NK cells, comprising the following steps:

[0057] (1) Collect peripheral blood, separate and collect human peripheral blood mononuclear cells using lymphocyte separation solution, and obtain human peripheral blood mononuclear cells (PBMCs) after washing; and separate NK cells from PBMC cells according to the NK cell sorting kit;

[0058] (2) NK cells were inoculated into T75 culture flasks and cultured for 1 day; the culture temperature was 36.5°C and the carbon dioxide concentration was 4.5%; activation medium was added to the culture flask; the inoculation density was 1×10 6 pieces / mL.

[0059] (3) The NK cells obtained in step (2) were transferred to a new G-Rex100 culture flask for expansion culture for 3 days; the culture temperature was 36.5°C and the carbon dioxide concentration was 4.5%; the G-Rex100 culture flask was added with expansion medium; the seeding density was 1×10 6 pieces / mL.

[0060] (4) The NK cells obtained in step (3) were transferred to a three-dimensional bioreactor for expansion culture for 10 days, and the culture medium was renewed by 5%, 50%, and 50% on the 3rd, 5th, and 7th days, respectively; the culture temperature was 36.5°C and the dissolved oxygen was 45%; the three-dimensional bioreactor was supplemented with expansion culture medium; the inoculation density was 1×10 6 pieces / mL.

[0061] The activation medium is composed of the following components: 3.5% (v / v) serum replacement, 0.8% penicillin-streptomycin, 18 mg / L transferrin, 25 mg / L human recombinant insulin, 12 mg / L adenosine triphosphate disodium, 20 ng / mL IL-4, 20 ng / mL IL-6, 25 ng / mL IL-15, and the rest is RPMI 1640; the pH is 7.2.

[0062] The expansion medium consists of the following components: 1.5% (v / v) serum replacement, 0.8% penicillin-streptomycin, 18 mg / L transferrin, 25 mg / L human recombinant insulin, 12 mg / L adenosine disodium triphosphate, 60 mg / L acetylcysteine, 55 nM salvianolic acid B, 40 ng / mL IL-4, 30 ng / ml IL-6, 45 ng / mL IL-15, and the rest is RPMI 1640, pH 7.2.

[0063] Example 2

[0064] A method for culturing NK cells, comprising the following steps:

[0065] (1) Collect peripheral blood, separate and collect human peripheral blood mononuclear cells using lymphocyte separation solution, and obtain human peripheral blood mononuclear cells (PBMCs) after washing; and separate NK cells from PBMC cells according to the NK cell sorting kit;

[0066] (2) NK cells were inoculated into T75 culture flasks and cultured for 4 days; the culture temperature was 37.5°C and the carbon dioxide concentration was 5.5%; activation medium was added to the culture flask; the inoculation density was 2×10 6 pcs / ml.

[0067] (3) The NK cells obtained in step (2) were transferred to a new G-Rex100 culture flask for expansion culture for 5 days; the culture temperature was 37.5°C and the carbon dioxide concentration was 5.5%; the G-Rex100 culture flask was added with expansion medium; the seeding density was 2×10 6 pcs / ml.

[0068] (4) The NK cells obtained in step (3) were transferred to a three-dimensional bioreactor for expansion culture for 10 days, and the culture medium was renewed by 5%, 50%, and 50% on the 3rd, 5th, and 7th days, respectively; the culture temperature was 37.5°C, the dissolved oxygen was 55%; the three-dimensional bioreactor was supplemented with expansion culture medium; the inoculation density was 1×10 6 pcs / ml.

[0069] The activation medium is composed of the following components: 4.5% (v / v) serum replacement, 1.2% penicillin-streptomycin, 22 mg / L transferrin, 29 mg / L human recombinant insulin, 18 mg / L adenosine triphosphate disodium, 30 ng / mL IL-4, 30 ng / ml IL-6, 35 ng / mL IL-15, and the rest is RPMI 1640; the pH is 7.4.

[0070] The expansion medium is composed of the following components: 2.5% (v / v) serum replacement, 1.2% penicillin-streptomycin, 22 mg / L transferrin, 29 mg / L human recombinant insulin, 18 mg / L adenosine disodium triphosphate, 70 mg / L acetylcysteine, 65 nM salvianolic acid B, 50 ng / mL IL-4, 40 ng / ml IL-6, 55 ng / mL IL-15, and the rest is RPMI 1640, pH 7.4.

[0071] Example 3

[0072] A method for culturing NK cells, comprising the following steps:

[0073] (1) Collect peripheral blood, separate and collect human peripheral blood mononuclear cells using lymphocyte separation solution, and obtain human peripheral blood mononuclear cells (PBMCs) after washing; and separate NK cells from PBMC cells according to the NK cell sorting kit;

[0074] (2) NK cells were inoculated into T75 culture flasks and cultured for 2 days; the culture temperature was 37°C and the carbon dioxide concentration was 5%; activation medium was added to the culture flask; the inoculation density was 1.5×10 6 pcs / ml.

[0075] (3) The NK cells obtained in step (2) were transferred to a new G-Rex100 culture flask for expansion culture for 5 days; the culture temperature was 37°C and the carbon dioxide concentration was 5%; the G-Rex100 culture flask was added with expansion medium; the seeding density was 1.5×10 6 pcs / ml.

[0076] (4) The NK cells obtained in step (3) were transferred to a three-dimensional bioreactor for expansion culture for 10 days, and the culture medium was renewed by 5%, 50%, and 50% on the 3rd, 5th, and 7th days, respectively; the culture temperature was 37°C and the dissolved oxygen was 50%; the three-dimensional bioreactor was supplemented with expansion culture medium; the inoculation density was 1.5×10 6 pcs / ml.

[0077] The activation medium is composed of the following components: 4% (v / v) serum replacement, 1% penicillin-streptomycin, 20 mg / L transferrin, 27 mg / L human recombinant insulin, 15 mg / L adenosine triphosphate disodium, 25 ng / mL IL-4, 25 ng / ml IL-6, 30 ng / mL IL-15, and the rest is DMEM; the pH is 7.3.

[0078] The expansion medium is composed of the following components: 2% (v / v) serum replacement, 1% penicillin-streptomycin, 20 mg / L transferrin, 27 mg / L human recombinant insulin, 15 mg / L adenosine disodium triphosphate, 65 mg / L acetylcysteine, 60 nM salvianolic acid B, 45 ng / mL IL-4, 35 ng / ml IL-6, 50 ng / mL IL-15, and the rest is DMEM, pH 7.3.

[0079] Experimental Example 1

[0080] In Examples 1 to 3, during the culture in the three-dimensional bioreactor, the cell concentration was measured by a Vi-CELL cell counter on the 3rd, 7th, and 10th day of culture, and the cell expansion multiple was calculated. The results are shown in Table 1.

[0081] Specific surface markers of NK cells were identified by flow cytometry. The identification results are shown in Table 2. Among them, NK cell markers: CD3 - CD56 + .

[0082] Table 1 Amplification fold

[0083] Group No. 0d 3d 7d 10d Example 1 1 15.2 25.4 56.6 Example 2 1 14.3 23.9 53.4 Example 3 1 16.7 28.3 58.1

[0084] Table 2 Cell positive rate

[0085] Group Cell positive rate% Example 1 94.2％ Example 2 93.5％ Example 3 95.7％

[0086] As shown in Tables 1 and 2, NK cells cultured using the culture method provided by the present invention have a fast proliferation rate and high purity. The cells can be expanded 58.1 times within 10 days, and a large number of NK cells can be cultured in a short time.

[0087] Experimental Example 2

[0088] Take the NK cells cultured in Example 3 and resuspend them in DMEM medium to a concentration of 1×10 6 Use K562 (human chronic myeloid leukemia cells) as target cells and resuspend the cells in DMEM medium containing 2% FBS to 1×10 5 The cell suspension of 100 cells / mL is ready for use;

[0089] Cells were inoculated into 96-well plates at effector-target ratios of 1:1, 5:1, and 10:1. Effector cell wells and target cell wells were set, and 5 replicate wells were plated and cultured at 37°C, 5% CO2 for 24 h. The absorbance value was measured using the MTT assay, and the tumor killing rate of NK cells was calculated. The average of the measurement results is shown in Table 3 below.

[0090] Table 3 Tumor killing rate

[0091] Group 1：1 5：1 10：1 Example 3 23.24％ 58.39％ 85.15％

[0092] As shown in Table 3, the NK cells cultured in the present invention have a good killing effect, which can reach up to 85.15%.

[0093] Experimental Example 3

[0094] The inventors continued to test the clinical safety of the cultured NK cells. Specific test indicators included hepatitis B surface antigen, biscuit antigen, human immunodeficiency virus antibody, Treponema pallidum-specific antibody, macrophage virus, as well as mycoplasma, bacteria and endotoxin. The test results were all negative, indicating that the NK cell product provided by the present invention is safe and no contamination is caused during the culture process. The specific test results are shown in Table 4.

[0095] Table 4 NK cell clinical safety test

[0096]

[0097]

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for culturing NK cells, characterized in that: The steps include: (1) separating peripheral blood to obtain PBMC cells; separating the PBMC cells to obtain NK cells; (2) inoculating NK cells into a culture flask for preliminary culture; the culture flask is supplemented with activation medium; (3) transferring the NK cells obtained in step (2) to a new culture flask for expansion culture; the culture flask is added with expansion medium; (4) transferring the NK cells obtained in step (3) to a three-dimensional bioreactor for expansion culture; the three-dimensional bioreactor is added with expansion culture medium; The activation culture medium comprises the following components: 3.5-4.5% (v / v) serum replacement, 0.8-1.2% penicillin-streptomycin, 18-22 mg / L transferrin, 25-29 mg / L human recombinant insulin, 12-18 mg / L adenosine triphosphate disodium, 20-30 ng / mL IL-4, 20-30 ng / mL IL-6, 25-35 ng / mL IL-15, and the rest is basal culture medium; The expansion culture medium includes the following components: 1.5-2.5% (v / v) serum replacement, 0.8-1.2% penicillin-streptomycin, 18-22 mg / L transferrin, 25-29 mg / L human recombinant insulin, 12-18 mg / L adenosine disodium triphosphate, 60-70 mg / L acetylcysteine, 55-65 nM salvianolic acid B, 40-50 ng / mL IL-4, 30-40 ng / mL IL-6, 45-55 ng / mL IL-15, and the rest is basal culture medium.

2. The culture method according to claim 1, wherein The basal culture medium is RPMI 1640 or DMEM.

3. The culture method according to claim 1, wherein The pH of the activation medium and the expansion medium are both 7.2-7.

4.

4. The culture method according to claim 1, wherein The time of the expanded culture is 3 to 5 days.

5. The culture method according to claim 1, wherein The initial culture time is 1 to 4 days; the inoculation density of the initial culture and the expanded culture is 1 to 2×10 6 pieces / mL.

6. The culture method according to claim 1, wherein The amplification culture time is 5 to 14 days.

7. The culture method according to claim 1, wherein The culture flask in step (2) is a T75 culture flask; the culture flask in step (3) is a G-Rex100 culture flask.

8. The culture method according to claim 1, wherein During the initial culture and expanded culture, the temperature is 36.5-37.5° C. and the carbon dioxide concentration is 4.5-5.5%.

9. The culture method according to claim 1, wherein During the amplification culture in step (4), the temperature is 36.5-37.5° C. and the dissolved oxygen is 45%-55%.

10. The culture method according to claim 1, wherein During the amplification culture in step (4), the seeding density is 1 to 2×10 6 pieces / mL.

Images