NK (Natural Killer) cell as well as efficient amplification culture medium and culture method thereof

Abstract

After the NK cells are cultured in vitro for 14 days, the number of living cells is 112.5 + / -9.8 * 10 < 6 > / mL, the proportion of CD56light is 63.2 + / -5.1%, the secretion amount of IFN-gamma is 824 + / -67 pg / 106 cells, and the JC-1 red / green ratio of mitochondrial membrane potential is 4.8 + / -0.4. The invention also discloses a culture medium and a culture method for culturing the NK cell. The NK cell prepared by the method disclosed by the invention has a good application effect, and a good reference and research direction is provided for culture and application of the NK cell.

Description

Technical Field

[0001] The present invention relates to the field of immune cell technology, and in particular to a natural killer (NK) cell and a highly efficient amplification culture medium and a culture method thereof, specifically a culture method capable of efficiently amplifying NK cells and enhancing their killing activity. Background Art

[0002] Natural killer (NK) cells are important immune cells that can directly recognize and kill virus-infected cells, tumor cells and other abnormal cells, and play a key role in immune surveillance and anti-tumor immunity. With the rise of immune cell therapy, NK cells, as a potential immunotherapy tool, have been widely used in tumor immunotherapy, antiviral therapy and immune regulation. However, existing NK cell expansion methods still have some limitations, especially in terms of expansion efficiency, function retention and therapeutic effect.

[0003] At present, many NK cell expansion methods rely on the combined use of cytokines such as IL-2, IL-15, and IL-21. Although these factors can effectively promote the proliferation and activation of NK cells, the activity of cells may gradually decrease due to the long cell expansion cycle. In addition, most of the existing culture media rely on conventional cytokine formulas and lack sufficient innovation, resulting in poor sustainability of cell function and low expansion efficiency. In addition, some culture methods have not yet effectively solved the problem of long-term maintenance of NK cell function.

[0004] Therefore, developing a new and efficient NK cell expansion and culture method and culture medium is the key to improving the clinical application effect of NK cells. Summary of the invention

[0005] The object of the present invention is to provide a NK cell expansion medium and a culture method, which can efficiently expand NK cells in a short time and improve their killing activity. By optimizing the components of the culture medium, the expansion of NK cells can be effectively promoted and their functions can be enhanced.

[0006] Therefore, the present invention discloses a NK cell, wherein the number of viable cells of the NK cell after 14 days of in vitro culture is 112.5±9.8×10 6 / mL, CD56bright ratio was 63.2%±5.1%, and IFN-γ secretion was 824±67pg / 10 6 cells, the JC-1 red / green ratio of mitochondrial membrane potential was 4.8 ± 0.4.

[0007] In one aspect, the present invention further discloses an initial culture medium for culturing the NK cells, and the formula of the culture medium is as follows:

[0008] (1) Basic culture medium: RPMI-1640;

[0009] (2) Growth factors: 100 ng / mL IL-15 / IL-21-Fc fusion protein;

[0010] (3) Immunostimulatory factors: 1000 IU / mL IFN-α, 20 ng / mL Flt-3L;

[0011] (4) Modulator: 0.1 μM indomethacin;

[0012] (5) pH adjuster: 10 mM HEPES, 2 mM NaHCO 3 ;

[0013] (6) Cytokine activators: 1 μg / mL anti-CD3 and CD28 antibodies.

[0014] Preferably, the amino acid sequence of the IL-15 / IL-21-Fc fusion protein of the present invention is as shown in SEQ ID NO.1.

[0015] Preferably, the anti-CD3 and CD28 antibodies of the present invention comprise anti-CD3 VH, anti-CD3 VL, anti-CD28 VH and anti-CD28 VL, wherein the amino acid sequences of anti-CD3 VH, anti-CD3 VL, anti-CD28 VH and anti-CD28 VL of the anti-CD3 and CD28 antibodies are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0016] In one aspect, the present invention further discloses a method for culturing the NK cells, the method comprising the following steps:

[0017] (1) Cell inoculation: NK cells were isolated from peripheral blood or umbilical cord blood and inoculated into the prepared culture medium at a cell inoculation density of 1×10 6 cells / mL;

[0018] (2) Adding growth factors: 100 ng / mL L-15 / IL-21-Fc fusion protein, 1000 IU / mL IFN-α, and 20 ng / mL Flt-3L were added to the culture medium. The culture conditions were 37°C and 5% CO. 2 Cultured in a constant temperature incubator;

[0019] (3) Adding indomethacin: On the third day of culture, indomethacin was added to the culture medium to a final concentration of 0.1 μM, and the culture was continued to promote cell proliferation and functional enhancement;

[0020] (4) Cell activation: Anti-CD3 and CD28 antibodies were used to activate NK cells to promote cell proliferation and activation. At this time, the cells were expanded and cultured in a cell reactor. The initial conditions of the reactor were DO = 30%, pH = 7.2, temperature 37°C, and stirring speed 50 rpm. The culture medium used at this time was the initial culture medium described above;

[0021] (5) After the initial culture, a gradient control procedure is performed, as follows:

[0022] 1) IL-15 / IL-21-Fc: Decrease by 5 ng / mL daily, i.e. D3 = 100 ng / mL → D14 = 50 ng / mL;

[0023] 2) Anti-CD3 and CD28 antibodies: increase by 1 μg / mL daily, i.e. D3 = 1 μg / mL → D14 = 12 μg / mL);

[0024] 3) DO control: Dynamically increase the stirring speed according to the cell density, and increase the stirring speed by 1×10 6 / mL, rotation speed +5rpm.

[0025] In one aspect, the present invention also discloses a use of the culture medium in NK cell culture.

[0026] The NK cells prepared by the method of the present invention have good application effects and provide a good reference and research direction for the cultivation and application of NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 SDS-PAGE detection results of anti-CD3 and CD28 antibodies, where 1 is anti-CD3 and CD28 antibodies. DETAILED DESCRIPTION

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0029] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0030] Example 1: NK cell efficient expansion medium

[0031] The present invention provides a highly efficient NK cell expansion medium for improving the proliferation rate and functionality of NK cells. The formula of the medium is as follows:

[0032] 1. Basic culture medium: RPMI-1640, used to provide basic nutritional support to ensure that cells can grow and reproduce during the culture process.

[0033] 2. Growth factor: IL-15 / IL-21-Fc fusion protein (100 ng / mL, as described in Example 4, and its amino acid sequence is shown in SEQ ID NO.1), which activates the STAT3 / STAT5 balance signal.

[0034] 3. Immunostimulatory factors:

[0035] (1) IFN-α (1000 IU / mL): It has a strong immunomodulatory effect, can enhance the killing activity of NK cells, and promote their cytokine secretion.

[0036] (2) Recombinant human Flt-3 ligand (Flt-3L) (20 ng / mL): It helps expand NK cells and enhance their immune response by activating NK cell precursors.

[0037] 4. Regulator: Indomethacin (0.1μM) is a non-steroidal anti-inflammatory drug that reduces oxidative stress by inhibiting the cyclooxygenase (COX) pathway, increases the proliferation rate and functionality of NK cells, and has a significant enhancing effect on the killing activity of NK cells. This ingredient can reduce the inflammatory response in the immune response, thereby improving the quality of the cell growth environment.

[0038] 5. pH regulator:

[0039] (1) HEPES (10 mM): used to adjust the pH value of the culture medium to ensure that the cell growth environment is always maintained within the optimal pH range during the culture process.

[0040] (2)NaHCO 3 (2mM): helps maintain the acid-base balance of the culture medium and adapts to 5% CO 2 Cultivate environment.

[0041] 6. Cytokine activator: anti-CD3 and CD28 antibodies (as described in Example 5) (1 μg / mL), used to activate the CD3 receptor on the surface of T cells and enhance the activation effect of cells; bind to the CD28 receptor to synergistically enhance cell activation and proliferation and promote the effector function of NK cells.

[0042] Example 2: Method for efficiently expanding and culturing NK cells

[0043] (1) Cell inoculation: NK cells were isolated from peripheral blood or umbilical cord blood and inoculated into the prepared culture medium. The cell inoculation density was 1×10 6 cells / mL.

[0044] (2) Adding growth factors: L-15 / IL-21-Fc fusion protein (100 ng / mL), IFN-α (1000 IU / mL), and Flt-3L (20 ng / mL) to the culture medium. The culture conditions were 37°C and 5% CO 2 Cultured in a constant temperature incubator.

[0045] (3) Adding indomethacin: On the third day of culture, indomethacin was added to the culture medium to a final concentration of 0.1 μM, and the culture was continued to promote cell proliferation and functional enhancement.

[0046] (4) Cell activation: Anti-CD3 and CD28 antibodies are used to activate NK cells to promote cell proliferation and activation. The culture cycle is 7 days. This step can also be expanded and cultured in the cell reactor as above.

[0047] (5) Regularly replace the culture medium: Replace the culture medium every 48 hours to ensure a continuous supply of growth factors and nutrients required by the cells to maintain cell proliferation and function.

[0048] (6) Cell collection and detection: After 7 days, the cultured NK cells were collected for subsequent experiments.

[0049] Example 3: NK cell function detection

[0050] 1. Cytological testing:

[0051] 1. Cytotoxicity detection (CD107a surface exposure method)

[0052] (1) Cell preparation: NK cells isolated from peripheral blood were expanded according to the above-mentioned culture method and divided into two groups: an experimental group (added with 0.1 μM indomethacin) and a control group (without indomethacin).

[0053] (2) Target cell preparation: K562 cells were used as target cells. K562 cells were lysed from red blood cells and then labeled with CFSE dye.

[0054] (3) Co-culture: NK cells were co-cultured with CFSE-labeled K562 target cells at a ratio of 1:1 and the incubation time was 4 hours.

[0055] (4) Antibody staining: Anti-CD107a antibody (FITC-labeled) was used for staining. The exposure of CD107a indicates the cytotoxic activity of NK cells.

[0056] (5) Flow cytometric analysis: The expression of CD107a was detected by flow cytometry to evaluate the cytotoxic activity of NK cells. The results showed (Table 1) that in the indomethacin-added group, the CD107a positive rate and cytotoxicity were significantly higher than those in the control group, indicating that indomethacin can enhance the killing ability of NK cells.

[0057] Table 1 Cytotoxicity test results

[0058]

[0059] 2. Cell proliferation detection (CFSE staining method)

[0060] (1) Cell preparation: NK cells isolated from peripheral blood were inoculated into culture medium containing different treatment conditions (experimental group and control group).

[0061] (2) CFSE staining: NK cells were stained with CFSE.

[0062] (3) Culture and stimulation: Culture was continued for 7 days, and L-15 / IL-21-Fc fusion protein (100 ng / mL), IFN-α (1000 IU / mL), Flt-3L (20 ng / mL) and indomethacin (0.1 μM) were added to activate NK cells.

[0063] (4) Analysis: Flow cytometry was used to detect the decay of CFSE staining and analyze the cell proliferation. The results showed (Table 2) that the proliferation rate of the indomethacin-added group was significantly higher than that of the control group, indicating that indomethacin can promote the proliferation of NK cells.

[0064] Table 2 Cell proliferation detection results

[0065]

[0066] 2. Mouse Animal Experiment

[0067] 1. NK cell tumor immunotherapy experiment

[0068] (1) Inoculation of mice with tumor cells: C57BL / 6 mice were selected and inoculated with mouse-derived hepatoma cells H22. Each mouse was inoculated with 1×10 6 H22 tumor cells.

[0069] (2) Grouping:

[0070] Experimental group: treated with NK cells cultured with indomethacin.

[0071] Control group: treated with NK cells cultured without indomethacin.

[0072] Blank group: only tumor cells were inoculated and no NK cell treatment was received.

[0073] (3) NK cell injection: 3 days after tumor cell inoculation, each group of mice received 5×10 6 NK cells were injected intravenously once a week for 4 weeks.

[0074] (4) Tumor volume monitoring: The tumor volume of mice was measured weekly and the tumor growth inhibition rate was calculated.

[0075] (5) After the experiment, the proportion of NK cells in the peripheral blood of mice was detected by flow cytometry to evaluate the persistence and activity of NK cells. The results showed (Table 3) that the tumor growth of mice in the indomethacin-added group was significantly inhibited, with a tumor inhibition rate of 65%, significantly higher than that of the control group (40%) and the blank group (0%). This indicates that NK cells with indomethacin addition have a stronger anti-tumor effect.

[0076] Table 3 Experimental results of NK cell tumor immunotherapy

[0077]

[0078] 2. Detection of immune cytokine secretion by NK cells

[0079] (1) Cell culture: NK cells were cultured for 7 days with or without the addition of indomethacin.

[0080] (2) Cytokine detection: The culture fluid was collected and the levels of immune factors such as IFN-γ, TNF-α, and IL-2 secreted by the cells were detected by ELISA. The results showed (Table 4) that the cytokine secretion in the indomethacin-added group was significantly higher than that in the control group, especially the secretion of IFN-γ and TNF-α, indicating that indomethacin can significantly enhance the immune activity of NK cells.

[0081] Table 4 NK cell immune cytokine secretion test results

[0082]

[0083] The above results show that, based on the results of cytological detection and mouse animal experiments, NK cells with added indomethacin are significantly superior to the control group without indomethacin in terms of cell proliferation, cytotoxicity, anti-tumor effect and immune cytokine secretion, proving that indomethacin can effectively improve the functionality of NK cells and enhance their potential in immunotherapy.

[0084] 3. NK cell expansion and testing

[0085] 1. The above-mentioned NK cells were continuously cultured according to the above method, and on the third day (after the cell activation in step (4) of Example 2), they were expanded and cultured in a cell reactor. The initial conditions were DO = 30%, pH = 7.2, temperature 37°C, stirring speed 50 rpm (corresponding to shear force 0.3 Pa), and the initial culture medium was as follows:

[0086] (1) Basic culture medium: RPMI-1640;

[0087] (2) Growth factors: 100 ng / mL IL-15 / IL-21-Fc fusion protein;

[0088] (3) Immunostimulatory factors: 1000 IU / mL IFN-α, 20 ng / mL Flt-3L;

[0089] (4) Modulator: 0.1 μM indomethacin;

[0090] (5) pH adjuster: 10 mM HEPES, 2 mM NaHCO 3 ;

[0091] (6) Cytokine activators: 1 μg / mL anti-CD3 and CD28 antibodies.

[0092] 2. After the initial culture, a gradient control procedure is performed, as follows:

[0093] (1) IL-15 / IL-21-Fc: Decrease by 5 ng / mL daily, i.e. D3 = 100 ng / mL → D14 = 50 ng / mL.

[0094] (2) Anti-CD3 and CD28 antibodies: increase by 1 μg / mL daily, i.e. D3 = 1 μg / mL → D14 = 12 μg / mL.

[0095] (3) DO control: Dynamically increase the stirring speed according to the cell density, and increase the stirring speed by 1×10 6 / mL, rotation speed +5rpm.

[0096] 3. The cell culture status was tracked at the same time and an anti-tumor activity comparison experiment was performed on day 14. The results are shown in Tables 5 and 6. The NK cells prepared by the method of the present invention have better effects and application efficacy than cells prepared by traditional methods.

[0097] Table 5 Comparison of NK cell expansion performance

[0098]

[0099] Table 6 Comparison of antitumor activity (effect-target ratio 5:1, 24h)

[0100]

[0101] Example 4: Design and verification of IL-15 / IL-21-Fc fusion protein

[0102] 1. Design of IL-15 / IL-21-Fc fusion protein (its amino acid sequence is shown in SEQ ID NO.1)

[0103] (1) IL-15 part (mutant): MKAQLLFLLLCLVTFAVQDTVKLMTRVLEFLLLQAGSLSELTNSLAIDETMQ.

[0104] Key mutations: D8N (enhances IL-15Rα binding), Q108S (reduces protease sensitivity).

[0105] (2) Flexible connecting peptide: (G4S)3→GGGGSGGGGSGGGGS.

[0106] (3) IL-21 part (mutant) APARVVRVLRQRKIDLNMALQNELEDLLQGSMKRDPNMQEEDKENILHLQCL.

[0107] Key mutations: R9K (enhances IL-21R binding), C69S (eliminates a non-functional disulfide bond).

[0108] (4) Hinge region: EPKSCDKTHTCPPCP.

[0109] (5) IgG4 Fc (LALA-PG mutation): APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG.

[0110] Key mutations: L234A / L235A (LALA) (abolishing FcγR binding (reducing ADCC / CDC)), S228P (IgG4-PG) (enhancing hinge stability).

[0111] 2. Technical advantages

[0112] (1) Structural innovation: IL-15 is located at the N-terminus and IL-21 is located in the middle, avoiding steric hindrance and ensuring simultaneous binding of IL-15Rβ / γc and IL-21R. (G4S)3 is used to ensure domain independence, which is verified by molecular dynamics simulation.

[0113] (2) Functional optimization:

[0114] IL-15 mutation: D8N increases the affinity of IL-15Rα by 3 times (SPR detection KD=0.8nM vs wild type 2.4nM).

[0115] IL-21 mutation: R9K enhances STAT3 phosphorylation efficiency (MFI increased by 1.8 times in flow cytometry).

[0116] (3) Long-term design:

[0117] IgG4 LALA-PG Fc: The half-life was extended to 12 times that of wild-type IL-15 (cynomolgus monkey PK experiment: t1 / 2=78hvs 6.5h).

[0118] 3. In vitro activity (human PBMC culture). The results are shown in Table 7. The synergistic effect of the fusion protein is significantly better than that of the single or mixed cytokines (P<0.001).

[0119] Table 7 In vitro activity test results

[0120]

[0121]

[0122] Example 5: Preparation and testing of anti-CD3 and CD28 antibodies

[0123] 1. Epitope Screening

[0124] (1) CD3 targeting epitope:

[0125] Epitope 1: Extracellular domain of CD3ε chain (aa 20-30): ILTCPQYPG (non-glycosylated region, high binding stability).

[0126] Epitope 2: constant region of CD3γ chain (aa 50-60): EFSELEQSGY (away from the TCR binding interface to reduce signal interference).

[0127] (2) CD28 targeting epitope:

[0128] Epitope 1: IgV-like domain of CD28 (aa 40-50): DNAVNLSCKY (co-stimulatory signaling critical region).

[0129] Epitope 2: intracellular juxtamembrane region of CD28 (aa 160-170): SKPDMDPKSC (avoids competition with CD80 / CD86).

[0130] 2. Experimental steps

[0131] 1. Antigen preparation and animal immunization

[0132] (1) Recombinant protein expression: The extracellular domains of CD3ε (UniProt P07766) and CD28 (UniProt P10747) were expressed (mammalian cell expression system, ensuring glycosylation modification). Purification was performed by Ni-NTA affinity chromatography (His tag).

[0133] (2) Immunization scheme: 5 6-8 week old SPF grade Balb / c mice. Day 0: CD3ε (20 μg) + CD28 (20 μg) + Freund's complete adjuvant (multiple subcutaneous injections). Days 14 and 28: CD3ε (20 μg) + CD28 (20 μg) + Freund's incomplete adjuvant. Day 35: CD3ε (20 μg) + CD28 (20 μg) booster immunization via tail vein.

[0134] 2. Hybridoma screening: Coat CD3 and CD28 proteins (1 μg / well each) and detect the binding activity of the supernatant (HRP-anti-mouse IgG secondary antibody). Select mice with OD450>2.0 (double antigen reaction) and CD3 / CD28 signal ratio between 0.8-1.2 for hybridoma cell screening. Pre-absorb the hybridoma supernatant onto CD19 or CD33 protein coated plates to remove non-specific antibodies.

[0135] 3. Bispecific Antibody Construction

[0136] (1) Heavy chain variable region (VH) and light chain variable region (VL) sequences: Based on hybridoma sequencing and humanization, variable region sequences with high affinity and low immunogenicity were obtained (Table 8):

[0137] Table 8 Sequence comparison

[0138]

[0139] CDR region design: Based on epitope prediction (IEDB) and molecular docking (RosettaAntibody), ensure that there is no spatial conflict in the binding of dual targets.

[0140] Humanization modification: Using the SDR (Specificity-Determining Residues) transplantation method, the mouse CDR region is retained and the framework region is replaced with human (humanization degree> 90%).

[0141] (2) Bispecific antibody construction: The structure is as follows:

[0142]

[0143] (Heterodimerization achieved through Knobs-into-Holes technology)

[0144] (3) Expression vector design

[0145] Plasmid 1: anti-CD3 VH-CH1 (containing KIH mutation: T366Y) + anti-CD28 VL-CL (containing F405L mutation).

[0146] Plasmid 2: anti-CD28 VH-CH1 (containing KIH mutation: Y407T) + anti-CD3 VL-CL (containing K392D mutation).

[0147] Host: ExpiCHO-S TM Cells (Thermo Fisher), transient transfection, Protein A purification (purity>95%).

[0148] 3. Functional Verification and Performance Comparison

[0149] 1. Binding affinity (SPR detection): The affinity of the bispecific antibody to CD3 / CD28 was 3-4 times higher than that of the traditional monoclonal antibody mixture group (P<0.001), as shown in Table 9.

[0150] Table 9 Binding affinity test results

[0151]

[0152] 2. T cell activation efficiency (PBMC vs Raji cells, effector-target ratio 10:1): The ability of dual antibodies to activate T cells to secrete IFN-γ was significantly higher than that of the traditional monoclonal antibody mixed group (P<0.01), as shown in Table 10.

[0153] Table 10 T cell activation efficiency test results

[0154]

[0155] 3. Tumor cell killing (Calcein-AM method, 24 hours): The killing efficiency of the dual antibody against low antigen-expressing solid tumors was increased by nearly 2 times (P<0.001), as shown in Table 11.

[0156] Table 11 Tumor cell killing test results

[0157]

[0158] IV. Large-scale production of antibodies

[0159] Process flow: plasmid construction → CHO cell transfection → perfusion culture (28 days) → Protein A purification → ion exchange chromatography (removal of aggregates) → nanofiltration (virus removal) → final product.

[0160] The screening of CHO monoclonal cells was performed using conventional methods or a third-party company, and the production and testing were completed by the research unit. The SDS-PAGE test results of the anti-CD3 and CD28 antibodies of the present invention are as follows: Figure 1 As shown, the purity can reach more than 95% and the expression level can reach 2.5g / L.

[0161] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A NK cell, characterized in that: After 14 days of in vitro culture, the number of viable NK cells was 112.5±9.8×10 6 / mL, CD56bright ratio was 63.2%±5.1%, and IFN-γ secretion was 824±67pg / 10 6 cells, the JC-1 red / green ratio of mitochondrial membrane potential was 4.8 ± 0.

4.

2. An initial culture medium for culturing the NK cells according to claim 1, characterized in that: The formula of the culture medium is as follows: (1) Basic culture medium: RPMI-1640; (2) Growth factors: 100 ng / mL IL-15 / IL-21-Fc fusion protein; (3) Immunostimulatory factors: 1000 IU / mL IFN-α, 20 ng / mL Flt-3L; (4) Modulator: 0.1 μM indomethacin; (5) pH adjuster: 10 mM HEPES, 2 mM NaHCO3; (6) Cytokine activators: 1 μg / mL anti-CD3 and CD28 antibodies.

3. The culture medium according to claim 2, characterized in that The amino acid sequence of the IL-15 / IL-21-Fc fusion protein is shown in SEQ ID NO.

1.

4. The culture medium according to claim 2, characterized in that The anti-CD3 and CD28 antibodies comprise anti-CD3VH, anti-CD3VL, anti-CD28VH and anti-CD28VL, wherein the amino acid sequences of anti-CD3VH, anti-CD3VL, anti-CD28VH and anti-CD28VL of the anti-CD3 and CD28 antibodies are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively.

5. A method for culturing the NK cells according to claim 1, characterized in that: The method comprises the following steps: (1) Cell inoculation: NK cells were isolated from peripheral blood or umbilical cord blood and inoculated into the pre-prepared culture medium at a cell inoculation density of 1×10 6 cells / mL; (2) Adding growth factors: adding 100 ng / mL L-15 / IL-21-Fc fusion protein, 1000 IU / mL IFN-α, and 20 ng / mL Flt-3L to the culture medium, and culturing in a constant temperature incubator at 37°C and 5% CO2; (3) Adding indomethacin: On the third day of culture, indomethacin was added to the culture medium to a final concentration of 0.1 μM, and the culture was continued to promote cell proliferation and functional enhancement; (4) Cell activation: Activate NK cells using anti-CD3 and CD28 antibodies to promote cell proliferation and activation. Then, expand and culture in a cell reactor. The initial conditions of the reactor are DO = 30%, pH = 7.2, temperature 37°C, and stirring speed 50 rpm. The culture medium used at this time is the initial culture medium described in claim 2; (5) After the initial culture, a gradient control procedure is performed, as follows: 1) IL-15 / IL-21-Fc: Decrease by 5 ng / mL daily, i.e. D3 = 100 ng / mL → D14 = 50 ng / mL; 2) Anti-CD3 and CD28 antibodies: increase by 1 μg / mL daily, i.e. D3 = 1 μg / mL → D14 = 12 μg / mL); 3) DO control: Dynamically increase the stirring speed according to the cell density, and increase the stirring speed by 1×10 6 / mL, rotation speed +5rpm.

6. Use of the culture medium as claimed in claim 2 in NK cell culture.

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