NK (Natural Killer) cell culture solution as well as culture and application of NK cells

Abstract

The invention discloses an NK cell culture solution and culture and application of NK cells, the NK cell culture solution comprises an activation culture solution and an amplification culture solution, the activation culture solution and the amplification culture solution respectively contain a basal culture medium and respective corresponding cell factors, and the activation culture solution also contains calycosin with the final concentration of 0.1-10 [mu] g / mL and formononetin with the final concentration of 1-20 [mu] g / mL; the amplification culture solution also contains astragaloside IV and soyasaponin I. The components such as calycosin, formononetin, astragaloside IV and soyasaponin I added into the NK cell culture fluid can maintain stable and lasting physiological activity of NK cells and slow down cell senescence, so that secretion of cell factors is promoted, the secretion level of the cell factors is higher, and the purposes of enhancing the persistence of cell activity and improving the killing rate of the NK cells are achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell culture protocols, in particular to a kind of NK cell culture medium preparation, and using the culture medium to carry out the culture method of NK cell and the application of the NK cell prepared in the preparation of the drug for treating tumor or cancer. BACKGROUND

[0002] Natural killer (NK) cells are an important cell type in the innate immune system, with the ability to recognize and kill tumor cells, virus-infected cells and other pathogenic microorganisms. Due to its important role in immune surveillance and tumor immunotherapy, NK cell immunotherapy is becoming a reliable anticancer therapy, suitable for the clinical treatment of various malignant tumors, and has little side effects and does not damage normal tissues. Therefore, in recent years, NK cells have become a research hotspot in the field of immunotherapy, especially in the application of cellular immunotherapy.

[0003] At present, in the culture method of NK cells, cytokine stimulation and receptor activator stimulation culture method requires a certain proportion of other lymphocytes (such as T lymphocytes, monocytes, etc.) in the early stage, which has the problems of difficult control of the proportion of other lymphocytes due to individual differences and uneven final NK purity, and high cell exhaustion; while the feeder cell stimulation culture method will cause the residue of foreign cells, which has certain exogenous pollution, reduces the cell activity and persistence; these deficiencies limit the feasibility of large-scale clinical application of NK cells. SUMMARY

[0004] Based on the above problems, the problem to be solved by the present application is to provide an NK cell culture medium with high purity, low consumption, good cell activity and safety and reliability.

[0005] The technical scheme of the present application is as follows:

[0006] An NK cell culture medium comprises an NK cell activation culture medium and an NK cell expansion culture medium, and the NK cell activation culture medium and the NK cell expansion culture medium both use a basic culture medium as a base liquid; wherein:

[0007] The NK cell activation culture solution contains IL-2 with a final concentration of 100-2000 IU / mL, IL-15 with a final concentration of 5-20 ng / mL, IL-18 with a concentration of 10-50 ng / mL, IL-27 with a final concentration of 5-15 ng / mL, SCM with a final concentration of 0.1-5 ng / mL, IFN-γ with a final concentration of 1-10 ng / mL, autologous blood plasma with a final concentration of 1-10 v / v%, Calycosin with a final concentration of 1-20 ug / mL, and Formononetin with a final concentration of 1-20 ug / mL in the basic culture medium.

[0008] The NK cell expansion culture solution contains IL-2 with a final concentration of 100-2000 IU / mL, IL-15 with a final concentration of 5-20 ng / mL, IL-18 with a concentration of 10-50 ng / mL, autologous blood plasma with a final concentration of 1-10 v / v%, Astragalosides with a final concentration of 5-50 ug / mL, and Soyasapogenin I with a final concentration of 1-10 ug / mL in the basic culture medium.

[0009] Preferably, the NK cell culture solution contains:

[0010] The basic culture medium of the NK cell activation culture solution contains IL-2 with a final concentration of 500-1000 IU / mL, IL-15 with a final concentration of 8-16 ng / mL, IL-18 with a concentration of 20-30 ng / mL, IL-27 with a final concentration of 8-12 ng / mL, SCM with a final concentration of 0.5-3 ng / mL, IFN-γ with a final concentration of 3-8 ng / mL, autologous blood plasma with a final concentration of 3-8 v / v%, Calycosin with a final concentration of 1-8 ug / mL, and Formononetin with a final concentration of 5-15 ug / mL.

[0011] The basic culture medium of the NK cell expansion culture solution contains IL-2 with a final concentration of 500-1000 IU / mL, IL-15 with a final concentration of 8-16 ng / mL, IL-18 with a concentration of 30-30 ng / mL, autologous blood plasma with a final concentration of 3-8 v / v%, Astragalosides with a final concentration of 10-30 ug / mL, and Soyasapogenin I with a final concentration of 3-8 ug / mL.

[0012] Preferably, the basic culture medium of the NK cell culture solution is one of DMEM / F12, IMDM, 581, and RPMI-1640 culture medium.

[0013] The application also provides a culture method using the above-mentioned NK cell culture solution, which comprises the following steps:

[0014] On day 0, first, resuspend mononuclear cells with the above-mentioned NK cell activation culture solution, and control the cell density to be 1.0 x 10 6 ~ 3.0 x 10 6 individuals / mL; then, inoculate the resuspended cells into the coated T75 culture flask; finally, incubate at 37℃ in a 5% CO2 incubator;

[0015] On day 3, add the above-mentioned NK cell expansion culture solution to the culture flask, and control the cell density to be 1.0 x 10 6 ~ 3.0 x 10 6 individuals / mL, and continue to culture;

[0016] On day 5, transfer the cells and culture solution in the culture flask to a 10L culture bag, add the above-mentioned NK cell expansion culture solution, and control the cell density to be 1.0 x 10 6 ~ 3.0 x 10 6 individuals / mL, and continue to culture at 37℃ in a 5% CO2 incubator;

[0017] From day 7 to day 13, every two days, add the above-mentioned NK cell expansion culture solution to the culture bag, and control the cell density to be 1.0 x 10 6 ~ 3.0 x 10 6 individuals / mL, and continue to culture;

[0018] On day 14, stop the culture, collect the cells and culture solution, centrifuge at 900g for 10min, remove the supernatant, and wash the cell pellet with PBS multiple times to obtain high-purity NK cells.

[0019] Preferably, in the NK cell culture method, on day 0, the coated T75 culture flask is obtained by the following steps:

[0020] First, prepare the coating solution: add and contain 20-80μg / mL heparin sodium and 0.1-20μg / mL CD16 monoclonal antibody in PBS to obtain a final concentration;

[0021] Second, take the coating solution and add it to the T75 culture flask, incubate in a 2-8℃ refrigerator, and end the incubation after the inner wall of the culture flask forms a coating film. Discard the remaining coating solution;

[0022] Finally, add PBS to the culture flask to wash the coating film, and remove the washed PBS to obtain a coated culture flask.

[0023] Preferably, in the above-mentioned NK cell culture method, the steps include:

[0024] On day 0, first, resuspend mononuclear cells with the NK cell activation culture solution as claimed in claim 1, and control the cell density to be 2.0*10 6 ML; then, inoculate the resuspended cells into a coated T75 culture flask; finally, incubate at 37℃ in a 5% CO2 incubator;

[0025] On day 3, add the NK cell expansion culture solution as claimed in claim 1 into the culture flask, and control the cell density to be 2.0*10 6 ML, and continue to incubate;

[0026] On day 5, transfer the cells and culture solution in the culture flask into a 10L culture bag, add the NK cell expansion culture solution as claimed in claim 1, and control the cell density to be 2.0*10 6 ML, and continue to incubate at 37℃ in a 5% CO2 incubator;

[0027] From day 7 to day 13, every two days, add the NK cell expansion culture solution as claimed in claim 1 into the culture bag, and control the cell density to be 2.0*10 6 ML, and continue to incubate;

[0028] On day 14, stop the incubation, collect the cells and culture solution, centrifuge at 900g for 10min, remove the supernatant, and wash the cell precipitate with PBS for multiple times to obtain high-purity NK cells.

[0029] The cultured NK cells can exist as a biological preparation or a component of a biological preparation, and the biological preparation can be widely applied to prepare a component of a drug for treating tumors or cancers.

[0030] In the NK culture, calycosin, formononetin, astragaloside IV and soybean saponin I are added into the activation culture solution and the expansion culture solution respectively, and the following technical advantages can be obtained:

[0031] 1. The components of astragaloside IV and soybean saponin I in the expansion culture solution can maintain the activity of NK cells, improve the cell purity (up to about 95%), promote the secretion of cytokines such as TNF-α, IL-1β, IL-6 and FasL, and slow down cell aging;

[0032] 2. The components of calycosin, formononetin, astragaloside IV and soybean saponin I in the NK cell culture solution can reduce the concentration of inhibitory receptors (TIM-3, TIGIT and KLGR1), resist NK cell aging, enhance the persistence of cell activity, and improve the tumor killing rate of NK cells;

[0033] 3. The activation culture solution and the expansion culture solution for the culture of NK cells do not contain exogenous cells, and are safe and reliable. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Flow cytometry diagram of the purity of CD3-CD56+ of the NK cells cultured in Example 1;

[0035] Figure 2 Flow cytometry diagram of the purity of CD3-CD56+ of the NK cells cultured in Example 6;

[0036] Figure 3 Columnar diagram of the concentration ratio of the secreted cytokines of the NK cells cultured in Examples 1 and 6;

[0037] Figure 4 Columnar diagram of the concentration ratio of the exhaustion markers of the NK cells cultured in Examples 1 and 6;

[0038] Figure 5 Curve diagram of the tumor killing rate of the NK cells cultured in Examples 1 and 6. DETAILED DESCRIPTION

[0039] The preferred embodiments of the present application will be further described in detail below with reference to the accompanying drawings.

[0040] The seed cells used in the in-vitro expansion culture of NK cells can be NK seed cells obtained through subculture, or mononuclear cells separated from umbilical cord blood or peripheral blood. In the present application, mononuclear cells are used as the seed cells for the in-vitro expansion culture of NK cells.

[0041] I. Obtaining mononuclear cells

[0042] In the culture of NK cells according to the present application, the mononuclear cells (PBMC) separated from peripheral blood are used as the seed cells, and the process is as follows:

[0043] S11. Collect 80 ml of peripheral blood from a volunteer in a sterile heparin sodium blood collection tube;

[0044] S12. Transfer the blood into a 50 ml centrifuge tube, and after centrifugation, obtain the plasma and whole blood cells;

[0045] S13. Combine the plasma obtained in step S12, and then place it in a 56℃ water bath for inactivation for 20 min, and then place it in a -20℃ environment for quick freezing for 15 min, and then centrifuge at 1500 g for 30 min to obtain clear plasma, which is stored in a 2-8℃ environment for standby use;

[0046] S14, dilute the whole blood cells obtained in step S12 with physiological saline at a ratio of 1:1, then slowly add into the lymph separation liquid at a ratio of 1:2, try to ensure clear layering, 650g, 1min, centrifugal 30min, absorb the white membrane layer, then wash with physiological saline twice, collect the mononuclear cells PBMC, and reserve for use.

[0047] In other embodiments of the present application, the mononuclear cells can also be obtained by umbilical cord blood separation, i.e. umbilical cord blood mononuclear cells (CMBC), and the separation process is the same as that of peripheral blood mononuclear cells.

[0048] II. Preparation of NK cell culture solution

[0049] The NK cell culture solution provided by the present application comprises two kinds of culture solutions, one is an activation culture solution for activating the growth of NK cells (also referred to as NK cell activation culture solution), and the other is a culture solution for promoting the rapid expansion of NK cells (also referred to as NK cell expansion culture solution), and both of the two culture solutions are based on a basic culture medium, and corresponding cytokines are added in the corresponding basic solution; the specific preparation of the culture solution is as follows:

[0050] Preparation of NK cell activation culture solution: add and contain 100-2000IU / mL of IL-2, 5-20ng / mL of IL-15, 10-50ng / mL of IL-18, 5-15ng / mL of IL-27, 0.1-5ng / mL of SCM, 1-10ng / mL of IFN-γ, 1-10v / v% of autologous plasma, 0.1-10ug / mL of Calycosin and 1-20ug / mL of Formononetin in the basic culture medium with final concentration;

[0051] Preparation of NK cell expansion culture solution: add and contain 100-2000IU / mL of IL-2, 5-20ng / mL of IL-15, 10-50ng / mL of IL-18, 1-10v / v% of autologous plasma, 5-50ug / mL of Astragalosides and 1-10ug / mL of Soybean saponin I in the basic culture medium with final concentration.

[0052] Preferably, when the above-mentioned NK cell culture solution is prepared, the preparation scheme can be as follows:

[0053] Preparation of NK cell activation culture medium: adding and containing final concentration of 500-1000 IU / mL IL-2, final concentration of 8-16 ng / mL IL-15, concentration of 20-30 ng / mL IL-18, final concentration of 8-12 ng / mL IL-27, final concentration of 0.5-3 ng / mL SCM, final concentration of 3-8 ng / mL IFN-γ, final concentration of 3-8 v / v% autologous plasma, final concentration of 1-8 ug / mL Calycosin and final concentration of 5-15 ug / mL Formononetin in the basic culture medium;

[0054] Preparation of NK cell expansion culture medium: adding and containing final concentration of 500-1000 IU / mL IL-2, final concentration of 8-16 ng / mL IL-15, concentration of 20-30 ng / mL IL-18, final concentration of 8-12 ng / mL IL-27, final concentration of 0.5-3 ng / mL SCM, final concentration of 3-8 ng / mL IFN-γ, final concentration of 3-8 v / v% autologous plasma, final concentration of 1-8 ug / mL Calycosin and final concentration of 5-15 ug / mL Formononetin in the basic culture medium;

[0055] Further, when the above-mentioned NK cell culture medium is prepared, the preparation scheme can be as follows:

[0056] Preparation of NK cell activation culture medium: adding and containing final concentration of 500-1000 IU / mL IL-2, final concentration of 8-16 ng / mL IL-15, concentration of 20-30 ng / mL IL-18, final concentration of 8-12 ng / mL IL-27, final concentration of 0.5-3 ng / mL SCM, final concentration of 3-8 ng / mL IFN-γ, final concentration of 3-8 v / v% autologous plasma, final concentration of 1-8 ug / mL Calycosin and final concentration of 5-15 ug / mL Formononetin in the basic culture medium;

[0057] Preparation of NK cell expansion culture medium: adding and containing final concentration of 500-1000 IU / mL IL-2, final concentration of 8-16 ng / mL IL-15, concentration of 20-30 ng / mL IL-18, final concentration of 8-12 ng / mL IL-27, final concentration of 0.5-3 ng / mL SCM, final concentration of 3-8 ng / mL IFN-γ, final concentration of 3-8 v / v% autologous plasma, final concentration of 1-8 ug / mL Calycosin and final concentration of 5-15 ug / mL Formononetin in the basic culture medium;

[0058] In the preparation of the above-mentioned NK cell culture medium, the basic culture medium can be selected from any one of DMEM / F12, IMDM, 581 or RPMI-1640 medium; and when the activation culture medium and the expansion culture medium of the same batch of NK cells are prepared, the selected basic culture medium is the same, that is, only any one of DMEM / F12, IMDM, 581 or RPMI-1640 medium can be selected.

[0059] The components of the above-mentioned NK cell culture medium, including basal culture medium, IL-2, IL-15, IL-18, IL-27, SCM, IFN-γ, and autologous plasma, are all conventional components used in NK cell culture.

[0060] III. Coated Culture Flasks

[0061] After preparing the NK cell culture medium, a coating medium must also be prepared to coat the inner wall of the culture flask and promote NK cell adhesion.

[0062] Coating solution: Based on PBS, with added heparin sodium at a final concentration of 20–80 μg / mL and CD16 monoclonal antibody at a final concentration of 0.1–20 μg / mL.

[0063] Preferably, the coating solution is prepared as follows:

[0064] Coating solution: Based on PBS, with added sodium heparin at a final concentration of 50 μg / mL and CD16 monoclonal antibody at a final concentration of 8 μg / mL.

[0065] After preparing the coating solution, the next step is to coat the culture flasks. The specific process is as follows:

[0066] First, take 30 mL of the above-prepared coating solution and add it to a T75 culture flask. Shake gently to coat the inner wall of the bottom of the T75 culture flask with the coating solution.

[0067] Subsequently, the culture flasks were placed in a refrigerator at 2–8°C for incubation. The incubation ended when a coating film formed on the inner wall of the culture flasks, and the remaining coating solution was aspirated.

[0068] Finally, add PBS to the culture flask, wash the coated membrane, and remove the washed PBS to obtain the coated culture flask for later use.

[0069] IV. NK Cell Culture

[0070] After completing the above preparations, the next step is to culture NK cells, and the process steps are as follows:

[0071] On day 0, firstly, the mononuclear cells isolated in step 2 were resuspended in the NK cell activation culture medium prepared in step 1 above, and the cell density was controlled at 1.0 × 10⁻⁶. 6 ~3.0×10 6 Cells / mL; then, the resuspended cells were seeded into the T75 culture flasks coated in the third step above; finally, they were incubated statically in an incubator at 37°C and 5% CO2.

[0072] On the 3rd day, the NK cell expansion culture solution prepared in the first item above was added to the culture bottle, and the cell density was controlled at 1.0 x 10 6 ~ 3.0 x 10 6 cells / mL, and the culture was continued.

[0073] On the 5th day, the cells and culture solution in the culture bottle were transferred to a 10L culture bag, the NK cell expansion culture solution prepared in the first item above was added, and the cell density was controlled at 1.0 x 10 6 ~ 3.0 x 10 6 cells / mL, and the culture was continued in a 37°C, 5% CO2 incubator.

[0074] On the 7th day to the 13th day, the NK cell expansion culture solution prepared in the first item above was added to the culture bag every two days, and the cell density was controlled at 1.0 x 10 6 ~ 3.0 x 10 6 cells / mL, and the culture was continued.

[0075] On the 14th day, the culture was stopped, the cells and culture solution were collected, centrifuged at 900g for 10 min, the supernatant was removed, and the cell pellet was washed with PBS multiple times to obtain high-purity NK cells.

[0076] The NK cells prepared by the above culture method can be used to prepare drugs for preventing or treating tumors or cancers. For example, the NK cells can be used as components of biological agents to prepare drugs for preventing or treating solid tumors such as gastric cancer, rectal cancer, liver cancer, and pancreatic cancer.

[0077] When the NK cells prepared by the above culture method are used as biological agents, they exist as components of biological agents, i.e., pharmaceutically acceptable diluents or excipients, etc.

[0078] The technical solutions of the present application will be further described in detail below through some specific examples.

[0079] In the following examples, the autologous plasma components used to prepare the culture solution and the PBMCs used to culture NK cells are derived from steps S13 and S14 of the first item above, and the PBMCs are derived from the same batch of peripheral blood.

[0080] (I) Cell culture

[0081] Example 1

[0082] 1.1 Preparation of culture solution and coating solution

[0083] Preparation of 30 mL NK cell activation culture solution: DMEM / F12 medium was taken and added with IL-2 at a final concentration of 1000 IU / mL, IL-15 at a final concentration of 13 ng / mL, IL-18 at a concentration of 20 ng / mL, IL-27 at a final concentration of 10 ng / mL, SCM at a final concentration of 2 ng / mL, IFN-γ at a final concentration of 6 ng / mL, autologous plasma at a final concentration of 5 v / v %, puerarin at a final concentration of 4 ug / mL, and formononetin at a final concentration of 10 ug / mL;

[0084] Preparation of 2000 mL NK cell expansion culture solution: DMEM / F12 medium was taken and added with IL-2 at a final concentration of 1000 IU / mL, IL-15 at a final concentration of 13 ng / mL, IL-18 at a concentration of 20 ng / mL, autologous plasma at a final concentration of 5 v / v %, astragaloside IV at a final concentration of 12 ug / mL, and soybean saponin I at a final concentration of 5 ug / mL;

[0085] Preparation of 40 mL coating solution: PBS was taken and added with heparin sodium at a final concentration of 45 ug / mL and CD16 monoclonal antibody at a final concentration of 10 ug / mL.

[0086] 1.2, culture bottle coating

[0087] First, 30 mL of the coating solution prepared in this embodiment was added to a T75 culture bottle, and the coating solution was coated on the inner wall of the bottom of the T75 culture bottle by gently shaking. Then, the culture bottle was incubated in a 4°C refrigerator for 16 hours. After the inner wall of the culture bottle formed a coating film, the remaining coating solution was removed by suction with a pipette. Finally, PBS was added to the culture bottle to wash the coating film, and the washed PBS was removed to obtain a coated culture bottle, which was ready for use.

[0088] 1.3, NK cell culture

[0089] On day 0, first, 20 mL of the NK cell activation culture solution prepared in step 1.1 of embodiment 1 was taken to resuspend 4.0 x 10 7 individual nucleated cells to obtain a cell suspension with a cell density of 2.0 x 10 6 individual nucleated cells / mL. Then, the cell suspension was inoculated into the T75 culture bottle coated in step 1.2 of embodiment 1. Finally, the cells were placed in a 37°C, 5% CO2 incubator for static culture.

[0090] On day 3, the NK cell expansion culture solution prepared in step 1.1 of embodiment 1 was added to the culture bottle, and the cell density was controlled at 2.0 x 10 6 individual nucleated cells / mL for continuous culture.

[0091] On day 5, the cells and culture solution in the culture flask were transferred to a 10-L culture bag, and the NK cell expansion culture solution prepared in step 1.1 of Example 1 was added to supplement, and the cell density was controlled at 2.0 x 10 6 The cells were cultured at 37°C in a 5% CO2 incubator.

[0092] On day 7 to day 13, the NK cell expansion culture solution prepared in step 1.1 of Example 1 was added to supplement the culture bag every two days, and the cell density was controlled at 2.0 x 10 6 The cells were cultured at 37°C in a 5% CO2 incubator.

[0093] On day 14, the culture was stopped, and the cells and culture solution were collected, centrifuged at 900g for 10 min, and the supernatant was removed. The cell pellet was washed with PBS multiple times to obtain high-purity NK cells.

[0094] Example 2

[0095] 2.1. Preparation of culture solution and coating solution

[0096] Preparation of 30 mL NK cell activation culture solution: Take 581 medium, add and contain IL-2 at a final concentration of 100 IU / mL, IL-15 at a final concentration of 5 ng / mL, IL-18 at a concentration of 10 ng / mL, IL-27 at a final concentration of 15 ng / mL, SCM at a final concentration of 0.1 ng / mL, IFN-γ at a final concentration of 1 ng / mL, autologous plasma at a final concentration of 1 v / v%, formononetin at a final concentration of 0.1 ug / mL, and calycosin at a final concentration of 1 ug / mL.

[0097] Preparation of 2000 mL NK cell expansion culture solution: Take 581 medium, add and contain IL-2 at a final concentration of 100 IU / mL, IL-15 at a final concentration of 5 ng / mL, IL-18 at a concentration of 10 ng / mL, autologous plasma at a final concentration of 10 v / v%, astragalus glycoside IV at a final concentration of 5 ug / mL, and soybean saponin I at a final concentration of 10 ug / mL.

[0098] Preparation of 40 mL coating solution: Take PBS, add and contain heparin sodium at a final concentration of 20 ug / mL, and CD16 monoclonal antibody at a final concentration of 0.1 ug / mL.

[0099] 2.2. Culture flask coating

[0100] First, take 30 mL of the coating solution prepared in this example, add it to a T75 culture flask, and gently shake it to coat the inner wall of the bottom of the T75 culture flask; then, place the culture flask in a 2°C refrigerator and incubate it for 16 hours; after the coating film forms on the inner wall of the culture flask, discard the remaining coating solution with a pipette; finally, add PBS to the culture flask to wash the coating film, and discard the PBS after washing to obtain a coated culture flask, which is ready for use.

[0101] 2.3, NK cell culture

[0102] On day 0, first, take 20 mL of the NK cell activation culture solution prepared in step 2.1 of Example 2, resuspend 2.0 x 10 7 cells / mL of the single nucleus cells to obtain a cell suspension; then, inoculate the cell suspension into the coated T75 culture flask prepared in step 2.2 of Example 2; finally, place the cells in a 37°C, 5% CO2 incubator for static culture; 6

[0103] On day 3, add the NK cell expansion culture solution prepared in step 2.1 of Example 2 to the culture flask, and control the cell density to be 1.0 x 10 6 cells / mL for continued culture;

[0104] On day 5, transfer the cells and culture solution in the culture flask to a 10L culture bag, add the NK cell expansion culture solution prepared in step 2.1 of Example 2, and control the cell density to be 1.0 x 10 6 cells / mL for continued culture in a 37°C, 5% CO2 incubator;

[0105] From day 7 to day 13, every two days, add the NK cell expansion culture solution prepared in step 2.1 of Example 2 to the culture bag, and control the cell density to be 2.0 x 10 6 cells / mL for continued culture;

[0106] On day 14, stop the culture, collect the cells and culture solution, centrifuge at 900g for 10 minutes, discard the supernatant, and wash the cell pellet with PBS multiple times to obtain high-purity NK cells.

[0107] Example 3

[0108] 3.1, Preparation of culture solution and coating solution

[0109] ​Preparation of 30 mL NK cell activation culture solution: take RPMI-1640 culture medium, add and contain final concentration of 2000 IU / mL IL-2, final concentration of 20 ng / mL IL-15, concentration of 50 ng / mL of IL-18, final concentration of 5 ng / mL IL-27, final concentration of 5 ng / mL SCM, final concentration of 10 ng / mL IFN-γ, final concentration of 10 v / v% autologous plasma, final concentration of 10 ug / mL calycosin and final concentration of 20 ug / mL formononetin;

[0110] Preparation of 2000 mL NK cell expansion culture solution: take RPMI-1640 culture medium, add and contain final concentration of 2000 IU / mL IL-2, final concentration of 20 ng / mL IL-15, concentration of 50 ng / mL of IL-18, final concentration of 1 v / v% autologous plasma, final concentration of 50 ug / mL astragaloside IV and final concentration of 1 ug / mL soybean saponin I;

[0111] Preparation of 40 mL coating solution: take PBS, add and contain final concentration of 80 ug / mL heparin sodium and final concentration of 20 ug / mL CD16 monoclonal antibody in PBS.

[0112] 3.2, culture bottle coating

[0113] First, take 30 mL of the coating solution prepared in this embodiment and add it to a T75 culture bottle. Gently shake the bottle to allow the coating solution to coat the inner wall of the bottom of the T75 culture bottle. Then, place the culture bottle in an 8℃ refrigerator for 16h of incubation. After the inner wall of the culture bottle forms a coating film, discard the remaining coating solution with a pipette. Finally, add PBS to the culture bottle to wash the coating film, and remove the washed PBS to obtain a coated culture bottle, which is ready for use.

[0114] 3.3, NK cell culture

[0115] On day 0, first, take 20 mL of the NK cell activation culture solution prepared in step 3.1 of embodiment 3, resuspend 6.0×10 7 individual nucleated cells to obtain a cell suspension with a cell density of 3.0×10 6 individual nucleated cells / mL; then, inoculate the cell suspension into the T75 culture bottle coated in step 3.2 of embodiment 3; finally, place the cells in a 37℃, 5% CO2 incubator for static culture;

[0116] On day 3, add the NK cell expansion culture solution prepared in step 3.1 of embodiment 3 to the culture bottle, and control the cell density to be 3.0×10 6 individual nucleated cells / mL for continuous culture;

[0117] On day 5, the cells and culture solution in the culture flask were transferred to a 10 L culture bag, and the NK cell expansion culture solution prepared in step 3.1 of Example 3 was added to supplement, and the cell density was controlled at 3.0 x 10 6 The culture was continued at 37°C in a 5% CO2 incubator.

[0118] On day 7 to day 13, the NK cell expansion culture solution prepared in step 3.1 of Example 3 was added to supplement the culture bag every two days, and the cell density was controlled at 3.0 x 10 6 The culture was continued.

[0119] On day 14, the culture was stopped, and the cells and culture solution were collected, centrifuged at 900g for 10 min, and the supernatant was removed. The cell pellet was washed with PBS multiple times to obtain high-purity NK cells.

[0120] Example 4 (Comparative Example 1)

[0121] 4.1. Preparation of culture solution and coating solution

[0122] 30 mL of NK cell activation culture solution was prepared by adding and containing 1000 IU / mL of IL-2, 13 ng / mL of IL-15, 20 ng / mL of IL-18, 10 ng / mL of IL-27, 2 ng / mL of SCM, 6 ng / mL of IFN-γ, 5 v / v% of autologous plasma, 4 ug / mL of calycosin, and 10 ug / mL of formononetin to DMEM / F12 medium to a final concentration.

[0123] 2000 mL of NK cell expansion culture solution was prepared by adding and containing 1000 IU / mL of IL-2, 13 ng / mL of IL-15, 20 ng / mL of IL-18, and 5 v / v% of autologous plasma to DMEM / F12 medium to a final concentration.

[0124] 40 mL of coating solution was prepared by adding and containing 45 ug / mL of heparin sodium and 10 ug / mL of CD16 monoclonal antibody to PBS to a final concentration.

[0125] 4.2. Culture flask coating

[0126] First, 30 mL of the coating solution prepared in this example was added to a T75 culture flask, and the culture flask was gently shaken to coat the inner wall of the bottom of the T75 culture flask with the coating solution. Then, the culture flask was incubated in a 4°C refrigerator for 16 h, and after the coating film was formed on the inner wall of the culture flask, the remaining coating solution was removed with a pipette. Finally, PBS was added to the culture flask to wash the coating film, and the PBS after washing was removed to obtain a coated culture flask, which was ready for use.

[0127] 4.3, NK cell culture

[0128] On day 0, first, take 20 mL of the NK cell activation culture solution prepared in step 4.1 of this Example 4, resuspend 4.0 x 10 7 cells to obtain a cell suspension with a cell density of 2.0 x 10 6 cells / mL; then, inoculate the cell suspension into the T75 culture flask coated in step 4.2 of this Example 4; finally, place the cells in a 37°C, 5% CO2 incubator for static culture;

[0129] On day 3, add the NK cell expansion culture solution prepared in step 4.1 of this Example 4 to the culture flask, and control the cell density to be 2.0 x 10 6 cells / mL for continued culture;

[0130] On day 5, transfer the cells and culture solution in the culture flask to a 10 L culture bag, add the NK cell expansion culture solution prepared in step 4.1 of this Example 4, and control the cell density to be 2.0 x 10 6 cells / mL for continued culture in a 37°C, 5% CO2 incubator;

[0131] From day 7 to day 13, every two days, add the NK cell expansion culture solution prepared in step 4.1 of this Example 4 to the culture bag, and control the cell density to be 2.0 x 10 6 cells / mL for continued culture;

[0132] On day 14, stop the culture, collect the cells and culture solution, centrifuge at 900g for 10 min, remove the supernatant, and wash the cell pellet with PBS multiple times to obtain high-purity NK cells.

[0133] Example 5 (Comparative Example 2)

[0134] 5.1, Preparation of culture solution and coating solution

[0135] Prepare 30 mL of NK cell activation culture solution: take DMEM / F12 culture medium, and add and contain IL-2 with a final concentration of 1000 IU / mL, IL-15 with a final concentration of 13 ng / mL, IL-18 with a concentration of 20 ng / mL, IL-27 with a final concentration of 10 ng / mL, SCM with a final concentration of 2 ng / mL, IFN-γ with a final concentration of 6 ng / mL, and autologous plasma with a final concentration of 5 v / v%;

[0136] Prepare 2000 mL NK cell expansion culture solution: take DMEM / F12 medium, add and contain final concentration of 1000 IU / mL IL-2, final concentration of 13 ng / mL IL-15, concentration of 20 ng / mL IL-18, final concentration of 5 v / v% autologous plasma, astragalus glycoside IV and final concentration of 1 ug / mL soybean saponin I;

[0137] Prepare 40 mL coating solution: take PBS, add and contain final concentration of 45 ug / mL heparin sodium and final concentration of 10 ug / mL CD16 monoclonal antibody in PBS.

[0138] 5.2, flask coating

[0139] First, take 30 mL of the coating solution prepared in this embodiment and add it to a T75 culture flask. Gently shake the flask to ensure that the coating solution covers the inner wall of the bottom of the T75 culture flask. Then, place the culture flask in a 4°C refrigerator for 16 hours of incubation. After the inner wall of the culture flask forms a coating film, discard the remaining coating solution with a pipette. Finally, add PBS to the culture flask to wash the coating film, and then remove the washed PBS to obtain a coated culture flask for later use.

[0140] 5.3, NK cell culture

[0141] On day 0, first, take 20 mL of the NK cell activation culture solution prepared in step 5.1 of embodiment 5, resuspend 4.0 x 10 7 individual nucleated cells to obtain a cell suspension with a cell density of 2.0 x 10 6 individual nucleated cells; then, inoculate the cell suspension into the T75 culture flask coated in step 5.2 of embodiment 5; finally, place the cells in a 37°C, 5% CO2 incubator for static culture.

[0142] On day 3, add the NK cell expansion culture solution prepared in step 5.1 of embodiment 5 to the culture flask, and control the cell density to be 2.0 x 10 6 individual nucleated cells / mL for continued culture.

[0143] On day 5, transfer the cells and culture solution in the culture flask to a 10 L culture bag, add the NK cell expansion culture solution prepared in step 5.1 of embodiment 5, and control the cell density to be 2.0 x 10 6 individual nucleated cells / mL for continued culture in a 37°C, 5% CO2 incubator.

[0144] From day 7 to day 13, every two days, add the NK cell expansion culture solution prepared in step 5.1 of embodiment 5 to the culture bag, and control the cell density to be 2.0 x 10 6 individual nucleated cells / mL for continued culture.

[0145] On day 14, culture was stopped, cells and culture medium were collected, centrifuged at 900g for 10 minutes, the supernatant was removed, and the cell pellet was washed multiple times with PBS to obtain high-purity NK cells.

[0146] Example 6 (Comparative Example 3)

[0147] 6.1 Preparation of culture medium and coating solution

[0148] Prepare 30 mL of NK cell activation culture medium: Take DMEM / F12 medium and add IL-2 with a final concentration of 1000 IU / mL, IL-15 with a final concentration of 13 ng / mL, IL-18 with a final concentration of 20 ng / mL, IL-27 with a final concentration of 10 ng / mL, IL-CM with a final concentration of 2 ng / mL, IFN-γ with a final concentration of 6 ng / mL, and autologous plasma with a final concentration of 5 v / v%.

[0149] Prepare 2000 mL of NK cell expansion culture medium: Take DMEM / F12 medium and add IL-2 with a final concentration of 1000 IU / mL, IL-15 with a final concentration of 13 ng / mL, IL-18 with a concentration of 20 ng / mL and autologous plasma with a final concentration of 5 v / v%.

[0150] Prepare 40 mL of coating solution: Take PBS and add heparin sodium at a final concentration of 45 μg / mL and CD16 monoclonal antibody at a final concentration of 10 μg / mL to the PBS.

[0151] 6.2 Coating of culture flasks

[0152] First, take 30 mL of the coating solution prepared in this example and add it to a T75 culture flask. Gently shake the flask to coat the inner wall of the bottom of the T75 culture flask with the coating solution. Then, place the culture flask in a refrigerator at 4°C and incubate for 16 hours. After the coating film forms on the inner wall of the culture flask, the incubation is over. Use a pipette to remove the remaining coating solution. Finally, add PBS to the culture flask to wash the coating film and remove the washed PBS to obtain the coated culture flask for later use.

[0153] 6.3 NK cell culture

[0154] On day 0, first, take 20 mL of the NK cell activation culture medium prepared in step 6.1 of Example 6 and resuspend it at 4.0 × 10⁻⁶. 7 A number of mononuclear cells were obtained, resulting in a cell density of 2.0 × 10⁶ cells. 6 Cell suspension of cells / mL; then, the cell suspension was seeded into the T75 culture flask coated in step 6.2 of this Example 6; finally, the cells were placed in an incubator at 37°C and 5% CO2 for static culture;

[0155] On day 3, the NK cell expansion culture solution prepared in step 6.1 of Example 6 was added to the culture bottle, and the cell density was controlled at 2.0 x 10 6 cells / mL, and the culture was continued.

[0156] On day 5, the cells and culture solution in the culture bottle were transferred to a 10 L culture bag, the NK cell expansion culture solution prepared in step 6.1 of Example 6 was added, and the cell density was controlled at 2.0 x 10 6 cells / mL, and the culture was continued in a 37°C, 5% CO2 incubator.

[0157] On day 7 to day 13, every two days, the NK cell expansion culture solution prepared in step 6.1 of Example 6 was added to the culture bag, and the cell density was controlled at 2.0 x 10 6 cells / mL, and the culture was continued.

[0158] On day 14, the culture was stopped, the cells and culture solution were collected, centrifuged at 900g for 10 min, the supernatant was removed, and the cell pellet was washed with PBS multiple times to obtain high-purity NK cells.

[0159] (II) Test detection

[0160] Example 7

[0161] 7.1, NK cell growth test detection

[0162] In the NK cell culture process of Examples 1 to 6, samples were taken on day 0, day 4, day 6, day 8, day 10, and day 13, and the number of cells was counted using a cell counter. The results are shown in Table 1. In Table 1, the cells counted on day 0 were PBMCs, and the cells counted from day 4 to day 13 were NK cells.

[0163] Table 1: NK cell growth number counting table

[0164]

[0165] As can be seen from Table 1, the NK cells cultured in Examples 1 to 6 had little change in the NK cell expansion fold detected by sampling on day 13; that is, the components in the NK cell culture solution, such as calycosin, formononetin, astragaloside IV, and soybean saponin I, had little effect on the expansion and growth of NK cells.

[0166] 7.2, NK cell CD3-CD56+ phenotype flow detection

[0167] NK cell samples cultured for 13 days in Example 1 and Example 6 (Comparative Example 3) were taken, cell suspensions were prepared, and the final concentration of cells was adjusted to 1 x 10 6Example 1 and Example 6 (Comparative Example 3) were taken on the 13th day of NK cell culture, and incubated at 37°C and 38°C for 6 min, respectively, to detect the secretion concentration of corresponding cytokines, such as TNF-a, IL-1β, IL-6, and FasL. The detection results are shown in Tables 2 and Figure 1 and 2 .

[0168] As can be seen from Figure 1 and 2 , the CD3-CD56+ purity of NK cells cultured for 13 days in Example 1 (94.80%) is much higher than that of NK cells cultured for 13 days in Example 6 (Comparative Example 3) (60.98%); this shows that the components added to the activation culture medium in the NK cell culture medium, such as calycosin, formononetin, astragaloside IV, and soybean saponin I, can maintain the activity of NK cells, promote the secretion of cytokines, and slow down cell aging, so as to improve the purity of cells.

[0169] 7.3, Cytokine secretion concentration detection

[0170] Example 1 and Example 6 (Comparative Example 3) were taken on the 13th day of NK cell culture, and incubated at 37°C and 38°C for 6 min, respectively, to detect the secretion concentration of corresponding cytokines, such as TNF-a, IL-1β, IL-6, and FasL. The detection results are shown in Tables 2 and Figure 3 . Figure 3 The data in Table 2 are consistent.

[0171] Table 2 Cytokine secretion concentration in NK cells

[0172]

[0173] As can be seen from Table 2 and Figure 3 , by intermittently simulating the temperature (37°C, 38°C) of immune inflammatory response in vivo, NK cells can be stimulated to achieve pro-inflammatory effect. The test detection of NK cells cultured for 13 days in Example 1 and Example 6 after incubation shows that NK cells can increase the secretion concentration of cytokines (such as TNF-a, IL-1β, IL-6, and FasL); this shows that the components added to the culture medium in Example 1, such as calycosin, formononetin, astragaloside IV, and soybean saponin I, can maintain the physiological activity of NK cells stably and durably, slow down cell aging, and then promote the secretion of cytokines, resulting in higher cytokine secretion level.

[0174] 7.4, NK cell exhaustion marker detection

[0175] The aging of NK cells leads to functional exhaustion, and one of the manifestations of cell exhaustion is the increase in the expression of inhibitory receptors; for example, the inhibitory receptor KLGR1 can inhibit NK cell proliferation by inhibiting AKT phosphorylation; TIGIT is a co-inhibitory or immune checkpoint receptor, which can inhibit NK cell activity; the immunosuppressive receptor TIM-3 can be expressed in various immune cells, and blocking TIM-3 can restore NK cell function in tumor patients and inhibit tumor growth; therefore, the expression level of inhibitory receptors (TIM-3, TIGIT, KLGR1, etc.) is increased on exhausted NK cells; therefore, by detecting the expression level of inhibitory receptors (TIM-3, TIGIT, KLGR1, etc.), the exhaustion state of NK cells can be analyzed, which helps to develop the most appropriate immune cell therapy treatment strategy for diseases.

[0176] The NK cells cultured for 13 days in Example 1 and Example 6 were taken as samples, and cell suspensions were prepared and adjusted to a final concentration of 1×10 6 / mL. The markers TIM-3, TIGIT, and KLGR1 monoclonal antibodies were added, respectively, and incubated at room temperature in the dark for 15 min. After incubation, the excess antibody was washed off, and flow cytometry was used for detection. The detection results are shown in Table 3 and Figure 4 Figure 4 The data in Table 3 are consistent.

[0177] Table 3: Proportion of NK cell exhaustion markers

[0178]

[0179] As shown in Table 3 and Figure 4 , the percentages of the NK cell exhaustion markers TIM-3, TIGIT, and KLGR1 cultured in Example 1 are much lower than those cultured in Example 6 (Comparative Example 3), that is, the expression of the exhaustion markers is reduced. Therefore, it can be known that the components such as irilone, formononetin, astragaloside IV, and soybean saponin I contained in the NK cell culture solution of the present application can reduce the concentration of inhibitory receptors (TIM-3, TIGIT, KLGR1), resist NK cell aging, and enhance the sustained ability of cell activity.

[0180] 7.5, NK cell killing rate test

[0181] The NK cells cultured for 13 days in Example 1 and Example 6 were taken as samples, and cell suspensions were prepared and adjusted to a final concentration of 1×10 6 / mL, 2.0×10 6 / mL, and 4.0×10​6 / mL of cell suspension as 3 groups of effector cells.

[0182] K562 cells as target cells, according to the effector-target ratio E:T = 1:1, 5:1, 10:1, 20:1, the target cells K562 and effector cells NK cells were plated in holes, 3 holes in each group. Each hole of NK cells and K562 target cells was 500 μL. 500 μL of NK cell expansion culture solution prepared in Example 1 and Example 6 was added to each hole.

[0183] Effector cell NK cell natural release group: 1.0 x 10 6 / mL, 2.0 x 10 6 / mL, 4.0 x 10 6 / mL of NK cells, 500 μl per hole, 3 holes in each group, 500 μL of medium was added to each hole, and cultured in a 37°C, 5% CO2 incubator for 40 min.

[0184] K562 target cell maximum release group: 1.0 x 10 5 / mL of target cells, 500 μL per hole, 3 holes in each group, 500 μL of medium was added to each hole, and cultured in a 37°C, 5% CO2 incubator for 40 min.

[0185] K562 target cell natural release group: 1.0 x 10 5 / mL of target cells, 500 μL per hole, 3 holes in each group, 500 μL was added to each hole.

[0186] Culture medium blank control test group: 500 μL per hole, 3 holes in each group, 500 μL was added to each hole.

[0187] After plating, the culture plate was centrifuged at 250g for 4 min and placed in a 37°C, 5% CO2 incubator for 4 h. 45 min before the end of the culture, the culture plate was removed, and the thawing solution was added to the target cell maximum release group, which was centrifuged at 250g for 4 min and continued to be cultured for 45 min. After that, the cells were gently mixed and centrifuged at 300g for 5 min, and the supernatant was collected. The absorbance value at 492 nm wavelength was determined by LDH (lactate dehydrogenase) assay.

[0188] The killing activity was calculated according to the following formula:

[0189] Killing activity = (A-B-C) / (D-C) x 100%;

[0190] Wherein:

[0191] A- represents the absorbance value of the test group corrected value;

[0192] B- represents the effector cell natural release group;

[0193] C- represents the natural release group of target cells;

[0194] D- represents the maximum release group of target cells.

[0195] The test results are shown in Table 4 and Figure 5 , and Table 4 is consistent with Figure 5 data.

[0196] Table 4 Detection of killing activity of NK cells on K562 target cells

[0197]

[0198] As can be seen from Table 4 and Figure 5 , the NK cells cultured in Example 1 have better killing effect on K562 cells than the NK cells cultured in Example 6, and the difference is obvious, which shows that the components such as stilbenes, formononetin, astragaloside IV and soyasaponin I in the NK cell culture solution of the application can resist the aging of NK cells, enhance the persistence of NK cell activity, achieve the effect of improving the killing of tumors, and in the application, the killing rate of K562 target cells is greater than 95.21% when the effector-target ratio E:T is 5:1, 10:1 and 20:1, which is much higher than the clinical application standard, i.e. greater than or equal to 40%.

[0199] It should be understood that the above description of the preferred embodiments of the application is more detailed, and therefore should not be considered as a limitation on the scope of patent protection of the application. The patent protection scope of the application should be subject to the appended claims.

Claims

1. An NK cell culture medium, characterized in that, It contains NK cell activation culture medium and NK cell expansion culture medium, both of which use basal culture medium as the base medium; wherein: The NK cell activation culture medium contains, in the basal culture medium, IL-2 at a final concentration of 100–2000 IU / mL, IL-15 at a final concentration of 5–20 ng / mL, IL-18 at a concentration of 10–50 ng / mL, IL-27 at a final concentration of 5–15 ng / mL, SCM at a final concentration of 0.1–5 ng / mL, IFN-γ at a final concentration of 1–10 v / v% autologous plasma at a final concentration of 1–10 ug / mL, verbenafil isoflavone at a final concentration of 0.1–10 ug / mL, and gentianin at a final concentration of 1–20 ug / mL. The NK cell expansion culture medium contains, in the basal culture medium, IL-2 at a final concentration of 100–2000 IU / mL, IL-15 at a final concentration of 5–20 ng / mL, IL-18 at a concentration of 10–50 ng / mL, autologous plasma at a final concentration of 1–10 v / v%, astragaloside IV at a final concentration of 5–50 ug / mL, and soybean saponin I at a final concentration of 1–10 ug / mL.

2. The NK cell culture medium according to claim 1, characterized in that, In this NK cell culture medium: The basal culture medium of the NK cell activation culture medium contains IL-2 at a final concentration of 500–1000 IU / mL, IL-15 at a final concentration of 8–16 ng / mL, IL-18 at a concentration of 20–30 ng / mL, IL-27 at a final concentration of 8–12 ng / mL, SCM at a final concentration of 0.5–3 ng / mL, IFN-γ at a final concentration of 3–8 v / v% autologous plasma at a final concentration of 3–8 ug / mL, verbascoside isoflavone at a final concentration of 1–8 ug / mL, and styracin at a final concentration of 5–15 ug / mL. The basal culture medium of the NK cell expansion culture medium contains IL-2 at a final concentration of 500-1000 IU / mL, IL-15 at a final concentration of 8-16 ng / mL, IL-18 at a concentration of 30-30 ng / mL, autologous plasma at a final concentration of 3-8 v / v%, astragaloside IV at a final concentration of 10-30 ug / mL, and soybean saponin I at a final concentration of 3-8 ug / mL.

3. The NK cell culture medium according to claim 1 or 2, characterized in that, The basal culture medium is any one of DMEM / F12, IMDM, 581 or RPMI-1640.

4. A method for culturing NK cells, characterized in that, The steps include the following: On day 0, firstly, mononuclear cells were resuspended in the NK cell activation culture medium as described in claim 1, and the cell density was controlled at 1.0 × 10⁻⁶. 6 ~3.0×10 6 Cells / mL; then, the resuspended cells were seeded into coated T75 culture flasks; finally, they were incubated statically in an incubator at 37°C and 5% CO2. On day 3, the NK cell expansion culture medium as described in claim 1 was added to the culture flask, and the cell density was controlled at 1.0 × 10⁶ cells / year. 6 ~3.0×10 6 Cells / mL, continue culturing; On day 5, the cells and culture medium in the culture flask were transferred to a 10L culture bag, and the NK cell expansion culture medium as described in claim 1 was added, with the cell density controlled at 1.0 × 10⁻⁶. 6 ~3.0×10 6 The cells / mL were incubated at 37°C in a 5% CO2 incubator. From day 7 to day 13, the NK cell expansion culture medium as described in claim 1 was added to the culture bag every two days, and the cell density was controlled at 1.0 × 10⁻⁶. 6 ~3.0×10 6 Cells / mL, continue culturing; On day 14, culture was stopped, cells and culture medium were collected, centrifuged at 900g for 10 minutes, the supernatant was removed, and the cell pellet was washed multiple times with PBS to obtain high-purity NK cells.

5. The NK cell culture method according to claim 3, characterized in that, On day 0, the coated T75 culture flask was obtained through the following steps: First, prepare the coating solution: using PBS as a base, add heparin sodium at a final concentration of 20–80 μg / mL and CD16 monoclonal antibody at a final concentration of 0.1–20 μg / mL to the PBS; Next, add the coating solution to the T75 culture flask and incubate it in a refrigerator at 2-8°C. After the coating film forms on the inner wall of the culture flask, the incubation ends and the remaining coating solution is discarded. Finally, PBS was added to the culture flask to wash the coated membrane, and the washed PBS was removed to obtain the coated culture flask.

6. The NK cell culture method according to claim 4, characterized in that, The steps include the following: On day 0, firstly, mononuclear cells were resuspended in the NK cell activation culture medium as described in claim 1, and the cell density was controlled at 2.0 × 10⁻⁶. 6 Cells / mL; then, the resuspended cells were seeded into coated T75 culture flasks; finally, they were incubated statically in an incubator at 37°C and 5% CO2. On day 3, the NK cell expansion culture medium as described in claim 1 was added to the culture flask, and the cell density was controlled at 2.0 × 10⁶ cells / year. 6 Cells / mL, continue culturing; On day 5, the cells and culture medium in the culture flask were transferred to a 10L culture bag, and the NK cell expansion culture medium as described in claim 1 was added, with the cell density controlled at 2.0 × 10⁻⁶. 6 The cells / mL were incubated at 37°C in a 5% CO2 incubator. From day 7 to day 13, the NK cell expansion culture medium as described in claim 1 was added to the culture bag every two days, and the cell density was controlled at 2.0 × 10⁻⁶. 6 Cells / mL, continue culturing; On day 14, culture was stopped, cells and culture medium were collected, centrifuged at 900g for 10 minutes, the supernatant was removed, and the cell pellet was washed multiple times with PBS to obtain high-purity NK cells.

7. A biological agent, characterized in that, The biological agent contains NK cells obtained by the culture method as described in claim 4.

8. The use of the biological agent as described in claim 7 in the preparation of drugs for treating tumors or cancer.

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