Combined culture medium of NK (Natural Killer) cells and culture method of NK cells

Abstract

The invention provides a combined culture medium of NK cells and a culture method of the NK cells, and relates to the technical field of NK cell culture. The NK cell combined culture medium comprises at least one of an NK cell induction culture medium, an NK cell amplification culture medium and an NK cell immune activation culture medium. By adopting the staged culture medium provided by the invention, the clustering quantity, the proliferation quantity and the immunocompetence of the NK cells can be improved.

Description

Technical Field

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

[0002] The human immune system is composed of immune organs, immune cells, and immune factors. The three have clear division of labor and cooperate with each other to maintain the body's homeostasis and resist the invasion of foreign pathogens. Among them, immune cells, as an important component, can not only act directly on diseased tissues or pathogens, but also kill by releasing cytokines. Therefore, immune cells play an important role in maintaining the health of the body. Immune cells include: T lymphocytes, B lymphocytes, macrophages and NK cells (natural killer cells). NK cells, as natural killer cells in the body, carry inherent recognition receptors on their own surface. They do not require prior antigen processing or presentation, and can directly act on tumor cells or pathogens, and kill by releasing granzymes, perforins, etc. Therefore, NK cells play a vital role in maintaining the normal homeostasis of the body.

[0003] As the human body ages, the vitality and number of its own immune cells are also decreasing, and as a result, the risk of disease is gradually increasing. Studies have shown that at the age of 50, the number and vitality of immune cells in the human body are only about half of those at the age of 20; at the age of 65, the number and vitality of immune cells are only about 1 / 10 of those at the age of 20, and the risk of disease at this time increases to more than 50%. Therefore, immune cell therapy has emerged and has become one of the mainstream research directions for tumor treatment. Immune cell therapy is a treatment method that collects immune cells from the body, expands and cultures them in vitro to multiply them, and then returns them to the body to exert immune function. At present, hot immune cell therapies include Car-T cells, NK cells, CIK cells, etc. As natural killer cells, NK cells can directly reach the lesions without the presentation of antigens, kill tumor cells and pathogens, and remove aging cells, thereby achieving the purpose of immune cell therapy. At present, there are still many problems in the in vitro culture of NK cells: (1) The expansion of NK cells depends on the formation of cell clusters. The number and size of cell clusters are proportional to the cell proliferation ability. Single NK cells have no proliferation ability. There is no good way to improve the problem of promoting the formation of NK cell clusters; (2) Due to differences in individual samples, the number and activity of NK cells isolated from the body vary greatly, and the differences between samples become more obvious during the subsequent culture process; (3) The NK cell culture cycle is relatively long, and cell aging is prone to occur during the culture process.

[0004] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute the prior art known to ordinary technicians in the field. Summary of the invention

[0005] In view of this, the present invention proposes a combination culture medium for NK cells and a culture method for NK cells, wherein NK cell induction medium, NK cell expansion medium and NK cell immune activation medium are used respectively in the three stages of NK cell growth, and different additives are added to the NK cells at different stages for culture, so as to improve the clustering amount, proliferation amount and immune activity of NK cells.

[0006] The technical solution of the present invention is implemented as follows: The present invention provides a NK cell induction culture medium, comprising a serum-free culture medium, quercetin-4-isobutyric acid, cyclocarya paliurus glycoside, IL-2, inositol and folic acid;

[0007] Among them, the concentration of quercetin-4-isobutyrate is 10-20 μg / L, the concentration of Cyclocarya paliurus glycoside is 25-30 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 50-70 mg / L, the concentration of folic acid is 5-10 μg / L, and the remainder is serum-free culture medium.

[0008] Based on the above technical scheme, preferably, the concentration of quercetin-4-isobutyrate is 15 μg / L, the concentration of Cyclocarya paliurus glycoside is 30 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 60 mg / L, the concentration of folic acid is 8 μg / L, and the remainder is serum-free culture medium.

[0009] The present invention also provides a NK cell expansion culture medium, comprising serum-free culture medium, kaempferol B, gossypol, IL-2, inositol and folic acid;

[0010] Among them, the concentration of kaempferol B is 40-50 μg / L, the concentration of gossypol is 30-40 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 50-70 mg / L, the concentration of folic acid is 5-10 μg / L, and the remainder is serum-free culture medium.

[0011] On the basis of the above technical scheme, preferably, the concentration of kaempferol B is 45 μg / L, the concentration of terrestris is 35 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 60 mg / L, the concentration of folic acid is 8 μg / L. L , the remainder was serum-free medium.

[0012] The present invention also provides a NK cell immune activation culture medium, comprising a serum-free culture medium, human recombinant C-type natriuretic peptide, Cyclocarya paliurus polysaccharide, IL2, inositol and folic acid;

[0013] Among them, the human recombinant C-type natriuretic peptide is 5-10 μg / L, the Cyclocarya paliurus polysaccharide is 100-120 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 50-70 mg / L, the concentration of folic acid is 5-10 μg / L, and the remainder is serum-free culture medium.

[0014] On the basis of the above technical scheme, preferably, the human recombinant C-type natriuretic peptide is 8 μg / L, the Cyclocarya paliurus polysaccharide is 110 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 60 mg / L, the concentration of folic acid is 8 μg / L, and the remainder is serum-free culture medium.

[0015] The present invention also provides a combined culture medium for NK cells, comprising at least one of (i), (ii) and (iii);

[0016] (i) the above-mentioned NK cell induction medium;

[0017] (ii) the above-mentioned NK cell expansion medium;

[0018] (iii) is the above-mentioned NK cell immune activation medium.

[0019] The present invention also provides a method for culturing NK cells, comprising the following steps:

[0020] S10, separating and obtaining peripheral blood mononuclear cells;

[0021] S20, mixing the peripheral blood mononuclear cells with the above-mentioned NK cell induction medium, and adding them to a cell culture bottle for NK cell induction culture; wherein the relationship between the number of peripheral blood mononuclear cells and the volume of the NK cell induction medium is (2.5-3.0)×10 6 Pieces / ml;

[0022] S30, after NK cell induction culture, adding the above-mentioned NK cell expansion medium, adding the NK cell expansion medium once every other day, and performing NK cell expansion culture; wherein the ratio of the volume of the NK cell expansion medium added each time to the total volume of the medium before each addition is (1-2):1;

[0023] S40, NK cell expansion culture, adding the above-mentioned NK cell immune activation medium, adding the NK cell immune activation medium once every other day, performing NK cell immune activation culture, and obtaining cultured cells, wherein the cultured cells include NK cells;

[0024] The ratio of the volume of the NK cell immune activation medium added each time to the total volume of the medium before each addition is (1-2):1.

[0025] On the basis of the above technical solution, preferably, among the cultured cells, at least 45% of the cells are NK cells, and at least 50% of the NK cells are clustered NK cells.

[0026] On the basis of the above technical solution, preferably, the NK cell induction culture is static culture at 37°C, 5% CO2 for 2 to 4 days;

[0027] The NK cell expansion culture is static culture at 37°C and 5% CO2 for 5 to 8 days;

[0028] The NK cell immune activation culture is statically cultured at 37° C. and 5% CO 2 for 4 to 6 days.

[0029] The NK cell culture medium culture method provided by the present invention has the following beneficial effects compared with the prior art:

[0030] 1. The NK cell culture medium provided by the present invention can ensure the activity and proliferation rate of NK cells by adding quercetin-4-isobutyric acid and Cyclocarya paliurus glycosides in the early stage of induction of NK cell culture. The addition of kaempferol glycoside B and terrestris glycosides in the expansion stage promoted the clustering of NK cells, and the number and size of clusters were significantly improved. The addition of human recombinant C-type natriuretic peptide and Cyclocarya paliurus polysaccharides in the immune enhancement stage can improve cell energy metabolism and vitality, and enhance the immune activity of NK cells. According to the growth characteristics of NK cells at different stages of culture, the present invention adds cell adhesion factors, active small molecules and biopolysaccharides to the NK cell culture medium, which can not only ensure the rapid and stable doubling of the number of NK cells, but also enhance the activity and lethality of NK cells.

[0031] 2. In the three main stages of NK cell growth, different substances are added to improve the characteristics of NK cells in this stage in a targeted manner, so as to achieve rapid proliferation of NK cells and enhancement of immune activity; according to the comparison results of the specific embodiments, it can be seen that the NK cell agglomeration rate, harvest volume, and secretion of TNF-α and IL-8 are significantly improved by using the three culture compositions provided by the present disclosure. At the same time, the culture method provided by the present disclosure adopts the method of directly adding culture medium midway to avoid excessive operation affecting the growth of NK cells. Therefore, the culture method provided by the present invention can well improve the number and immune activity of NK cells and provide an effective cell culture scheme for immune cell therapy.

[0032] 3. The NK cell culture method provided by the present invention can achieve rapid expansion of NK cells in vitro, maintain high cell viability, improve immunomodulatory activity, and achieve stable production of NK cells, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 The figure shows the microscopic characterization of NK cells in some embodiments of the present invention.

[0035] Figure 2 This is a counting curve diagram of NK cell culture process in some embodiments of the present invention.

[0036] Figure 3 This is a schematic diagram of the results of flow cytometry detection of the proportion of NK cells in cultured cells in some embodiments of the present invention.

[0037] Figure 4 This is a schematic diagram of the results of detecting the concentration of TNF-α in cultured cells in some embodiments of the present invention.

[0038] Figure 5 This is a schematic diagram of the results of detecting the concentration of IL-8 in cultured cells in some embodiments of the present invention. DETAILED DESCRIPTION

[0039] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The NK cell culture medium and the NK cell culture method provided by the present disclosure are further described below in conjunction with specific examples.

[0041] Material:

[0042] Serum-free medium was purchased from NC0102, Youkang Biotechnology (Beijing) Co., Ltd.;

[0043] Quercetin-4-isobutanol and gossypol were purchased from Chengdu Pufeide Co., Ltd.;

[0044] Cyclocarya paliurus glycosides and Cyclocarya paliurus polysaccharides were purchased from Shaanxi Jinkangtai Biological Co., Ltd.;

[0045] IL-2 and IL-21 were purchased from Shuanglu Pharmaceutical;

[0046] Kaempferol B, inositol, and folic acid were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0047] Human recombinant C-type natriuretic peptide was purchased from Shanghai Chunmai Biotechnology Co., Ltd.

[0048] Different NK cell induction medium, NK cell expansion medium and NK cell immune activation medium were prepared.

[0049] Induction medium 1: the concentration of quercetin-4-isobutyrate is 15 μg / L, the concentration of cyclocarya paliurus glycoside is 30 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 60 mg / L, the concentration of folic acid is 8 μg / L, and the remainder is NK cell serum-free medium.

[0050] Induction medium 2: the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 60 mg / L, the concentration of folic acid is 8 μg / L, and the remainder is NK cell serum-free medium.

[0051] Induction medium 3: the concentration of quercetin-4-isobutyrate is 15 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 60 mg / L, the concentration of folic acid is 8 μg / L, and the remainder is NK cell serum-free medium.

[0052] Induction medium 4: the concentration of Cyclocarya paliurus glycoside is 30 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 60 mg / L, the concentration of folic acid is 8 μg / L, and the remainder is NK cell serum-free medium.

[0053] Expansion medium 1: the concentration of kaempferol B is 45 μg / L, the concentration of gossypol is 35 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 60 mg / L, the concentration of folic acid is 8 μg / L, and the remainder is NK cell serum-free medium.

[0054] Expansion medium 2: the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 60 mg / L, the concentration of folic acid is 8 μg / L, and the remainder is NK cell serum-free medium.

[0055] Expansion medium 3: the concentration of kaempferol B is 45 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 60 mg / L, the concentration of folic acid is 8 μg / L, and the remainder is NK cell serum-free medium.

[0056] Expansion medium 4: the concentration of upland gossypol is 35 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 60 mg / L, the concentration of folic acid is 8 μg / L, and the remainder is NK cell serum-free medium.

[0057] Immune activation medium 1: human recombinant C-type natriuretic peptide 8 μg / L, Cyclocarya paliurus polysaccharide 110 μg / L, IL-2 concentration of 1000 IU / ml, inositol concentration of 60 mg / L, folic acid concentration of 8 μg / L, and the remainder is NK cell serum-free medium.

[0058] Immune activation medium 2: the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 60 mg / L, the concentration of folic acid is 8 μg / L, and the remainder is NK cell serum-free medium.

[0059] Immune activation culture medium 3: Cyclocarya paliurus polysaccharide 110 μg / L, IL-2 concentration of 1000 IU / ml, inositol concentration of 60 mg / L, folic acid concentration of 8 μg / L, and the remainder is NK cell serum-free culture medium.

[0060] Immune activation medium 4: human recombinant C-type natriuretic peptide 8 μg / L, IL-2 concentration of 1000 IU / ml, inositol concentration of 60 mg / L, folic acid concentration of 8 μg / L, and the remainder is NK cell serum-free medium.

[0061] Example 1

[0062] The NK cell culture method of this embodiment comprises the following steps:

[0063] 1. Peripheral blood sample collection: Add heparin sodium to the blood collection tube to prevent blood coagulation, collect 40ml of peripheral blood from healthy volunteers in a sterile centrifuge tube, and centrifuge at 800×g for 25min. Pipette the upper pale yellow plasma into a 50mL centrifuge tube, place it in a 56℃ water bath for 30min to inactivate, and place it at -20℃ for 10min. Take the bottom blood cell layer and dilute it with physiological saline 1:1 to obtain a blood cell solution.

[0064] PBMC separation: Add 20 ml of lymphocyte separation solution (Ficoll) to a 50 ml centrifuge tube, take 20 ml of blood cell solution and slowly add it to the lymphocyte separation solution, taking care not to destroy the layers. Centrifuge at 900 × g for 30 min, aspirate the middle white film layer, add physiological saline to wash twice and count, centrifuge at 400 × g for 10 min, aspirate the supernatant, and obtain PBMC cells.

[0065] 2. Resuspend PBMC cells in induction medium 1 at (2.0-3.0)×10 6The cells were inoculated into T25 cell culture flasks at a density of 1.5 cells / ml and cultured at 37°C and 5% CO2 for 3 days.

[0066] 3. Observe the cell growth status and add induction medium 1 every other day. The volume of induction medium 1 added each time should be in a ratio of (1-2):1 to the total volume of medium before each addition. After 4 days of culture, if the total volume of medium exceeds 30 ml, transfer the culture medium to a T225 cell culture flask for further culture. The cultured cells obtained are defined as cultured cells 1.

[0067] Example 2

[0068] The difference between Example 2 and Example 1 is that induction culture medium 2 is used, and the remaining steps are the same as those of Example 1. The obtained cultured cells are defined as cultured cells 2.

[0069] Example 3

[0070] The difference between Example 3 and Example 1 is that induction culture medium 3 is used, and the remaining steps are the same as those of Example 1. The cultured cells obtained are defined as cultured cells 3.

[0071] Example 4

[0072] The difference between Example 4 and Example 1 is that induction culture medium 4 is used, and the remaining steps are the same as those of Example 1. The cultured cells obtained are defined as cultured cells 4.

[0073] The cultured cells 1, 2, 3 and 4 obtained in Examples 1 to 4 were observed under a microscope. Figure 1 , A is a cell microscopic characterization diagram of Example 1, B is a cell microscopic characterization diagram of Example 2, C is a cell microscopic characterization diagram of Example 3, and D is a cell microscopic characterization diagram of Example 4. It can be seen that although only the induction medium was used to induce the NK cells, the NK cell clusters in the cultured cells prepared in Example 1 were significantly more than those in Examples 2-4. This shows that the use of the NK cell induction medium provided in this example can improve the activity and proliferation rate of NK cells.

[0074] Example 5

[0075] 1. Peripheral blood sample collection: Add heparin sodium to the blood collection tube to prevent blood coagulation, collect 40ml of peripheral blood from healthy volunteers in a sterile centrifuge tube, and centrifuge at 800×g for 25min. Pipette the upper pale yellow plasma into a 50mL centrifuge tube, place it in a 56℃ water bath for 30min to inactivate, and place it at -20℃ for 10min. Take the bottom blood cell layer and dilute it with physiological saline 1:1 to obtain a blood cell solution.

[0076] PBMC separation: Add 20 ml of lymphocyte separation solution (Ficoll) to a 50 ml centrifuge tube, take 20 ml of blood cell solution and slowly add it to the lymphocyte separation solution, taking care not to destroy the layers. Centrifuge at 900 × g for 30 min, aspirate the middle white film layer, add physiological saline to wash twice and count, centrifuge at 400 × g for 10 min, aspirate the supernatant, and obtain PBMC cells.

[0077] 2. Resuspend PBMC cells in induction medium 1 at (2.5-3.0)×10 6 The cells were inoculated into T25 cell culture flasks at a density of 1.5 cells / ml and cultured at 37°C and 5% CO2 for 3 days.

[0078] 3. Observe the cell growth status and add induction medium 1. Then add induction medium 1 every other day. The volume of induction medium 1 added each time should be in the ratio of (1-2):1 to the total volume of medium before each addition. If the total volume of medium exceeds 30 ml, transfer it to T225 cell culture flask and continue to culture for 4 days.

[0079] 4. Observe the cell growth status and add expansion medium 1. Then, add expansion medium 1 every other day. The volume of expansion medium 1 added each time should be in the ratio of (1-2):1 to the total volume of medium before each addition. When the total volume of medium exceeds 200 ml, transfer it to lymphocyte culture bag and continue to culture. Culture for 7-8 days to obtain cultured cells 5.

[0080] Example 6

[0081] The difference between Example 6 and Example 5 is that in step 4, expansion culture medium 2 is used, and the remaining steps are the same as those in Example 5. The obtained cultured cells are defined as cultured cells 6.

[0082] Example 7

[0083] The difference between Example 7 and Example 5 is that in step 4, expansion culture medium 3 is used, and the remaining steps are the same as those in Example 5. The obtained cultured cells are defined as cultured cells 7.

[0084] Example 8

[0085] The difference between Example 8 and Example 5 is that in step 4, expansion culture medium 4 is used, and the remaining steps are the same as those in Example 5. The obtained cultured cells are defined as cultured cells 8.

[0086] The trypan blue staining method was used to record the number of cells per day during the culture process of Examples 5-8. Specifically, 100ul of cell suspension was taken and mixed evenly with 100ul of 0.4% trypan blue every day, and 20ul was added to the cell counting plate, and counted and statistically analyzed using a cell counter (Shanghai Mengwei Co., Ltd.). The results are shown in Figure 2 , A is a cell count curve diagram of Example 5 during NK cell culture, B is a cell count curve diagram of Example 6 during NK cell culture, C is a cell count curve diagram of Example 7 during NK cell culture, and D is a cell count curve diagram of Example 8 during NK cell culture. The maximum number of cells in Example 5 is 4.98 billion, the maximum number of cells in Example 6 is 2.76 billion, the maximum number of cells in Example 7 is 3.7 billion, and the maximum number of cells in Example 8 is 3.21 billion. It shows that after culturing with the same NK cell induction medium, the number of NK cells is greatly increased after using the NK expansion medium provided by the present disclosure.

[0087] Example 9

[0088] 1. Peripheral blood sample collection: Add heparin sodium to the blood collection tube to prevent blood coagulation, collect 40ml of peripheral blood from healthy volunteers in a sterile centrifuge tube, and centrifuge at 800×g for 25min. Pipette the upper pale yellow plasma into a 50mL centrifuge tube, place it in a 56℃ water bath for 30min to inactivate, and place it at -20℃ for 10min. Take the bottom blood cell layer and dilute it with physiological saline 1:1 to obtain a blood cell solution.

[0089] PBMC separation: Add 20 ml of lymphocyte separation solution (Ficoll) to a 50 ml centrifuge tube, take 20 ml of blood cell solution and slowly add it to the lymphocyte separation solution, taking care not to destroy the layers. Centrifuge at 900 × g for 30 min, aspirate the middle white film layer, add physiological saline to wash twice and count, centrifuge at 400 × g for 10 min, aspirate the supernatant, and obtain PBMC cells.

[0090] Resuspend PBMC cells in induction medium 1 at a rate of (2.5-3.0) × 10 6 The cells were inoculated into T25 cell culture flasks at a density of 1.5 cells / ml and cultured at 37°C and 5% CO2 for 4 days.

[0091] 2. Observe the cell growth status and add induction medium 1. Then add induction medium 1 every other day. The volume of induction medium 1 added each time should be (1-2):1 to the total volume of medium before each addition. If the total volume of medium exceeds 30 ml, transfer it to T225 cell culture flask and continue to culture for 4 days.

[0092] 3. Observe the cell growth status and add expansion medium 1. Then, add expansion medium 1 every other day. The volume of expansion medium 1 added each time should be in a ratio of (1-2):1 to the total volume of the medium before each addition. When the total volume of the medium exceeds 30 ml, transfer it to a T225 cell culture flask and continue culturing. Cultivate for 7-8 days to obtain cultured cells 5.

[0093] 4. Observe the cell growth status, add immune activation medium 1, and then add immune activation medium 1 every other day. The ratio of the volume of immune activation medium 1 added each time to the total volume of the medium before each addition is (1-2): 1. When the total volume of the medium exceeds 200 ml, transfer it to a lymphocyte culture bag and continue culturing for a total of 5 consecutive days; obtain cultured cells 9.

[0094] Example 10

[0095] The difference between Example 10 and Example 9 is that in step 4, immune activation culture medium 2 is used, and the remaining steps are the same as those in Example 9. The cultured cells obtained are defined as cultured cells 10.

[0096] Embodiment 11

[0097] The difference between Example 11 and Example 9 is that in step 4, immune activation culture medium 3 is used, and the remaining steps are the same as those in Example 9. The obtained cultured cells are defined as cultured cells 11.

[0098] Example 12

[0099] The difference between Example 12 and Example 9 is that in step 4, immune activation culture medium 4 is used, and the remaining steps are the same as those in Example 9. The obtained cultured cells are defined as cultured cells 12.

[0100] The cultured cells 9, 10, 11 and 12 obtained in Examples 9-12 were measured by flow cytometry, and the flow cytometry markers were CD3 and CD56. The measurement results are shown in FIG. Figure 3 As shown. In the cultured cell 9 obtained in Example 9, the proportion of NK cells was the highest, reaching 96.83%, which was much greater than the proportion of NK cells in the cultured cells 10 (65.76%), cultured cells 11 (72.35%) and cultured cells 12 (55.24%). It shows that the combination of the NK cell induction medium, the NK cell expansion medium and the NK cell immune activation medium provided by the present disclosure can greatly improve the proliferation capacity of NK cells.

[0101] The concentrations of TNF-α and IL-8 in cultured cells 9, 10, 11, and 12 obtained in Examples 9-12 were detected using a detection kit (Shanghai ELISA Biotechnology Co., Ltd.). The measurement results are as follows: Figure 4 and Figure 5 The secretion of TNF-α and IL-8 by the cultured cells obtained in Example 9 was significantly higher than that of other groups, with the secretion increasing by about 30%-50%, indicating that the combination of NK cell induction medium, NK cell expansion medium and NK cell immune activation medium provided by the present disclosure can better enhance the immune activity of NK cells.

[0102] Combined with the above results, the present disclosure provides three different culture media according to the growth habits of NK cells at different stages of the culture process. In the early stage of induction of culture, the addition of quercetin-4-isobutyric acid and Cyclocarya paliurus glycosides promoted the clustering of NK cells, and the number and size of clusters were significantly improved. The addition of kaempferol B and terrestris glycosides in the amplification stage increased the number and proportion of NK cells. Adding human recombinant C-type natriuretic peptide and Cyclocarya paliurus polysaccharides in the immune enhancement stage can enhance cell energy metabolism and vitality, and enhance the immune activity of NK cells. According to the growth characteristics of NK cells at different stages of culture, the present disclosure adds Chinese medicine extracts, ketone organic matter and recombinant proteins to the NK cell culture medium, which not only ensures the rapid and stable doubling of the number of NK cells, but also enhances the vitality and lethality of NK cells.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A NK cell induction medium, characterized in that Includes serum-free medium, quercetin-4-isobutyrate, cyclocarya paliurus glycosides, IL-2, inositol and folic acid.

2. The NK cell induction medium according to claim 1, characterized in that The concentration of quercetin-4-isobutyrate is 10-20 μg / L, the concentration of cyclocarya paliurus glycoside is 25-30 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 50-70 mg / L, the concentration of folic acid is 5-10 μg / L, and the remainder is serum-free culture medium.

3. A NK cell expansion medium, characterized in that Includes serum-free medium, kaempferol B, gossypium hirsutum, IL-2, inositol and folic acid.

4. The NK cell expansion medium according to claim 3, characterized in that The concentration of kaempferol B is 40-50 μg / L, the concentration of gossypol is 30-40 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 50-70 mg / L, the concentration of folic acid is 5-10 μg / L, and the remainder is serum-free culture medium.

5. A NK cell immune activation culture medium, characterized in that It includes serum-free culture medium, human recombinant C-type natriuretic peptide, Cyclocarya paliurus polysaccharide, IL2, inositol and folic acid.

6. The NK cell immune activation culture medium according to claim 5, characterized in that The human recombinant C-type natriuretic peptide is 5-10 μg / L, the Cyclocarya paliurus polysaccharide is 100-120 μg / L, the concentration of IL-2 is 1000 IU / ml, the concentration of inositol is 50-70 mg / L, the concentration of folic acid is 5-10 μg / L, and the remainder is serum-free culture medium.

7. A combined culture medium for NK cells, characterized in that: including at least one of (i), (ii) and (iii); (i) the NK cell induction medium according to claim 1 or 2; (ii) the NK cell expansion medium according to claim 3 or 4; (iii) the NK cell immune activation medium according to claim 5 or 6.

8. A method for culturing NK cells, characterized in that: The following steps are involved: S10, separating and obtaining peripheral blood mononuclear cells; S20, mixing peripheral blood mononuclear cells with the NK cell induction medium according to claim 1 or 2, and adding the cells to a cell culture bottle for NK cell induction culture; wherein the relationship between the number of peripheral blood mononuclear cells and the volume of the NK cell induction medium is (2.5-3.0)×10 6 Pieces / ml; S30, after NK cell induction culture, adding the NK cell expansion medium according to claim 3 or 4, and adding the NK cell expansion medium every other day to carry out NK cell expansion culture; wherein the ratio of the volume of the NK cell expansion medium added each time to the total volume of the medium before each addition is (1-2):1; S40, NK cell expansion culture, adding the NK cell immune activation medium according to claim 5 or 6, adding the NK cell immune activation medium once every other day, performing NK cell immune activation culture, and obtaining cultured cells, wherein the cultured cells include NK cells; The ratio of the volume of the NK cell immune activation medium added each time to the total volume of the medium before each addition is (1-2):

1.

9. The method for culturing NK cells according to claim 8, characterized in that: Among the cultured cells, at least 45% of the cells are NK cells, and at least 50% of the NK cells are clustered NK cells.

10. The method for culturing NK cells according to claim 8 or 9, characterized in that: The NK cell induction culture is performed by static culture at 37°C and 5% CO2 for 2 to 4 days; The NK cell expansion culture is static culture at 37°C and 5% CO2 for 5 to 8 days; The NK cell immune activation culture is statically cultured at 37° C. and 5% CO 2 for 4 to 6 days.

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