Serum-free culture system and culture method of NK (Natural Killer) cells

Abstract

The invention discloses a serum-free culture system and culture method of NK cells. The invention firstly discloses a serum-free culture system of the NK cells, wherein the serum-free culture system comprises a coating solution, an amplification culture medium, an activation culture medium and autologous plasma; the invention further discloses a culture method for NK cells by using the serum-free culture system. The method for culturing the NK cells by using the serum-free culture system disclosed by the invention reduces the culture cost of the NK cells, can simply, quickly, efficiently and safely obtain a large number of NK cells with high activity, high purity and strong killing ability in vitro, provides a better cell source for clinical application of the NK cells, and promotes further development of an NK cell treatment technology.

Description

Technical Field

[0001] The present invention relates to the technical field of cell and tissue culture, and more specifically, to a serum-free culture system and a culture method for NK cells. Background Art

[0002] Natural Killer cells (NK cells) are key members of the innate immune system and play an irreplaceable role in the body's immune defense and immune surveillance. NK cells originate from bone marrow hematopoietic stem cells, and their development process is finely regulated by a variety of cytokines and transcription factors, and they gradually mature in the bone marrow and other lymphoid organs.

[0003] NK cells are significantly different from lymphocytes such as T cells and B cells. They can launch rapid and efficient immune attacks against a variety of target cells without pre-sensitization with antigens. This unique immune activity stems from its complex and precise recognition mechanism. The surface of NK cells is equipped with a series of activating receptors and inhibitory receptors, which have a keen sense of subtle changes in molecules on the surface of target cells. For example, normal cells usually express major histocompatibility complex (MHC) class I molecules, and the inhibitory receptors on the surface of NK cells can recognize them, thereby effectively avoiding accidental injury to normal cells. However, when the expression of MHC class I molecules in target cells (such as tumor cells or virus-infected cells) is downregulated or abnormal due to various reasons, the activation receptor signal of the NK cell will prevail, thereby initiating the killing program.

[0004] The killing mechanism of NK cells is rich and diverse and highly efficient, among which the release of perforin and granzyme is one of the main killing methods. Perforin can form holes in the cell membrane of target cells, allowing granzymes to enter the target cells smoothly, activating apoptosis-related signaling pathways in target cells, and ultimately inducing apoptosis of target cells. In addition, NK cells can also induce apoptosis of target cells through the Fas / FasL pathway, that is, the Fas ligand on the surface of NK cells interacts with the Fas receptor on the surface of target cells, triggering programmed cell death of target cells.

[0005] With the continuous deepening of research on the immune system, NK cells have shown extremely broad application prospects in the field of clinical treatment. In the field of tumor treatment, NK cells have a natural killing ability against various types of tumor cells, covering blood system malignancies (such as leukemia, lymphoma, etc.) and solid tumors (such as lung cancer, liver cancer, melanoma, etc.). Compared with traditional tumor treatment methods (such as surgery, chemotherapy and radiotherapy), NK cell therapy has unique advantages. It can specifically identify and kill tumor cells while causing less damage to normal tissue cells. Moreover, in certain specific cases, NK cell therapy can also overcome the drug resistance of tumor cells, bringing new hope to those tumor patients who are insensitive to traditional treatment methods. In the treatment of viral infectious diseases, NK cells also perform well. For example, in diseases such as chronic hepatitis B, hepatitis C and AIDS, NK cells can accurately identify and eliminate virus-infected cells, effectively inhibit the replication and spread of viruses, and thus play a positive role in controlling viral infection and disease progression.

[0006] However, despite the huge potential of NK cells in clinical applications, their current application is still limited by many factors. Among them, the quantity and quality of NK cells are one of the most critical limiting factors. It is not easy to obtain a sufficient number of highly active NK cells in vitro for clinical treatment, which places extremely high demands on the in vitro culture technology of NK cells. As the core part of NK cell in vitro culture technology, the importance of NK cell culture medium is self-evident. The ideal NK cell culture medium needs to meet multiple conditions. It must not only provide NK cells with sufficient nutrients, including various amino acids, sugars mainly based on glucose, vitamins and inorganic salts, etc., to ensure the basic metabolic needs of cells, but also create a suitable growth environment for them, including appropriate pH value, osmotic pressure and gas environment.

[0007] Currently, although the NK cell culture media on the market can support the growth of NK cells to a certain extent, there are still many shortcomings. Some culture media have low efficiency in NK cell proliferation, which will lead to a longer culture cycle, thereby increasing the culture cost and the risk of cell contamination. At the same time, NK cells cultured in some culture media have defects in activity and functional integrity, which has a negative impact on their efficacy in clinical treatment. In addition, existing culture media also have problems with component stability and batch consistency, which poses great challenges to the standardization and scale of NK cell culture. How to achieve large-scale, efficient and high-quality expansion of NK cells in vitro has become a key issue in the current field of NK cell therapy.

[0008] At present, there are three main technologies for in vitro expansion of NK cells: first, using immunomagnetic cell sorting to obtain purified NK cells from peripheral blood mononuclear cells, and then adding factors in vitro for expansion and culture; second, using trophoblast cells (K562 cells) to expand primary NK cells; third, directly using factor stimulation to expand and culture NK cells from peripheral blood. However, the safety of the first two methods of expanding NK cells in clinical use has not been verified, and because K562 cells are tumor cells, there are certain risks, so these two methods are mostly used in scientific research. The third method is currently the main method for NK cell expansion. It uses factors to stimulate the expansion and culture of NK cells with better safety and higher clinical application value, but this method has problems such as low amplification multiples and purity and instability, and needs to be further optimized and upgraded.

[0009] In summary, given the important value of NK cells in clinical treatment and the many problems existing in existing NK cell culture media, there is an urgent need to develop a new culture system that can efficiently support the growth of NK cells and maintain their high activity. Summary of the invention

[0010] The purpose of the present invention is to provide an optimized serum-free culture system and culture method for NK cells, which can effectively overcome the problems of low amplification efficiency, low cell activity and insufficient functional integrity, as well as poor component stability and batch consistency in existing culture media when culturing NK cells, thereby achieving efficient and stable culture of NK cells in vitro and providing a high-quality cell source for the application of NK cells in clinical immunotherapy.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] The present invention first provides a serum-free culture system for NK cells, including a coating solution, an expansion culture medium, an activation culture medium and autologous plasma;

[0013] Wherein, the coating solution contains Human CD16; the final concentration of Human CD16 is 1-10 μg / mL;

[0014] The expansion medium comprises a basal medium and interleukin-2 (IL-2), wherein the final concentration of the IL-2 is 200-3000 IU / mL;

[0015] The activation medium comprises a basal medium, IL-2, interleukin-15 (IL-15), interleukin-21 (IL-21) and Tacrolimus; the final concentrations of the IL-2, IL-15, IL-21 and Tacrolimus are 200-3000 IU / mL, 10-100 ng / mL, 10-100 ng / mL and 0.1-5 ng / mL, respectively.

[0016] Furthermore, the autologous plasma is heat-inactivated autologous plasma. If heat-inactivated autologous plasma is not available, human AB serum can be used instead.

[0017] Furthermore, the final concentration of the Human CD16 antibody in the coating solution is 5-10 μg / mL; preferably, the final concentration of the Human CD16 antibody is 8 μg / mL.

[0018] Furthermore, the final concentration of the IL-2 in the expansion medium is 1000-3000 IU / mL; preferably, the final concentration of the IL-2 is 2000 IU / mL.

[0019] Furthermore, the final concentration of the IL-2 in the activation medium is 1000-3000 IU / mL, the final concentration of IL-15 is 50-100 ng / mL, the final concentration of IL-21 is 10-50 ng / mL and the final concentration of Tacrolimus is 1-5 ng / mL; preferably, the final concentration of the IL-2 is 1000 IU / mL, the final concentration of IL-15 is 80 ng / mL, the final concentration of IL-21 is 10 ng / mL and the final concentration of Tacrolimus is 3 ng / mL.

[0020] Furthermore, the basic culture medium includes amino acids, vitamins, inorganic salts, hormones, lipids, proteins other than hormones, energy substances, buffer systems, nitrogenous bases and other substances;

[0021] in,

[0022] The amino acids include glycine, alanine, arginine, asparagine, aspartic acid, cystine, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine and hydroxyproline;

[0023] The vitamins include biotin, choline chloride, D-calcium pantothenate, folic acid, niacinamide, pyridoxine, riboflavin, thiamine hydrochloride, cobalamin, inositol, p-aminobenzoic acid, ascorbic acid, vitamin D2, α-tocopherol, vitamin K2 and vitamin K3;

[0024] The inorganic salts include calcium chloride, magnesium sulfate, potassium chloride, potassium nitrate, ferric sulfate, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium selenite, copper sulfate, zinc sulfate, ferrous citrate, manganese sulfate, sodium silicate, ammonium molybdate, ammonium metavanadate, nickel sulfate, stannous chloride, aluminum chloride, silver nitrate, barium acetate, potassium bromide, cadmium chloride, cobalt chloride, chromium trichloride, sodium fluoride, germanium oxide, potassium iodide, rubidium chloride and zirconium oxide;

[0025] The hormones include insulin, estradiol, progesterone, dexamethasone and methylprednisolone;

[0026] The lipids include arachidonic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, myristic acid, cholesterol, phosphatidylcholine and triglycerides;

[0027] The proteins other than hormones include human serum albumin and transferrin;

[0028] The energy substances include glucose, sodium pyruvate, α-ketoglutarate and glutamine;

[0029] The buffer system includes HEPES, sodium dihydrogen phosphate and disodium hydrogen phosphate;

[0030] The nitrogenous bases include thymine, uracil, hypoxanthine, adenine and guanine hydrochloride;

[0031] The other substances include reduced glutathione, ethanolamine, mercaptoethanol, heparin sodium, indomethacin, putrescine, spermine, spermidine, mevalonic acid, GGPP, taurine and lipoic acid.

[0032] Further, the final concentrations of the amino acid components in the basal medium are: glycine 10-50 mg / L, alanine 10-50 mg / L, arginine 50-100 mg / L, asparagine 10-50 mg / L, aspartic acid 10-50 mg / L, cystine 50-100 mg / L, cysteine ​​1-20 mg / L, glutamic acid 50-80 mg / L, glutamine 1-10 mM, histidine 10-50 mg / L, isoleucine 80-120 mg / L, leucine 80-120 mg / L, lysine 100-200 mg / L, methionine 10-50 mg / L, phenylalanine 30-80 mg / L / L, proline 30-80mg / L, serine 30-80mg / L, threonine 10-100mg / L, tryptophan 10-50mg / L, tyrosine 80-120mg / L, valine 10-100mg / L and hydroxyproline 10-100mg / L; preferably, the final concentrations of the amino acids in the basal medium are: glycine 25-35mg / L, alanine 20-30mg / L, arginine 60-100mg / L, asparagine 30-50mg / L, aspartic acid 20-30mg / L, cystine 60-100mg / L, cysteine ​​5-20mg / L, glutamic acid 60- 80mg / L, glutamine 5-10mM, histidine 20-50mg / L, isoleucine 100-120mg / L, leucine 100-120mg / L, lysine 150-200mg / L, methionine 20-50mg / L, phenylalanine 40-80mg / L, proline 30-50mg / L, serine 30-50mg / L, threonine 50-100mg / L, tryptophan 10-20mg / L, tyrosine 100-120mg / L, valine 50-100mg / L and hydroxyproline 50-100mg / L; more preferably, the final concentration of each component in the basal medium is They are: glycine 30mg / L, alanine 25mg / L, arginine 94mg / L, asparagine 35mg / L, aspartic acid 30mg / L, cystine 93mg / L, cysteine ​​18.5mg / L, glutamate 75mg / L, glutamine 5mM, histidine 42mg / L, isoleucine 105mg / L, leucine 105mg / L, lysine 166mg / L, methionine 30mg / L, phenylalanine 46mg / L, proline 40mg / L, serine 42mg / L, threonine 95mg / L, tryptophan 20mg / L, tyrosine 120mg / L, valine 96mg / L and hydroxyproline 80m g / L.

[0033] Further, the final concentrations of the components of the vitamins in the basal medium are: biotin 0.1-0.3 mg / L, choline chloride 100-250 mg / L, D-calcium pantothenate 1-10 mg / L, folic acid 1-10 mg / L, nicotinamide 1-10 mg / L, pyridoxine 1-10 mg / L, riboflavin 0.5-1 mg / L, thiamine hydrochloride 1-10 mg / L, cobalamin 1-3 mg / L, inositol 100-200 mg / L, p-aminobenzoic acid 0.1-1.5 mg / L, ascorbic acid 50-120 mg / L, vitamin D2 1-100 mg / L, α-tocopherol 50-100 mg / L, vitamin K2 1-100 mg / L and vitamin K3 1-100mg / L; preferably, the final concentrations of the components of the vitamins in the basal medium are: biotin 0.1-0.2mg / L, choline chloride 100-150mg / L, D-calcium pantothenate 5-10mg / L, folic acid 5-10mg / L, nicotinamide 5-10mg / L, pyridoxine 6-10mg / L, riboflavin 0.6-1mg / L, thiamine hydrochloride 5-10mg / L, cobalamin 1-2mg / L, inositol 100-150mg / L, p-aminobenzoic acid 1-1.5mg / L, ascorbic acid 50-100mg / L, vitamin D2 10-30mg / L, α-tocopherol 50-80mg / L, vitamin K2 More preferably, the final concentrations of the vitamin components in the basal medium are: biotin 0.19 mg / L, choline chloride 140.2 mg / L, D-calcium pantothenate 10 mg / L, folic acid 8 mg / L, nicotinamide 8.62 mg / L, pyridoxine 9.05 mg / L, riboflavin 1 mg / L, thiamine hydrochloride 7.4 mg / L, cobalamin 1.25 mg / L, inositol 134.8 mg / L, p-aminobenzoic acid 1.5 mg / L, ascorbic acid 88 mg / L, vitamin D2 20 mg / L, α-tocopherol 75 mg / L, vitamin K2 50 mg / L and vitamin K3 20 mg / L.

[0034] Further, the final concentrations of the components of the inorganic salts in the basal culture medium are: calcium chloride 100-200 mg / L, magnesium sulfate 50-100 mg / L, potassium chloride 300-500 mg / L, potassium nitrate 0.05-0.1 mg / L, iron sulfate 0.1-0.5 mg / L, sodium chloride 2000-6000 mg / L, sodium dihydrogen phosphate 100-200 mg / L, disodium hydrogen phosphate 50-100 mg / L, sodium selenite 0.01-0.06 mg / L, copper sulfate 0.001-0.006 mg / L, zinc sulfate 0.5-1 mg / L, ferrous citrate 1-2 mg / L, manganese sulfate 0.0001-0.0002 mg / L, sodium silicate 0.1-0.5 mg / L. g / L, ammonium molybdate 0.001-0.003mg / L, ammonium metavanadate 0.0005-0.001mg / L, nickel sulfate 0.0001-0.0005mg / L, stannous chloride 0.0001-0.0005mg / L, aluminum chloride 0.001-0.005mg / L, silver nitrate 0.0001-0.0005mg / L, barium acetate 0.0001-0.0005mg / L, potassium bromide 0.0001-0.0005mg / L, cadmium chloride 0.001-0.005mg / L, cobalt chloride 0.001-0.005mg / L, chromium trichloride 0.0001-0.0005mg / L, sodium fluoride 0.001-0.005mg / L, germanium oxide 0.0001-0.0005 mg / L, potassium iodide 0.0001-0.0005 mg / L, rubidium chloride 0.001-0.005 mg / L and zirconium oxide 0.0001-0.0006 mg / L; preferably, the final concentrations of the components in the basal medium are: calcium chloride 150-200 mg / L, magnesium sulfate 60-100 mg / L, potassium chloride 300-400 mg / L, potassium nitrate 0.05-0.1 mg / L, iron sulfate 0.2-0.5 mg / L, sodium chloride 3000-6000 mg / L, sodium dihydrogen phosphate 100-200 mg / L, disodium hydrogen phosphate 60-100 mg / L, sodium selenite 0.03-0 .06mg / L, copper sulfate 0.001-0.005mg / L, zinc sulfate 0.6-1mg / L, ferrous citrate 1-1.5mg / L, manganese sulfate 0.0001-0.0002mg / L, sodium silicate 0.1-0.2mg / L, ammonium molybdate 0.001-0.003mg / L, ammonium metavanadate 0.0005-0.001mg / L, nickel sulfate 0.0001-0.0005mg / L, stannous chloride 0.0001-0.0002mg / L, aluminum chloride 0.001-0.005mg / L, silver nitrate 0.0001-0.0005mg / L, barium acetate 0.0001-0.0005mg / L, potassium bromide 0.0001-0.0.0005mg / L, cadmium chloride 0.001-0.005mg / L, cobalt chloride 0.001-0.005mg / L, chromium trichloride 0.0001-0.0005mg / L, sodium fluoride 0.001-0.005mg / L, germanium oxide 0.0001-0.0006mg / L, potassium iodide 0.0001-0.0005mg / L, rubidium chloride 0.001-0.005mg / L and zirconium oxide 0.0001-0.0005 mg / L; More preferably, the final concentrations of the components of the inorganic salts in the basal medium are: calcium chloride 175 mg / L, magnesium sulfate 100 mg / L, potassium chloride 350 mg / L, potassium nitrate 0.1 mg / L, iron sulfate 0.34 mg / L, sodium chloride 5500 mg / L, sodium dihydrogen phosphate 100 mg / L, disodium hydrogen phosphate 60.42 mg / L, sodium selenite 0.051 mg / L g / L, copper sulfate 0.005mg / L, zinc sulfate 0.913mg / L, ferrous citrate 1.25mg / L, manganese sulfate 0.0002mg / L, sodium silicate 0.15mg / L, ammonium molybdate 0.00224mg / L, ammonium metavanadate 0.00095mg / L, nickel sulfate 0.00024mg / L, stannous chloride 0.00012mg / L, aluminum chloride 0.0012mg / L, silver nitrate 0.00 012mg / L, barium acetate 0.0029mg / L, potassium bromide 0.00012mg / L, cadmium chloride 0.0033mg / L, cobalt chloride 0.0025mg / L, chromium trichloride 0.00032mg / L, sodium fluoride 0.0042mg / L, germanium oxide 0.0006mg / L, potassium iodide 0.00017mg / L, rubidium chloride 0.0012mg / L and zirconium oxide 0.00032mg / L.

[0035] Further, the final concentrations of the hormone components in the basal culture medium are: insulin 1-30 mg / L, estradiol 1-10 mg / L, progesterone 1-10 mg / L, dexamethasone 1-5 mM and methylprednisolone 1-10 mM; preferably, the final concentrations of the hormone components in the basal culture medium are: insulin 5-20 mg / L, estradiol 5-10 mg / L, progesterone 5-10 mg / L, dexamethasone 2-5 mM and methylprednisolone 1-5 mM; more preferably, the final concentrations of the hormone components in the basal culture medium are: insulin 15 mg / L, estradiol 6 mg / L, progesterone 10 mg / L, dexamethasone 3 mM and methylprednisolone 3 mM.

[0036] Further, the final concentrations of the components in the lipids in the basal medium are: arachidonic acid 1-10 mg / L, palmitic acid 1-100 mg / L, palmitoleic acid 1-100 mg / L, stearic acid 1-100 mg / L, oleic acid 1-100 mg / L, linoleic acid 1-100 mg / L, linolenic acid 1-100 mg / L, myristic acid 1-100 mg / L, cholesterol 100-500 mg / L, phosphatidylcholine 0.5-5 mg / L and triglyceride 0.1-1 mM; preferably, the final concentrations of the components in the basal medium are: arachidonic acid 5-10 mg / L, palmitic acid 1-30 mg / L, palmitoleic acid 1-20 mg / L, stearic acid 1-100 mg / L, oleic acid 1-100 mg / L, linoleic acid 1-100 mg / L, linolenic acid 1-100 mg / L, myristic acid 1-100 mg / L, cholesterol 100-500 mg / L, phosphatidylcholine 0.5-5 mg / L and triglyceride 0.1-1 mM. Fatty acid 1-30mg / L, oleic acid 1-15mg / L, linoleic acid 5-30mg / L, linolenic acid 5-30mg / L, myristic acid 1-30mg / L, cholesterol 100-300mg / L, phosphatidylcholine 1-5mg / L and triglyceride 0.5-1mM; more preferably, the final concentrations of each component in the lipid class in the basal culture medium are: arachidonic acid 10mg / L, palmitic acid 10mg / L, palmitoleic acid 10mg / L, stearic acid 10mg / L, oleic acid 10mg / L, linoleic acid 10mg / L, linolenic acid 10mg / L, myristic acid 10mg / L, cholesterol 220mg / L, phosphatidylcholine 2mg / L and triglyceride 0.6mM.

[0037] Furthermore, the final concentrations of the components in the protein class other than hormones in the basal culture medium are: human albumin 1500-3000 mg / L and transferrin 1-50 mg / L; preferably, the final concentrations of the components in the protein class other than hormones in the basal culture medium are: human albumin 2000-3000 mg / L and transferrin 1-25 mg / L; more preferably, the final concentrations of the components in the protein class other than hormones in the basal culture medium are: human albumin 2600 mg / L and transferrin 15 mg / L.

[0038] Furthermore, the final concentrations of the components in the energy substance category in the basal culture medium are: glucose 10-50mM, sodium pyruvate 100-200mg / L, α-ketoglutaric acid 1-10mg / L, and glutamine 1-10mM; preferably, the final concentrations of the components in the energy substance category in the basal culture medium are: glucose 10-30mM, sodium pyruvate 100-150mg / L, α-ketoglutaric acid 5-8mg / L, and glutamine 1-5mM; more preferably, the final concentrations of the components in the energy substance category in the basal culture medium are: glucose 25mM, sodium pyruvate 110mg / L, α-ketoglutaric acid 7.3mg / L, and glutamine 3mM.

[0039] Further, the final concentrations of the components in the buffer system in the basal culture medium are: HEPES 4000-8000 mg / L, sodium dihydrogen phosphate 10-50 mg / L, and disodium hydrogen phosphate 1-20 mg / L; preferably, the final concentrations of the components in the buffer system in the basal culture medium are: HEPES 4000-6500 mg / L, sodium dihydrogen phosphate 20-50 mg / L, and disodium hydrogen phosphate 10-20 mg / L; more preferably, the final concentrations of the components in the buffer system in the basal culture medium are: HEPES 5958 mg / L, sodium dihydrogen phosphate 25 mg / L, and disodium hydrogen phosphate 15 mg / L.

[0040] Further, the final concentrations of the components of the nitrogenous bases in the basal culture medium are: thymine 10-20 mg / L, uracil 1-100 mg / L, hypoxanthine 1-20 mg / L, adenine 1-100 mg / L and guanine hydrochloride; preferably, the final concentrations of the components of the nitrogenous bases in the basal culture medium are: thymine 10-18 mg / L, uracil 50-100 mg / L, hypoxanthine 5-10 mg / L, adenine 1-50 mg / L and guanine hydrochloride 1-50 mg / L; more preferably, the final concentrations of the components of the nitrogenous bases in the basal culture medium are: thymine 14 mg / L, uracil 55 mg / L, hypoxanthine 5 mg / L, adenine 45 mg / L and guanine hydrochloride 50 mg / L.

[0041] Further, the final concentrations of the components in the other substances in the basal medium are: reduced glutathione 0.1-2 mg / L, ethanolamine 50-150 mg / L, mercaptoethanol 10-100 M, heparin sodium 1-100 mg / L, indomethacin 1-3 mg / L, putrescine 1-10 mg / L, spermine 1-10 mg / L, spermidine 1-10 mg / L, mevalonate 1-5 mg / L, GGPP 1-10mg / L, taurine 1-10mg / L and lipoic acid 0.1-0.5mg / L; preferably, the final concentrations of the components in the other substances in the basal medium are: reduced glutathione 0.5-1.5mg / L, ethanolamine 100-150mg / L, mercaptoethanol 50-80mM, heparin sodium 50-100mg / L, indomethacin 1-2mg / L, putrescine 5-10mg / L, spermine 1-5mg / L, spermidine 1-5mg / L, mevalonate 1-3mg / L, GGPP 1-5mg / L, taurine 1-5mg / L and lipoic acid 0.1-0.2mg / L; more preferably, the final concentrations of each component in the other substances in the basal culture medium are: reduced glutathione 1.2mg / L, ethanolamine 101mg / L, mercaptoethanol 55mM, heparin sodium 63mg / L, indomethacin 1.8mg / L, putrescine 7.4mg / L, spermine 3.5mg / L, spermidine 3.5mg / L, mevalonate 1.34mg / L, GGPP 4mg / L, taurine 5mg / L and lipoic acid 0.105mg / L.

[0042] Furthermore, the other substances also include one or both of phenol red and antibiotics; preferably, the final concentration of the phenol red in the basal culture medium is 10-20 mg / L, and the final concentration of the antibiotic in the basal culture medium is 10%; more preferably, the final concentration of the phenol red in the basal culture medium is 10-15 mg / L, and the final concentration of the antibiotic in the basal culture medium is 10%; most preferably, the final concentration of the phenol red in the basal culture medium is 15 mg / L, and the final concentration of the antibiotic in the basal culture medium is 10%.

[0043] Further, the pH of the basal culture medium is 7.10-7.30 and the osmotic pressure is 280-320 mOSM; preferably, the pH of the basal culture medium is 7.20-7.30 and the osmotic pressure is 290-320 mOSM; more preferably, the pH of the basal culture medium is 7.20 and the osmotic pressure is 300 mOSM.

[0044] The present invention further provides a method for culturing NK cells using the above serum-free culture system, comprising the following steps:

[0045] Coating the cell culture flask: adding the coating solution to the cell culture flask, making the coating solution evenly dispersed on the bottom of the flask, coating, and obtaining the coated cell culture flask;

[0046] Cell inoculation: discard the coating solution in the cell culture flask after coating, resuspend the mononuclear cells in the activation medium and inoculate them in the cell culture flask, add heat-inactivated autologous plasma, and culture. The day of inoculation is recorded as day 0;

[0047] Fluid replenishment culture: On the third day of culture, activated culture medium and heat-inactivated autologous plasma are added and cultured;

[0048] Expanded bottle culture: On the 5th day of culture, take samples and count, adjust the cell density, and transfer the cell suspension into a new cell culture bottle or cell culture bag according to the volume of the cell suspension for culture;

[0049] Expanded culture: On the 7th day of culture, add expansion medium and heat-inactivated autologous plasma, take samples and count, adjust the cell density, transfer to a new cell culture bottle or cell culture bag according to the volume of the cell suspension, and culture dynamically;

[0050] Continue culturing: add expansion medium and heat-inactivated autologous plasma every one or two days, take samples and count, adjust cell density, transfer to new cell culture bottles or cell culture bags according to the volume of cell suspension, and culture dynamically;

[0051] Harvesting cells: NK cells were harvested on day 14 of culture.

[0052] Furthermore, the new cell culture flask or cell culture bag refers to a cell culture flask or cell culture bag that has never been coated.

[0053] Furthermore, the dynamic culture can be cultured on a seesaw shaker; preferably, the conditions for the culture on the seesaw shaker are: a swing frequency of 20-60 times / min, 37°C, 5% CO 2 More preferably, the conditions for the seesaw shaker culture are: a swing frequency of 30-60 times / min, 37°C, 5% CO 2 Most preferably, the conditions for the seesaw shaker culture are: a swing frequency of 30 times / min, 37°C, 5% CO 2 Dynamic culture can better simulate the physiological environment in the body, provide more uniform nutrient supply and gas exchange, and is conducive to the growth and function maintenance of NK cells. Compared with traditional static culture, it may increase the activity of NK cells.

[0054] Furthermore, in the cell inoculation step, the heat-inactivated autologous plasma accounts for 7-10% of the total volume of the activation medium and the heat-inactivated autologous plasma; preferably, 10%.

[0055] Furthermore, in the fluid replacement culture step, the heat-inactivated autologous plasma accounts for 7-10% of the total volume of the activation culture medium and the heat-inactivated autologous plasma; preferably, it is 10%.

[0056] Furthermore, in the bottle expansion culture step, the heat-inactivated autologous plasma accounts for 4-7% of the total volume of the expansion medium and the heat-inactivated autologous plasma; preferably, 5%.

[0057] Furthermore, in the expansion culture step, the heat-inactivated autologous plasma accounts for 1-3% of the total volume of the expansion medium and the heat-inactivated autologous plasma; preferably, 1%.

[0058] Furthermore, the cell inoculation step has an inoculation density of 2.0-3.0×10 6 cells / mL; preferably, 2.0×10 6 cells / mL.

[0059] Furthermore, the cell density is adjusted in the steps of expanding the culture, expanding the culture and continuing the culture so that the cell concentration is maintained at 0.5-1.0×10 6 cells / mL; preferably, 1.0×10 6 cells / mL.

[0060] Furthermore, in the step of coating the culture flask, the coating is performed at 2-8°C overnight; preferably, the coating is performed at 4°C overnight.

[0061] Furthermore, the culture was carried out at 37°C and 5% CO 2 Medium cultivation.

[0062] Furthermore, the heat-inactivated autologous plasma is obtained by inactivating the plasma separated from human blood at 56° C. for 30 minutes and then centrifuging it.

[0063] In a specific embodiment of the present invention, the method for preparing heat-inactivated autologous plasma and mononuclear cells is:

[0064] Preparation of heat-inactivated autologous plasma: anticoagulated blood (from human blood) was centrifuged at room temperature at 650×g for 15 minutes, and the upper yellow plasma portion was inactivated in a 56°C water bath for 30 minutes; after inactivation, it was centrifuged at 900×g for 10 minutes; the supernatant was taken and frozen at -20°C for 15 minutes; it was centrifuged again at 900×g for 10 minutes, and the supernatant was taken to obtain heat-inactivated autologous plasma;

[0065] Isolation of mononuclear cells: centrifuge anticoagulated blood (from human blood) at room temperature at 650×g for 15 minutes, remove the red precipitate, add PBS with the same volume as the original blood and mix thoroughly, and name the fully mixed solution as diluted blood; slowly add diluted blood to the lymphocyte separation solution; centrifuge at 800×g for 20 minutes at room temperature (speed slowly increased or decreased); after centrifugation, carefully aspirate the white film layer into a new centrifuge tube, add PBS to mix, and centrifuge at 300×g for 10 minutes; discard the supernatant, resuspend with PBS again, count the cells, and centrifuge at 300×g for 10 minutes; discard the supernatant to obtain mononuclear cells.

[0066] In the present invention, the human blood source includes human peripheral blood or umbilical cord blood, and the mononuclear cells are human peripheral blood mononuclear cells (PBMC) or umbilical cord blood mononuclear cells.

[0067] The beneficial effects of the present invention are as follows:

[0068] The serum-free culture system of NK cells of the present invention has the following advantages:

[0069] 1. Efficient proliferation: Through optimized culture medium components, especially unique growth factor combinations and the addition of nutrients, the serum-free culture system of NK cells of the present invention can significantly improve the proliferation efficiency of NK cells. In in vitro culture experiments, compared with existing commonly used culture media, the number of NK cells can be significantly increased within the same culture time, greatly shortening the culture cycle, making it possible to quickly obtain a large number of NK cells in clinical applications.

[0070] 2. Enhance cell activity and functional integrity: The components in each culture medium in the serum-free culture system help maintain the high activity and functional integrity of NK cells. The cultured NK cells showed stronger killing ability in the cytotoxic activity test, and the killing efficiency of tumor cells was also significantly improved. At the same time, the expression levels of activation receptors and inhibitory receptors on the surface of NK cells are more stable, ensuring the accuracy of cell recognition of target cells and the precision of immune response.

[0071] 3. Improve the stability of ingredients and consistency between batches: The use of new serum substitutes and precise control of the concentration range of each component effectively solves the problems of unstable ingredients and poor consistency between batches of existing culture media. In multiple batch production and experimental verification, the performance of the culture medium showed a high degree of stability, ensuring the repeatability of NK cell culture results, which is conducive to the standardization and scale of NK cell culture and provides stable quality cell products for clinical immunotherapy.

[0072] The NK cell culture method using the serum-free culture system of the present invention reduces the cost of NK cell culture, and can simply, quickly, efficiently and safely obtain a large number of NK cells with high activity, high purity and strong killing ability in vitro, providing a better source of cells for the clinical application of NK cells and promoting the further development of NK cell therapy technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0074] Figure 1 It is the cell viability during the cell culture process of Examples 1-3 and the comparative example, wherein the horizontal axis represents different time periods and the vertical axis represents the cell viability in %.

[0075] Figure 2 It is the cumulative expansion of cells during the cell culture process of Examples 1-3 and the comparative example, wherein the horizontal axis represents different time periods, and the vertical axis represents the cumulative expansion multiples of cells, in times.

[0076] Figure 3 CD3 cells cultured for 14 days in Example 1 - CD56 + / CD3 - CD16 + / CD3 - CD16 + CD56 + The proportion of cell populations.

[0077] Figure 4 CD3 cells cultured for 14 days in Example 2 - CD56 + / CD3 - CD16 + / CD3 - CD16 + CD56 + The proportion of cell populations.

[0078] Figure 5 CD3 cells cultured for 14 days in Example 3 - CD56 + / CD3 - CD16 + / CD3 - CD16 + CD56 + The proportion of cell populations.

[0079] Figure 6 For the control group, CD3 - CD56 + / CD3 - CD16+ / CD3 - CD16 + CD56 + The proportion of cell populations.

[0080] Figure 7 It is the killing rate of the cells cultured for 14 days in Examples 1-3 and the comparative example, wherein the abscissa is the effect-target ratio and the ordinate is the killing ability, and the unit is %. DETAILED DESCRIPTION

[0081] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0082] The consumables and reagents used in the present invention are all common commercial products and can be purchased on the market. Specifically, the reagents for preparing the basal culture medium were purchased from Sigma-Aldrich; Human CD16 antibody, IL-2, IL-15 and IL-21 were purchased from R&D Systems; Tacrolimus was purchased from Merk; FITC anti-human CD3 antibody (clone UCHT1), PE anti-human CD56 antibody (clone HCD56) and APC anti-human CD16 antibody (clone B73.1) were all purchased from BioLegend.

[0083] Example 1 A serum-free culture system and culture method for natural killer cells

[0084] 1. Preparation of serum-free culture system

[0085] The serum-free culture system includes coating solution, expansion medium, activation medium and heat-inactivated autologous plasma, as follows:

[0086] 1. Preparation of basal culture medium:

[0087] The basic culture medium includes amino acids, vitamins, inorganic salts, hormones, lipids, proteins other than hormones, energy substances, buffer systems, nitrogenous bases and other substances;

[0088] Amino acids include glycine, alanine, arginine, asparagine, aspartic acid, cystine, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and hydroxyproline;

[0089] Vitamins include biotin, choline chloride, D-calcium pantothenate, folic acid, niacinamide, pyridoxine, riboflavin, thiamine hydrochloride, cobalamin, inositol, para-aminobenzoic acid, ascorbic acid, vitamin D2, alpha-tocopherol, vitamin K2, and vitamin K3;

[0090] Inorganic salts include calcium chloride, magnesium sulfate, potassium chloride, potassium nitrate, ferric sulfate, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium selenite, copper sulfate, zinc sulfate, ferrous citrate, manganese sulfate, sodium silicate, ammonium molybdate, ammonium metavanadate, nickel sulfate, stannous chloride, aluminum chloride, silver nitrate, barium acetate, potassium bromide, cadmium chloride, cobalt chloride, chromium trichloride, sodium fluoride, germanium oxide, potassium iodide, rubidium chloride and zirconium oxide;

[0091] Hormones include insulin, estradiol, progesterone, dexamethasone, and methylprednisolone;

[0092] Lipids include arachidonic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, myristic acid, cholesterol, phosphatidylcholine, and triglycerides;

[0093] Proteins other than hormones include human serum albumin and transferrin;

[0094] Energy substances include glucose, sodium pyruvate, α-ketoglutarate and glutamine;

[0095] Buffer systems include HEPES, sodium dihydrogen phosphate, and disodium hydrogen phosphate;

[0096] Nitrogenous bases include thymine, uracil, hypoxanthine, adenine, and guanine hydrochloride;

[0097] Other substances include reduced glutathione, ethanolamine, mercaptoethanol, heparin sodium, indomethacin, putrescine, spermine, spermidine, mevalonate, GGPP, taurine, lipoic acid and phenol red.

[0098] The final concentrations of the components in the basal medium are: glycine 30 mg / L, alanine 25 mg / L, arginine 94 mg / L, asparagine 35 mg / L, aspartic acid 30 mg / L, cystine 93 mg / L, cysteine ​​18.5 mg / L, glutamic acid 75 mg / L, glutamine 8 mM, histidine 42 mg / L, isoleucine 105 mg / L, leucine 105 mg / L, lysine 166 mg / L, methionine 30 mg / L, phenylalanine 46 mg / L, proline 40 mg / L, serine 42 mg / L, threonine 100 mg / L, 40 mg / L, 10 mg / L, 10 mg / L, 2 ... Amine 95mg / L, tryptophan 20mg / L, tyrosine 120mg / L, valine 96mg / L, hydroxyproline 80mg / L, biotin 0.19mg / L, choline chloride 140.2mg / L, D-calcium pantothenate 10mg / L, folic acid 8mg / L, niacinamide 8.62mg / L, pyridoxine 9.05mg / L, riboflavin 1mg / L, thiamine hydrochloride 7.4mg / L, cobalamin 1.25mg / L, inositol 134.8mg / L, p-aminobenzoic acid 1.5mg / L, ascorbic acid 88mg / L, vitamin D2 20mg / L, alpha-tocopherol 75mg / L, vitamin K2 50mg / L, vitamin K3 20mg / L, calcium chloride 175mg / L, magnesium sulfate 100mg / L, potassium chloride 350mg / L, potassium nitrate 0.1mg / L, ferrous sulfate 0.34mg / L, sodium chloride 5500mg / L, sodium dihydrogen phosphate 125mg / L, disodium hydrogen phosphate 75.42mg / L, sodium selenite 0.051mg / L, copper sulfate 0.005mg / L, zinc sulfate 0.913mg / L, ferrous citrate 1.25mg / L, manganese sulfate 0.0002mg / L, sodium silicate 0.15mg / L, ammonium molybdate 0.00224mg / L, ammonium metavanadate 0. 0.00095mg / L, nickel sulfate 0.00024mg / L, stannous chloride 0.00012mg / L, aluminum chloride 0.0012mg / L, silver nitrate 0.00012mg / L, barium acetate 0.0029mg / L, potassium bromide 0.00012mg / L, cadmium chloride 0.0033mg / L, cobalt chloride 0.0025mg / L, chromium trichloride 0.00032mg / L, sodium fluoride 0.0042mg / L, germanium oxide 0.0006mg / L, potassium iodide 0.00017mg / L, rubidium chloride 0.0012mg / L, zirconium oxide 0.00032mg / L, insulin 15mg / L, estradiol 6mg / L, progesterone 10mg / L, dexamethasone 3mM, methylprednisolone 3mM, arachidonic acid 10mg / L, palmitic acid 10mg / L, palmitoleic acid 10mg / L, stearic acid 10mg / L, oleic acid 10mg / L, linoleic acid 10mg / L, linolenic acid 10mg / L, myristic acid 10mg / L, cholesterol 220mg / L, phosphatidylcholine 2mg / L, triglyceride 0.6mM, human serum albumin 2600mg / L, transferrin 15mg / L, glucose 25mM, sodium pyruvate 110mg / L, α-ketoglutaric acid 7.3mg / L, HEPES 5958mg / L, thymine 14mg / L, uracil 55mg / L, hypoxanthine 5mg / L, adenine 45mg / L, guanine hydrochloride 50mg / L, reduced glutathione 1.2mg / L, ethanolamine 101mg / L, mercaptoethanol 55mM, heparin sodium 63mg / L, indomethacin 1.8mg / L, putrescine 7.4mg / L, spermine 3.5mg / L, spermidine 3.5mg / L, mevalonate 1.34mg / L, GGPP 4mg / L, taurine 5mg / L, lipoic acid 0.105mg / L, phenol red 15mg / L.

[0099] Prepare the final concentrations of the above components, mix the components thoroughly, filter through a 0.22 μm filter membrane, adjust the pH to 7.20 and the osmotic pressure to 300 mOSM, and obtain a basal culture medium for later use.

[0100] 2. Preparation of activation medium

[0101] Add IL-2, IL-15, IL-21 and Tacrolimus (all powders) to the basal medium and mix well to make the final concentrations of IL-2, IL-15, IL-21 and Tacrolimus 1000 IU / mL, 80 ng / mL, 10 ng / mL and 3 ng / mL to obtain an activated medium for use.

[0102] 3. Preparation of expansion medium

[0103] IL-2 (powder) was added to the basal medium and mixed to make the final concentration of IL-2 2000 IU / mL to obtain an expansion medium for use.

[0104] 4. Preparation of coating solution

[0105] Human CD16 antibody was melted at room temperature, added to 5 mL PBS buffer, and mixed by inversion to make the final concentration of Human CD16 8 μg / mL to obtain coating solution for use.

[0106] 5. Obtaining autologous plasma and PBMC cells

[0107] Extraction and inactivation of autologous plasma: centrifuge the anticoagulated blood at 650×g for 15 minutes at room temperature, take the upper yellow plasma portion (the lower red precipitate is the blood cell component, which is used for the subsequent extraction of PBMC) and inactivate it in a 56°C water bath for 30 minutes; after inactivation, centrifuge at 900×g for 10 minutes; take the supernatant and freeze it at -20°C for 15 minutes; centrifuge again at 900×g for 10 minutes, take the supernatant, and obtain the heat-inactivated autologous plasma and store it at 4°C for later use.

[0108] PBMC separation: Take the lower red precipitate from the plasma extraction step, add PBS of the same volume as the original blood and mix thoroughly, and name the fully mixed solution as diluted blood; slowly add the diluted blood to the lymphocyte separation solution. Centrifuge at 800×g for 20 minutes at room temperature (speed increases and decreases slowly); after centrifugation, carefully aspirate the white film layer into a new centrifuge tube, add PBS to mix, and centrifuge at 300×g for 10 minutes; discard the supernatant, resuspend with PBS again, count the cells, and centrifuge at 300×g for 10 minutes; discard the supernatant to obtain PBMC cells for use.

[0109] 2. Cultivation of NK cells

[0110] Pretreatment of culture flask (-1 day): Add coating solution to T25 cell culture flask, shake it back and forth and left and right to make the coating solution evenly dispersed on the bottom of the flask, and coat it at 4℃ overnight.

[0111] Cell inoculation (Day 0): Take out the T25 cell culture flask coated overnight at 4°C and equilibrate it to room temperature. Discard the coating solution, resuspend the PBMC cells obtained in the above step in 9 mL of activation medium and inoculate them in a T25 cell culture flask (inoculation density 2×10 6 cells / mL), and add 1 mL of heat-inactivated autologous plasma (10%), shake it back and forth, and place it in 37°C, 5% CO 2 The cells were cultured in an incubator and the day of inoculation was recorded as day 0.

[0112] Rehydration culture: On the third day of culture, slowly add 18 mL of activation culture medium and 2 mL of heat-inactivated autologous plasma (i.e., heat-inactivated autologous plasma accounts for 10% of the total volume of activation culture medium and heat-inactivated autologous plasma, i.e., the content of heat-inactivated autologous plasma is 10%) along the side wall of the T25 cell culture flask. The total volume of liquid in the T25 cell culture flask is 30 mL. Avoid touching the bottom of the culture flask, do not blow the cells, and minimize counting, observation and other operations to avoid affecting the initial growth of cells. 37°C, 5% CO 2 Culture in an incubator.

[0113] Bottle expansion culture: On the fifth day of culture, take samples for counting, add expansion medium and 5% heat-inactivated autologous plasma (heat-inactivated autologous plasma accounts for 5% of the total volume of expansion medium and heat-inactivated autologous plasma, that is, the content of heat-inactivated autologous plasma is 5%), and adjust the cell density to 1.0×10 6 cells / mL, expand the flask according to the volume of the cell suspension (i.e. transfer to a new T175 cell culture flask or transfer to a cell culture bag for culture. Neither the T175 cell culture flask nor the cell culture bag for transfer flask culture needs to be coated with coating solution), 37°C, 5% CO 2 Continue culturing in the incubator. Minimize cell counting, observation, pipetting and other operations during the first 5 days to avoid affecting the initial growth of cells.

[0114] Expansion culture: On the 7th day of culture, add expansion medium and heat-inactivated autologous plasma (the content of heat-inactivated autologous plasma is reduced to 1%), take samples and count, and adjust the cell density to 1.0×10 6 cells / mL, expand the bottle according to the volume of cell suspension, and transfer to a rocker incubator at 37°C and 5% CO 2 , 30 times / min and continue to culture.

[0115] Continue culturing: add expansion medium and heat-inactivated autologous plasma every other day or two, take samples and count, and adjust the cell density to 1.0×10 6 cells / mL, expand the bottle according to the volume of cell suspension, and place in a rocker in 37°C, 5% CO 2 , 30 times / min and continue to culture.

[0116] Harvesting cells: NK cells were harvested on day 14 of culture.

[0117] 3. Evaluation of NK cells

[0118] Record the cell viability at each stage of the culture process. Figure 1 As shown, the results showed that the cell viability was high overall, and the cell viability remained above 94% during the entire culture process.

[0119] Record the number of viable cells at each stage of the culture process. Figure 2 As shown, the results show that the initial cell number is 2×10 7 cells, 20.7134 billion cells were harvested in 14 days, and the cells expanded by 1035.67 times.

[0120] Purity testing of cells at each stage of the culture process, i.e. CD3 - CD56 + / CD3 - CD16 + / CD3 - CD16+ CD56 + The percentage of cell populations is shown in Figure 3 CD3 of NK cells obtained by culturing for 14 days - CD56 + / CD3 - CD16 + / CD3 - CD16 + CD56 + The results of the cell population ratio test are as follows Figure 6 The results showed that after 14 days of culture, the NK cells obtained had CD3 - CD56 + 95.55%, CD3 - CD16 + 97.05%, CD3 - CD16 + CD56 + It is 95.83%.

[0121] The results of the test on the killing ability of NK cells obtained after 14 days of culture are as follows Figure 7 As shown, the results showed that when the effector-target ratio was 1:1, the killing rate of NK cells was 94.64%; when the effector-target ratio was 5:1, the killing rate of NK cells was 95.39%; when the effector-target ratio was 10:1, the killing rate of NK cells was 96.38%; when the effector-target ratio was 20:1, the killing rate of NK cells was 98.20%.

[0122] Example 2 A serum-free culture system and culture method for natural killer cells

[0123] 1. Preparation of serum-free culture system

[0124] The serum-free culture system includes coating solution, expansion medium, activation medium and heat-inactivated autologous plasma, as follows:

[0125] 1. The preparation of the basal medium is different from that of the basal medium in Example 1 in that the final concentrations of the components in the basal medium are different, specifically:

[0126] Glycine 25mg / L, Alanine 20mg / L, Arginine 60mg / L, Asparagine 30mg / L, Aspartic acid 20mg / L, Cystine 60mg / L, Cysteine ​​5mg / L, Glutamic acid 60mg / L, Glutamine 7mM, Histidine 20mg / L, Isoleucine 100mg / L, Leucine 100mg / L, Lysine 150mg / L, Methionine 20mg / L, Phenylalanine 40mg / L, Proline 30mg / L, Serine 30mg / L, Threonine 50mg / L, Tryptophan 10mg / L, Tyrosine 100mg / L, Valine 50mg / L, Hydroxyproline 50mg / L, Biotin 0.1mg / L, Choline Chloride 100mg / L, D-Calcium Pantothenate 5mg / L, Folic Acid 5mg / L, Nicotinamide 5mg / L, Pyridoxine 6mg / L, Riboflavin 0.6mg / L, Thiamine Hydrochloride 5mg / L, Cobalamin 1mg / L, Inositol 100mg / L, Para-Aminobenzoic Acid 1mg / L, Ascorbic Acid 50mg / L, Vitamin D2 10mg / L, α-Tocopherol 50mg / L, Vitamin K2 60mg / L, Vitamin K310mg / L, Calcium Chloride 150mg / L, Magnesium Sulfate 60mg / L, Potassium Chloride 300mg / L, Potassium Nitrate 0.05mg / L, Ferrous Sulfate 0.2mg / L, Sodium Chloride 3000mg / L, Sodium Dihydrogen Phosphate 130mg / L, Sodium Hydrogen Phosphate 70mg / L, Sodium Selenite 0.03mg / L, Copper Sulfate 0.001mg / L, Zinc Sulfate 0.6mg / L, Ferrous Citric Acid 1mg / L, Manganese Sulfate 0.0001mg / L, Sodium Silicate 0.1mg / L, Ammonium Molybdate 0.001mg / L, Ammonium Metavanadate 0.0005mg / L, Nickel Sulfate 0.0001mg / L, Stannous Chloride 0.0001mg / L, Aluminum Chloride 0.001mg / L, Silver Nitrate 0.0001mg / L, Barium Acetate 0.0 01mg / L, potassium bromide 0.0001mg / L, cadmium chloride 0.001mg / L, cobalt chloride 0.001mg / L, chromium trichloride 0.0001mg / L, sodium fluoride 0.001mg / L, germanium oxide 0.0001mg / L, potassium iodide 0.0001mg / L, rubidium chloride 0.001mg / L, zirconium oxide 0.0001mg / L, insulin 5mg / L, estradiol 5mg / L, progesterone 5mg / L, dexamethasone 2mM, methylprednisolone 1mM, arachidonic acid 5mg / L, palmitic acid 1mg / L, palmitoleic acid 1mg / L, stearic acid 1mg / L, oleic acid 1mg / L, linoleic acid 5mg / L, linolenic acid 5mg / L, myristic acid 1mg / L, cholesterol 100mg / L, phosphatidylcholine 1mg / L, triglycerides 0.5mM, human albumin 2000mg / L, transferrin 1mg / L, glucose 10mM, sodium pyruvate 100mg / L, α-ketoglutaric acid 5mg / L, HEPES 4000mg / L, thymine 10mg / L, uracil 50mg / L, hypoxanthine 6mg / L, adenine 1mg / L, guanine hydrochloride 1mg / L, reduced glutathione 0.5mg / L, ethanolamine 100mg / L, mercaptoethanol 50mM, heparin sodium 50mg / L, indomethacin 1mg / L, putrescine 5mg / L, spermine 1mg / L, spermidine 1mg / L, mevalonate 1mg / L, GGPP 1mg / L, taurine 1mg / L, lipoic acid 0.1mg / L, phenol red 15mg / L. .

[0127] Prepare the final concentrations of the above components, mix the components thoroughly, filter through a 0.22 μm filter membrane, adjust the pH to 7.20 and the osmotic pressure to 300 mOSM, and obtain a basal culture medium for later use.

[0128] 2. The preparation of the activation medium is different from that of the activation medium in Example 1 in that the final concentrations of IL-2, IL-15, IL-21 and Tacrolimus are different, namely 1000 IU / mL, 50 ng / mL, 20 ng / mL and 1 ng / mL, respectively.

[0129] 3. The preparation of the expansion medium is the same as in Example 1.

[0130] 4. The difference between the preparation of the coating solution and the preparation of the coating solution in Example 1 is that the final concentration of Human CD16 is different, which is 5 μg / mL.

[0131] 5. The acquisition of autologous plasma and PBMC cells is the same as in Example 1.

[0132] 2. The culture of NK cells was the same as in Example 1.

[0133] 3. Evaluation of NK cells

[0134] Record the cell viability at each stage of the culture process. Figure 1 As shown, the results showed that the cell viability was high overall, and the cell viability remained above 93% during the entire culture process.

[0135] Record the number of viable cells at each stage of the culture process. Figure 2 As shown, the results show that the initial cell number is 2×10 7 cells, 18.7912 billion cells were harvested in 14 days, and the cells expanded by 939.56 times.

[0136] Purity testing of cells at each stage of the culture process, i.e. CD3- CD56 + / CD3 - CD16 + / CD3 - CD16 + CD56 + The percentage of cell populations is shown in Figure 4 CD3 of NK cells obtained by culturing for 14 days - CD56 + / CD3 - CD16 + / CD3 - CD16 + CD56 + The results of the cell population ratio test are as follows Figure 6 The results showed that after 14 days of culture, the NK cells obtained had CD3 - CD56 + 95.38%, CD3 - CD16 + 96.08%, CD3 - CD16 + CD56 + It is 94.56%.

[0137] The results of the test on the killing ability of NK cells obtained after 14 days of culture are as follows Figure 7 As shown, the results showed that when the effector-target ratio was 1:1, the killing rate of NK cells was 90.21%; when the effector-target ratio was 5:1, the killing rate of NK cells was 92.54%; when the effector-target ratio was 10:1, the killing rate of NK cells was 93.65%; when the effector-target ratio was 20:1, the killing rate of NK cells was 94.12%.

[0138] Example 3 A serum-free culture system and culture method for natural killer cells

[0139] 1. Preparation of serum-free culture system

[0140] The serum-free culture system includes coating solution, expansion medium, activation medium and heat-inactivated autologous plasma, as follows:

[0141] 1. The preparation of the basal medium is different from that of the basal medium in Example 1 in that the final concentrations of the components in the basal medium are different, specifically:

[0142] Glycine 35mg / L, Alanine 30mg / L, Arginine 100mg / L, Asparagine 50mg / L, Aspartic acid 30mg / L, Cystine 100mg / L, Cysteine ​​20mg / L, Glutamic acid 80mg / L, Glutamine 15mM, Histidine 50mg / L, Isoleucine 120mg / L, Leucine 120mg / L, Lysine 200mg / L, Methionine 50mg / L, Phenylalanine 80mg / L, Proline 50mg / L, Serine 50mg / L, Threonine 10 0mg / L, Tryptophan 20mg / L, Tyrosine 120mg / L, Valine 100mg / L, Hydroxyproline 100mg / L, Biotin 0.2mg / L, Choline Chloride 150mg / L, D-Calcium Pantothenate 10mg / L, Folic Acid 10mg / L, Nicotinamide 10mg / L, Pyridoxine 10mg / L, Riboflavin 1mg / L, Thiamine Hydrochloride 10mg / L, Cobalamin 2mg / L, Inositol 150mg / L, Para-Aminobenzoic Acid 1.5mg / L, Ascorbic Acid 100mg / L, Vitamin D2 30mg / L, α-Tocopherol 80mg / L, Vitamin K2 100mg / L, Vitamin K3 50mg / L, calcium chloride 200mg / L, magnesium sulfate 100mg / L, potassium chloride 400mg / L, potassium nitrate 0.1mg / L, ferrous sulfate 0.5mg / L, sodium chloride 6000mg / L, sodium dihydrogen phosphate 250mg / L, disodium hydrogen phosphate 120mg / L, sodium selenite 0.06mg / L, copper sulfate 0.005mg / L, zinc sulfate 1mg / L, ferrous citrate 1.5mg / L, manganese sulfate 0.0002mg / L, sodium silicate 0.2mg / L, ammonium molybdate 0.003mg / L, ammonium metavanadate 0. 001mg / L, nickel sulfate 0.0005mg / L, stannous chloride 0.0005mg / L, aluminum chloride 0.005mg / L, silver nitrate 0.0005mg / L, barium acetate 0.0005mg / L, potassium bromide 0.0005mg / L, cadmium chloride 0.005mg / L, cobalt chloride 0.005mg / L, chromium trichloride 0.0005mg / L, sodium fluoride 0.005mg / L, germanium oxide 0.0006mg / L, potassium iodide 0.0005mg / L, rubidium chloride 0.005mg / L, zirconium oxide 0.0005mg / L, insulin 20mg / L, estradiol 10mg / L, progesterone 10mg / L, dexamethasone 5mM, methylprednisolone 5mM, arachidonic acid 10mg / L, palmitic acid 30mg / L, palmitoleic acid 20mg / L, stearic acid 30mg / L, oleic acid 15mg / L, linoleic acid 30mg / L, linolenic acid 30mg / L, myristic acid 30mg / L, cholesterol 300mg / L, phosphatidylcholine 5mg / L, triglyceride 1mM, human serum albumin 3000mg / L, transferrin 25mg / L, glucose 30mM, sodium pyruvate 150mg / L, α-ketoglutaric acid 8mg / L, HEPES 6500mg / L, thymine 18mg / L, uracil 100mg / L, hypoxanthine 10mg / L, adenine 50mg / L, guanine hydrochloride 50mg / L, reduced glutathione 1.5mg / L, ethanolamine 150mg / L, mercaptoethanol 80mM, heparin sodium 100mg / L, indomethacin 2mg / L, putrescine 10mg / L, spermine 5mg / L, spermidine 5mg / L, mevalonic acid 3mg / L, GGPP 5mg / L, taurine 5mg / L, lipoic acid 0.2mg / L, phenol red 15mg / L.

[0143] Prepare the final concentrations of the above components, mix the components thoroughly, filter through a 0.22 μm filter membrane, adjust the pH to 7.20 and the osmotic pressure to 300 mOSM, and obtain a basal culture medium for later use.

[0144] 2. The preparation of the activation medium is different from that of the activation medium in Example 1 in that the final concentrations of IL-2, IL-15, IL-21 and Tacrolimus are different, namely 1000 IU / mL, 100 ng / mL, 50 ng / mL and 5 ng / mL, respectively.

[0145] 3. The preparation of the expansion medium is the same as in Example 1.

[0146] 4. The difference between the preparation of the coating solution and the preparation of the coating solution in Example 1 is that the final concentration of Human CD16 is different, which is 10 μg / mL.

[0147] 5. The acquisition of autologous plasma and PBMC cells is the same as in Example 1.

[0148] 2. The culture of NK cells was the same as in Example 1.

[0149] 3. Evaluation of NK cells

[0150] Record the cell viability at each stage of the culture process. Figure 1 As shown, the results showed that the cell viability was high overall, and the cell viability remained above 92% during the entire culture process.

[0151] Record the number of viable cells at each stage of the culture process. Figure 2 As shown, the results show that the initial cell number is 2×10 7 cells, 19.8936 billion cells were harvested in 14 days, and the cells expanded by 994.68 times.

[0152] Purity testing of cells at each stage of the culture process, i.e. CD3 - CD56 + / CD3 - CD16 + / CD3 - CD16 + CD56 + The percentage of cell populations is shown in Figure 3 CD3 of NK cells obtained by culturing for 14 days - CD56 + / CD3 - CD16 + / CD3 - CD16 + CD56 + The results of the cell population ratio test are as follows Figure 6 The results showed that after 14 days of culture, the NK cells obtained had CD3 - CD56 + 95.32%, CD3 - CD16 + 97.43%, CD3 - CD16 + CD56 + It is 94.70%.

[0153] The results of the test on the killing ability of NK cells obtained after 14 days of culture are as follows Figure 7 As shown, the results showed that when the effector-target ratio was 1:1, the killing rate of NK cells was 89.12%; when the effector-target ratio was 5:1, the killing rate of NK cells was 93.26%; when the effector-target ratio was 10:1, the killing rate of NK cells was 94.78%; when the effector-target ratio was 20:1, the killing rate of NK cells was 95.03%.

[0154] Comparative Example A serum-free culture system and culture method for natural killer cells

[0155] A commonly used NK cell culture kit on the market was used for comparative experiments. The evaluation of its NK cells is as follows:

[0156] Record the cell viability at each stage of the culture process. Figure 1As shown, the results showed that the cell viability was high overall, and the cell viability remained above 88% during the entire culture process.

[0157] Record the number of viable cells at each stage of the culture process. Figure 2 As shown, the results show that the initial cell number is 2×10 7 cells, 14.5431 billion cells were harvested in 14 days, and the cells expanded 727.16 times.

[0158] Purity testing of cells at each stage of the culture process, i.e. CD3 - CD56 + / CD3 - CD16 + / CD3 - CD16 + CD56 + The percentage of cell populations is shown in Figure 3 CD3 of NK cells obtained by culturing for 14 days - CD56 + / CD3 - CD16 + / CD3 - CD16 + CD56 + The results of the cell population ratio test are as follows Figure 6 The results showed that after 14 days of culture, the NK cells obtained had CD3 - CD56 + 86.43%, CD3 - CD16 + 85.89%, CD3 - CD16 + CD56 + It is 86.09%.

[0159] The results of the test on the killing ability of NK cells obtained after 14 days of culture are as follows Figure 7 As shown, the results showed that when the effector-target ratio was 1:1, the killing rate of NK cells was 80.12%; when the effector-target ratio was 5:1, the killing rate of NK cells was 85.24%; when the effector-target ratio was 10:1, the killing rate of NK cells was 89.74%; when the effector-target ratio was 20:1, the killing rate of NK cells was 93.12%.

[0160] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A serum-free culture system for NK cells, characterized in that: The serum-free culture system comprises a coating solution, an expansion culture medium, an activation culture medium and autologous plasma; in, The coating solution contains Human CD16; the final concentration of Human CD16 is 1-10 μg / mL; The expansion medium comprises a basal medium and IL-2, wherein the final concentration of the IL-2 is 200-3000 IU / mL; The activation medium comprises a basal medium, IL-2, IL-15, IL-21 and Tacrolimus; the final concentrations of the IL-2, IL-15, IL-21 and Tacrolimus are 200-3000 IU / mL, 10-100 ng / mL, 10-100 ng / mL and 0.1-5 ng / mL, respectively.

2. The serum-free culture system according to claim 1, characterized in that The autologous plasma is heat-inactivated autologous plasma; and / or, The final concentration of the Human CD16 antibody in the coating solution is 5-10 μg / mL; preferably, the final concentration of the Human CD16 antibody is 8 μg / mL; and / or, The final concentration of the IL-2 in the expansion medium is 1000-3000 IU / mL; preferably, the final concentration of the IL-2 is 2000 IU / mL; and / or, The final concentration of IL-2 in the activation medium is 1000-3000 IU / mL, the final concentration of IL-15 is 50-100 ng / mL, the final concentration of IL-21 is 10-50 ng / mL and the final concentration of Tacrolimus is 1-5 ng / mL; more preferably, the final concentration of IL-2 is 1000 IU / mL, the final concentration of IL-15 is 80 ng / mL, the final concentration of IL-21 is 10 ng / mL and the final concentration of Tacrolimus is 3 ng / mL.

3. The serum-free culture system according to claim 1, characterized in that: The basic culture medium includes amino acids, vitamins, inorganic salts, hormones, lipids, proteins other than hormones, energy substances, buffer systems, nitrogenous bases and other substances; in, The amino acids include glycine, alanine, arginine, asparagine, aspartic acid, cystine, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine and hydroxyproline; The vitamins include biotin, choline chloride, D-calcium pantothenate, folic acid, niacinamide, pyridoxine, riboflavin, thiamine hydrochloride, cobalamin, inositol, p-aminobenzoic acid, ascorbic acid, vitamin D2, α-tocopherol, vitamin K2 and vitamin K3; The inorganic salts include calcium chloride, magnesium sulfate, potassium chloride, potassium nitrate, ferric sulfate, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium selenite, copper sulfate, zinc sulfate, ferrous citrate, manganese sulfate, sodium silicate, ammonium molybdate, ammonium metavanadate, nickel sulfate, stannous chloride, aluminum chloride, silver nitrate, barium acetate, potassium bromide, cadmium chloride, cobalt chloride, chromium trichloride, sodium fluoride, germanium oxide, potassium iodide, rubidium chloride and zirconium oxide; The hormones include insulin, estradiol, progesterone, dexamethasone and methylprednisolone; The lipids include arachidonic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, myristic acid, cholesterol, phosphatidylcholine and triglycerides; The proteins other than hormones include human serum albumin and transferrin; The energy substances include glucose, sodium pyruvate, α-ketoglutarate and glutamine; The buffer system includes HEPES, sodium dihydrogen phosphate and disodium hydrogen phosphate; The nitrogenous bases include thymine, uracil, hypoxanthine, adenine and guanine hydrochloride; The other substances include reduced glutathione, ethanolamine, mercaptoethanol, heparin sodium, indomethacin, putrescine, spermine, spermidine, mevalonic acid, GGPP, taurine and lipoic acid.

4. The serum-free culture system according to claim 3, characterized in that: The final concentrations of the amino acid components in the basal medium are: glycine 10-50 mg / L, alanine 10-50 mg / L, arginine 50-100 mg / L, asparagine 10-50 mg / L, aspartic acid 10-50 mg / L, cystine 50-100 mg / L, cysteine ​​1-20 mg / L, glutamic acid 50-80 mg / L, glutamine 1-10 mM, histidine 10-50 mg / L, isoleucine 80-120 mg / L, leucine 80-120 mg / L, lysine 100-200 mg / L, methionine 10-50 mg / L, phenylalanine 30-80 mg / L, proline 100-200 mg / L, methionine 10-50 mg / L, phenylalanine 30-80 mg / L, Preferably, the final concentrations of the amino acids in the basal medium are: glycine 25-35 mg / L, alanine 20-30 mg / L, arginine 60-100 mg / L, asparagine 30-50 mg / L, aspartic acid 20-30 mg / L, cystine 60-100 mg / L, cysteine ​​5-20 mg / L, glutamic acid 60-80 mg / L. / L, glutamine 5-10mM, histidine 20-50mg / L, isoleucine 100-120mg / L, leucine 100-120mg / L, lysine 150-200mg / L, methionine 20-50mg / L, phenylalanine 40-80mg / L, proline 30-50mg / L, serine 30-50mg / L, threonine 50-100mg / L, tryptophan 10-20mg / L, tyrosine 100-120mg / L, valine 50-100mg / L and hydroxyproline 50-100mg / L; more preferably, the final concentrations of the amino acids in the basal medium are: glycine Acid 30mg / L, alanine 25mg / L, arginine 94mg / L, asparagine 35mg / L, aspartic acid 30mg / L, cystine 93mg / L, cysteine ​​18.5mg / L, glutamate 75mg / L, glutamine 5mM, histidine 42mg / L, isoleucine 105mg / L, leucine 105mg / L, lysine 166mg / L, methionine 30mg / L, phenylalanine 46mg / L, proline 40mg / L, serine 42mg / L, threonine 95mg / L, tryptophan 20mg / L, tyrosine 120mg / L, valine 96mg / L and hydroxyproline 80mg / L; and / or, The final concentrations of the vitamin components in the basic culture medium are: biotin 0.1-0.3 mg / L, choline chloride 100-250 mg / L, D-calcium pantothenate 1-10 mg / L, folic acid 1-10 mg / L, nicotinamide 1-10 mg / L, pyridoxine 1-10 mg / L, riboflavin 0.5-1 mg / L, thiamine hydrochloride 1-10 mg / L, cobalamin 1-3 mg / L, inositol 100-200 mg / L, p-aminobenzoic acid 0.1-1.5 mg / L, ascorbic acid 50-120 mg / L, vitamin D2 1-100 mg / L, α-tocopherol 50-100 mg / L, vitamin K2 1-100 mg / L and vitamin K3 1-100 mg / L; preferably, the final concentrations of the components of the vitamins in the basal medium are: biotin 0.1-0.2 mg / L, choline chloride 100-150 mg / L, D-calcium pantothenate 5-10 mg / L, folic acid 5-10 mg / L, nicotinamide 5-10 mg / L, pyridoxine 6-10 mg / L, riboflavin 0.6-1 mg / L, thiamine hydrochloride 5-10 mg / L, cobalamin 1-2 mg / L, inositol 100-150 mg / L, p-aminobenzoic acid 1-1.5 mg / L, ascorbic acid 50-100 mg / L, vitamin D2 10-30 mg / L, α-tocopherol 50-80 mg / L, vitamin K2 50-100 mg / L and vitamin K3 10-50 mg / L; More preferably, the final concentrations of the components of the vitamins in the basal medium are: biotin 0.19 mg / L, choline chloride 140.2 mg / L, D-calcium pantothenate 10 mg / L, folic acid 8 mg / L, nicotinamide 8.62 mg / L, pyridoxine 9.05 mg / L, riboflavin 1 mg / L, thiamine hydrochloride 7.4 mg / L, cobalamin 1.25 mg / L, inositol 134.8 mg / L, p-aminobenzoic acid 1.5 mg / L, ascorbic acid 88 mg / L, vitamin D2 20 mg / L, α-tocopherol 75 mg / L, vitamin K2 50 mg / L and vitamin K3 20 mg / L; and / or, The final concentrations of the inorganic salt components in the basic culture medium are: calcium chloride 100-200 mg / L, magnesium sulfate 50-100 mg / L, potassium chloride 300-500 mg / L, potassium nitrate 0.05-0.1 mg / L, iron sulfate 0.1-0.5 mg / L, sodium chloride 2000-6000 mg / L, sodium dihydrogen phosphate 100-200 mg / L, disodium hydrogen phosphate 50-100 mg / L, sodium selenite 0.01-0.06 mg / L, copper sulfate 0.001-0.006 mg / L, zinc sulfate 0.5-1 mg / L, ferrous citrate 1-2 mg / L, manganese sulfate 0.0001-0.0002 mg / L, sodium silicate 0.1-0.5 mg / L, molybdate Ammonium salt 0.001-0.003mg / L, ammonium metavanadate 0.0005-0.001mg / L, nickel sulfate 0.0001-0.0005mg / L, stannous chloride 0.0001-0.0005mg / L, aluminum chloride 0.001-0.005mg / L, silver nitrate 0.0001-0.0005mg / L, barium acetate 0.0001-0.0005mg / L, potassium bromide 0.0001-0.0005mg / L, cadmium chloride 0.001-0.005mg / L, cobalt chloride 0.001-0.005mg / L, chromium trichloride 0.0001-0.0005mg / L, sodium fluoride 0.001-0.005mg / L, germanium oxide 0.00 01-0.0005mg / L, potassium iodide 0.0001-0.0005mg / L, rubidium chloride 0.001-0.005mg / L and zirconium oxide 0.0001-0.0006mg / L; preferably, the final concentrations of the components in the basal medium are: calcium chloride 150-200mg / L, magnesium sulfate 60-100mg / L, potassium chloride 300-400mg / L, potassium nitrate 0.05-0.1mg / L, iron sulfate 0.2-0.5mg / L, sodium chloride 3000-6000mg / L, sodium dihydrogen phosphate 100-200mg / L, disodium hydrogen phosphate 60-100mg / L, sodium selenite 0.03-0.06mg / L, copper sulfate 0.001-0.005mg / L, zinc sulfate 0.6-1mg / L, ferrous citrate 1-1.5mg / L, manganese sulfate 0.0001-0.0002mg / L, sodium silicate 0.1-0.2mg / L, ammonium molybdate 0.001-0.003mg / L, ammonium metavanadate 0.0005-0.001mg / L, nickel sulfate 0.0001-0.0005mg / L, stannous chloride 0.0001-0.0002mg / L, aluminum chloride 0.001-0.005mg / L, silver nitrate 0.0001-0.0005mg / L, barium acetate 0.0001-0.0005mg / L, potassium bromide 0.0001-0.0005mg / L, cadmium chloride 0.001-0.005mg / L, cobalt chloride 0.001-0.005mg / L, chromium trichloride 0.0001-0.0005mg / L, sodium fluoride 0.001-0.005mg / L, germanium oxide 0.0001-0.0006mg / L, potassium iodide 0.0001-0.0005mg / L, rubidium chloride 0.001-0.005mg / L and zirconium oxide 0.0001-0.00 05mg / L; More preferably, the final concentrations of the components of the inorganic salts in the basal medium are: calcium chloride 175mg / L, magnesium sulfate 100mg / L, potassium chloride 350mg / L, potassium nitrate 0.1mg / L, iron sulfate 0.34mg / L, sodium chloride 5500mg / L, sodium dihydrogen phosphate 100mg / L, sodium hydrogen phosphate 60.42mg / L, sodium selenite 0.051mg / L, copper sulfate 0 0.005mg / L, zinc sulfate 0.913mg / L, ferrous citrate 1.25mg / L, manganese sulfate 0.0002mg / L, sodium silicate 0.15mg / L, ammonium molybdate 0.00224mg / L, ammonium metavanadate 0.00095mg / L, nickel sulfate 0.00024mg / L, stannous chloride 0.00012mg / L, aluminum chloride 0.0012mg / L, silver nitrate 0.00012mg / L g / L, barium acetate 0.0029mg / L, potassium bromide 0.00012mg / L, cadmium chloride 0.0033mg / L, cobalt chloride 0.0025mg / L, chromium trichloride 0.00032mg / L, sodium fluoride 0.0042mg / L, germanium oxide 0.0006mg / L, potassium iodide 0.00017mg / L, rubidium chloride 0.0012mg / L and zirconium oxide 0.00032mg / L;. and / or, The final concentrations of the hormone components in the basal culture medium are: insulin 1-30 mg / L, estradiol 1-10 mg / L, progesterone 1-10 mg / L, dexamethasone 1-5 mM and methylprednisolone 1-10 mM; preferably, the final concentrations of the hormone components in the basal culture medium are: insulin 5-20 mg / L, estradiol 5-10 mg / L, progesterone 5-10 mg / L, dexamethasone 2-5 mM and methylprednisolone 1-5 mM; more preferably, the final concentrations of the hormone components in the basal culture medium are: insulin 15 mg / L, estradiol 6 mg / L, progesterone 10 mg / L, dexamethasone 3 mM and methylprednisolone 3 mM; and / or, The final concentrations of the components in the lipids in the basal medium are: arachidonic acid 1-10 mg / L, palmitic acid 1-100 mg / L, palmitoleic acid 1-100 mg / L, stearic acid 1-100 mg / L, oleic acid 1-100 mg / L, linoleic acid 1-100 mg / L, linolenic acid 1-100 mg / L, myristic acid 1-100 mg / L, cholesterol 100-500 mg / L, phosphatidylcholine 0.5-5 mg / L and triglyceride 0.1-1 mM; preferably, the final concentrations of the components in the basal medium are: arachidonic acid 5-10 mg / L, palmitic acid 1-30 mg / L, palmitoleic acid 1-20 mg / L, stearic acid 1-30mg / L, oleic acid 1-15mg / L, linoleic acid 5-30mg / L, linolenic acid 5-30mg / L, myristic acid 1-30mg / L, cholesterol 100-300mg / L, phosphatidylcholine 1-5mg / L and triglyceride 0.5-1mM; More preferably, the final concentrations of the components in the basal medium are: arachidonic acid 10mg / L, palmitic acid 10mg / L, palmitoleic acid 10mg / L, stearic acid 10mg / L, oleic acid 10mg / L, linoleic acid 10mg / L, linolenic acid 10mg / L, myristic acid 10mg / L, cholesterol 220mg / L, phosphatidylcholine 2mg / L and triglyceride 0.6mM; and / or, The final concentrations of the components of the protein except hormones in the basal culture medium are: 1500-3000 mg / L human albumin and 1-50 mg / L transferrin; preferably, the final concentrations of the components of the protein except hormones in the basal culture medium are: 2000-3000 mg / L human albumin and 1-25 mg / L transferrin; more preferably, the final concentrations of the components of the protein except hormones in the basal culture medium are: 2600 mg / L human albumin and 15 mg / L transferrin; and / or, The final concentrations of the components in the energy substance category in the basal culture medium are: glucose 10-50mM, sodium pyruvate 100-200mg / L, α-ketoglutaric acid 1-10mg / L, glutamine 1-10mM; preferably, the final concentrations of the components in the energy substance category in the basal culture medium are: glucose 10-30mM, sodium pyruvate 100-150mg / L, α-ketoglutaric acid 5-8mg / L, glutamine 1-5mM; more preferably, the final concentrations of the components in the energy substance category in the basal culture medium are: glucose 25mM, sodium pyruvate 110mg / L, α-ketoglutaric acid 7.3mg / L, glutamine 3mM; and / or, The final concentrations of the components in the buffer system in the basal culture medium are: HEPES 4000-8000 mg / L, sodium dihydrogen phosphate 10-50 mg / L, disodium hydrogen phosphate 1-20 mg / L; preferably, the final concentrations of the components in the buffer system in the basal culture medium are: HEPES 4000-6500 mg / L, sodium dihydrogen phosphate 20-50 mg / L, disodium hydrogen phosphate 10-20 mg / L; more preferably, the final concentrations of the components in the buffer system in the basal culture medium are: HEPES 5958 mg / L, sodium dihydrogen phosphate 25 mg / L, disodium hydrogen phosphate 15 mg / L; and / or, The final concentrations of the components of the nitrogenous bases in the basal culture medium are: thymine 10-20 mg / L, uracil 1-100 mg / L, hypoxanthine 1-20 mg / L, adenine 1-100 mg / L and guanine hydrochloride; preferably, the final concentrations of the components of the nitrogenous bases in the basal culture medium are: thymine 10-18 mg / L, uracil 50-100 mg / L, hypoxanthine 5-10 mg / L, adenine 1-50 mg / L and guanine hydrochloride 1-50 mg / L; more preferably, the final concentrations of the components of the nitrogenous bases in the basal culture medium are: thymine 14 mg / L, uracil 55 mg / L, hypoxanthine 5 mg / L, adenine 45 mg / L and guanine hydrochloride 50 mg / L; and / or, The final concentrations of the components in the other substances in the basal medium are: reduced glutathione 0.1-2 mg / L, ethanolamine 50-150 mg / L, mercaptoethanol 10-100 M, heparin sodium 1-100 mg / L, indomethacin 1-3 mg / L, putrescine 1-10 mg / L, spermine 1-10 mg / L, spermidine 1-10 mg / L, mevalonate 1-5 mg / L, GGPP 1-10mg / L, taurine 1-10mg / L and lipoic acid 0.1-0.5mg / L; preferably, the final concentrations of the components in the other substances in the basal medium are: reduced glutathione 0.5-1.5mg / L, ethanolamine 100-150mg / L, mercaptoethanol 50-80mM, heparin sodium 50-100mg / L, indomethacin 1-2mg / L, putrescine 5-10mg / L, spermine 1-5mg / L, spermidine 1-5mg / L, mevalonate 1-3mg / L, GGPP 1-5mg / L, taurine 1-5mg / L and lipoic acid 0.1-0.2mg / L; More preferably, the final concentrations of the components in the other substances in the basal medium are: reduced glutathione 1.2mg / L, ethanolamine 101mg / L, mercaptoethanol 55mM, heparin sodium 63mg / L, indomethacin 1.8mg / L, putrescine 7.4mg / L, spermine 3.5mg / L, spermidine 3.5mg / L, mevalonate 1.34mg / L, GGPP 4mg / L, taurine 5mg / L and lipoic acid 0.105mg / L; and / or, The other substances also include one or two of phenol red and antibiotics; preferably, the final concentration of the phenol red in the basal culture medium is 10-20 mg / L, and the final concentration of the antibiotic in the basal culture medium is 10%; more preferably, the final concentration of the phenol red in the basal culture medium is 10-15 mg / L, and the final concentration of the antibiotic in the basal culture medium is 10%; most preferably, the final concentration of the phenol red in the basal culture medium is 15 mg / L, and the final concentration of the antibiotic in the basal culture medium is 10%.

5. The serum-free culture system according to any one of claims 1 to 4, characterized in that: The pH of the basal medium is 7.10-7.30 and the osmotic pressure is 280-320 mOSM; preferably, the pH of the basal medium is 7.20-7.30 and the osmotic pressure is 290-320 mOSM; more preferably, the pH of the basal medium is 7.20 and the osmotic pressure is 300 mOSM.

6. A method for culturing NK cells using the serum-free culture system according to any one of claims 1 to 5, characterized in that: The steps include: Coating the cell culture flask: adding the coating solution to the cell culture flask, making the coating solution evenly dispersed on the bottom of the flask, coating, and obtaining the coated cell culture flask; Cell inoculation: discard the coating solution in the cell culture flask after coating, resuspend the mononuclear cells in the activation medium and inoculate them in the cell culture flask, add heat-inactivated autologous plasma, and culture. The day of inoculation is recorded as day 0; Fluid replenishment culture: On the third day of culture, activated culture medium and heat-inactivated autologous plasma are added and cultured; Expanded bottle culture: On the 5th day of culture, take samples and count, adjust the cell density, and transfer the cell suspension into a new cell culture bottle or cell culture bag according to the volume of the cell suspension for culture; Expanded culture: On the 7th day of culture, add expansion medium and heat-inactivated autologous plasma, take samples and count, adjust the cell density, transfer to a new cell culture bottle or cell culture bag according to the volume of the cell suspension, and culture dynamically; Continue culturing: add expansion medium and heat-inactivated autologous plasma every one or two days, take samples and count, adjust cell density, transfer to new cell culture bottles or cell culture bags according to the volume of cell suspension, and culture dynamically; Harvesting cells: NK cells were harvested on day 14 of culture.

7. The culture method according to claim 6, characterized in that: The dynamic culture is cultured on a seesaw shaker; preferably, the conditions for culture on the seesaw shaker are: a swing frequency of 20-60 times / min, 37°C, and 5% CO2; more preferably, the conditions for culture on the seesaw shaker are: a swing frequency of 30-60 times / min, 37°C, and 5% CO2; most preferably, the conditions for culture on the seesaw shaker are: a swing frequency of 30 times / min, 37°C, and 5% CO2.

8. The culture method according to claim 6, characterized in that: In the cell inoculation step, the heat-inactivated autologous plasma accounts for 7-10% of the total volume of the activation medium and the heat-inactivated autologous plasma; preferably, 10%; and / or, In the rehydration culture step, the heat-inactivated autologous plasma accounts for 7-10% of the total volume of the activation culture medium and the heat-inactivated autologous plasma; preferably, 10%; and / or, In the bottle expansion culture step, the heat-inactivated autologous plasma accounts for 4-7% of the total volume of the expansion medium and the heat-inactivated autologous plasma; preferably, 5%; and / or, In the expansion culture step, the heat-inactivated autologous plasma accounts for 1-3% of the total volume of the expansion medium and the heat-inactivated autologous plasma; preferably, 1%.

9. The culture method according to claim 6, characterized in that: The cell inoculation step has an inoculation density of 2.0-3.0×10 6 cells / mL; preferably, 2.0×10 6 cells / mL; and / or, The cell density is adjusted in the steps of expanding the flask culture, expanding the culture and continuing the culture to maintain the cell concentration at 0.5-1.0×10 6 cells / mL; preferably, 1.0×10 6 cells / mL.

10. The culture method according to claim 6, characterized in that: In the step of coating the culture flask, the coating is carried out at 2-8°C overnight; preferably, at 4°C overnight; and / or, The culture is carried out at 37°C and 5% CO2; and / or, The heat-inactivated autologous plasma is obtained by separating plasma from human blood and inactivating it at 56° C. for 30 minutes and then centrifuging it; and / or, The human blood source includes human peripheral blood or umbilical cord blood, and the mononuclear cells are human peripheral blood mononuclear cells or umbilical cord blood mononuclear cells.

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