NK cell culture medium and NK cell culture method

Abstract

The invention relates to an NK cell culture medium and an NK cell culture method, and relates to the technical field of cell culture.The NK cell culture medium comprises a basic culture medium and an additive, and the additive is prepared from, by concentration, 200-400 IU / L of cytokines, 3-6 mmol / L of nutritional supplements, 1-2 mmol / L of metabolism regulators, 90-110 mg / L of vitamin C, 150-300 micrograms / mL of plant polysaccharides, 1-2 micrograms / mL of 1, 2-propylene glycol, 1-2 micrograms / L of 1, 3-propylene glycol and 1, 3-propylene glycol. The medicine is prepared from 50-100 [mu] mol / L of 1, 3-dicaffeoylquinic acid, 48-52 [mu] g / mL of nano-scale hydroxyapatite, 14-16 [mu] mol / L of a signal channel regulator, 5-10 IU / L of heparin sodium and 10-80 [mu] g / mL of astragaloside IV-liposome. The culture medium and the culture method can efficiently amplify the NK cells and maintain the activity and the functional stability of the NK cells.

Description

Technical Field

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

[0002] Natural killer cells (NK cells), as an important part of the immune system, play a key role in tumor immune surveillance and anti-viral infection. Due to their characteristic of directly killing tumor cells without prior sensitization, they have become immune therapy cells with great potential. However, NK cells face problems such as low proliferation efficiency and difficulty in maintaining activity during in vitro culture. Currently, traditional NK cell culture media usually use animal-derived components such as fetal bovine serum. However, such components have large differences between batches, may carry pathogens and other risks, and will bring safety hazards such as immunogenicity to clinical applications. Existing serum-free culture media also have technical defects such as unsatisfactory culture effects and the need to improve the amplification efficiency and activity maintenance of NK cells.

[0003] To solve the above problems, a Chinese invention patent with the authorization announcement number CN114196630B discloses an NK cell culture medium and a method for culturing NK cells. In the NK cell culture medium of this invention, IL-2, IL-15, and IL-21 have synergistic effects and act together on mononuclear cells derived from umbilical cord blood to obtain a large number of NK cells with high purity, high killing activity, and high cytotoxic activity. The NK cell culture method of this invention has the advantages of high induction efficiency and fast amplification speed; during the process of culturing NK cells using the culture medium of this invention, there is no need to introduce tumor cells and no need to add animal serum, avoiding safety hazards and having the advantage of high safety; the NK cell culture medium of this invention has few added factors, low cost, and the culture method is simple, efficient, and can meet the clinical requirements for NK cells. However, the NK cell amplification efficiency and activity maintenance of this culture medium and culture method still need to be further improved.

[0004] Therefore, it is of great practical significance to develop a culture medium and a culture method that can efficiently amplify NK cells, maintain their activity and functional stability, and have safe components and controllable costs. Summary of the Invention

[0005] The purpose of the present invention is to provide an NK cell culture medium and a method for culturing NK cells that can efficiently amplify NK cells, maintain their activity and functional stability, and have safe components and controllable costs in order to overcome the deficiencies of the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is a NK cell culture medium, which includes a basal medium and additives. The components and concentrations of the additives are as follows: cytokines 200 - 400 IU / L, nutritional supplements 3 - 6 mmol / L, metabolic regulators 1 - 2 mmol / L, vitamin C 90 - 110 mg / L, plant polysaccharides 150 - 300 μg / mL, 1,3-dicaffeoylquinic acid 50 - 100 μmol / L, nano-hydroxyapatite 48 - 52 μg / mL, signal pathway regulators 14 - 16 μmol / L, heparin sodium 5 - 10 IU / L, astragaloside-IV-liposomes 10–80 μg / mL.

[0007] Preferably, the cytokines are a mixture of IL-2, IL-15, IL-21, and IL-33 in a mass ratio of (15 - 20):(7 - 9):(2 - 4):1.

[0008] Preferably, the nutritional supplements are a mixture of glutamine and sodium pyruvate in a molar ratio of (2 - 4):(1 - 2).

[0009] Preferably, the metabolic regulator is ethyl pyruvate.

[0010] Preferably, the plant polysaccharides are a mixture of astragalus polysaccharides, ginseng polysaccharides, ganoderma polysaccharides, lentinan, poria cocos polysaccharides, wolfberry polysaccharides, and tea polysaccharides in a mass ratio of (1 - 3):1:(0.8 - 1.2):(1 - 2):(0.5 - 0.8):1:(0.3 - 0.5).

[0011] Preferably, the particle size of the nano-hydroxyapatite is 10 - 80 nm.

[0012] Preferably, the signal pathway regulators are a mixture of Y-27632 and AZD8055 in a molar ratio of 3000:1.

[0013] Preferably, the preparation method of the astragaloside-IV-liposomes includes the following steps: mixing soybean phospholipids and cholesterol to form a lipid raw material, and dissolving it together with astragaloside-IV in a mixed solvent; rotating and evaporating at 45 °C and 150 rpm for 30 minutes to form a uniform lipid film, and then vacuum drying for 2 hours to remove the residual solvent; adding PBS buffer solution with a pH of 7.4 preheated to 45 °C to the flask containing the lipid film, hydrating in a water bath at 45 °C for 30 minutes, and then ultrasonically treating for 3 - 5 minutes to form a primary emulsion suspension; subjecting the primary emulsion suspension to high-pressure homogenization treatment at 15000 psi for 5 - 8 cycles to obtain astragaloside-IV-liposomes.

[0014] Preferably, the molar ratio of soybean phospholipids to cholesterol is 7:3.

[0015] Preferably, the mass ratio of astragaloside-IV, lipid raw material, and mixed solvent is 1:9:(40 - 50).

[0016] Preferably, the mixed solvent is a mixture of chloroform and methanol in a volume ratio of 2:1.

[0017] Preferably, the amount ratio of the lipid film to the PBS buffer solution is 100 mg:10 mL.

[0018] Preferably, the basal medium is IMDM medium.

[0019] Another object of the present invention is to provide a method for preparing the NK cell medium, comprising the following steps: mixing the basal medium with each component in proportion and adjusting the pH to 7.2 - 7.4; sterilizing with a 0.22 μm filter membrane, aliquoting, and storing at -20°C.

[0020] Another object of the present invention is to provide a method for culturing NK cells using the above NK cell medium, comprising the following steps: Step S1: Using a 3D suspension culture system, inoculate peripheral blood mononuclear cells at an inoculation density of cells / mL into the NK cell medium and culture at 37°C, 5% CO2, and 95% humidity conditions; Step S2: After counting every day, replace half of the medium according to the situation; at the same time, adjust the cell concentration according to the cell growth situation and maintain it at cells / mL; Step S3: Collect the cells after culturing for 10 - 14 days.

[0021] Preferably, in step S1, low-frequency pulsed electromagnetic fields are used for auxiliary treatment during the culture process. The frequency of the low-frequency pulsed electromagnetic fields is 50 - 100 Hz, the intensity is 50 - 100 mT, the pulse width is 1 - 10 ms, the treatment time is 15 - 25 min each time, and it is performed 3 - 5 times a day.

[0022] Due to the application of the above technical solutions, the present invention has the following beneficial effects: (1) The NK cell medium disclosed by the present invention has a simple and easy preparation method, convenient operation and control, high preparation efficiency and finished product qualification rate, low dependence on equipment, is easy to be mass-produced industrially, and has high popularization and application value.

[0023] (2) The NK cell culture medium disclosed by the present invention comprises a basal medium and additives. The components and concentrations of the additives are as follows: cytokine 200 - 400 IU / L, nutritional supplement 3 - 6 mmol / L, metabolic regulator 1 - 2 mmol / L, vitamin C 90 - 110 mg / L, plant polysaccharide 150 - 300 μg / mL, 1,3 - dicaffeoylquinic acid 50 - 100 μmol / L, nano - hydroxyapatite 48 - 52 μg / mL, signal pathway regulator 14 - 16 μmol / L, heparin sodium 5 - 10 IU / L, astragaloside - IV - liposome 10–80 μg / mL. Through the mutual cooperation and joint action of each component, the prepared culture medium can efficiently expand NK cells, maintain their activity and functional stability, and has safe components and controllable costs. By synergistically combining various components such as cytokines, signal pathway regulators, and nano - hydroxyapatite, a three - dimensional technical system of "signal regulation - metabolic optimization - microenvironment improvement" is formed, breaking through the limitation of single - component optimization of existing culture media, and significantly improving the expansion effect and killing rate of NK cells. The plant polysaccharide is a mixture of astragalus polysaccharide, ginseng polysaccharide, ganoderma polysaccharide, lentinan, poria cocos polysaccharide, wolfberry polysaccharide, and tea polysaccharide in a mass ratio of (1 - 3):1:(0.8 - 1.2):(1 - 2):(0.5 - 0.8):1:(0.3 - 0.5). Through such a formulation selection, the addition of the plant polysaccharide can effectively promote the proliferation and activation of NK cells, enhance the cytotoxicity of NK cells, regulate the immune microenvironment, and inhibit NK cell exhaustion.

[0024] (3) The NK cell culture medium disclosed by the present invention, when adding astragaloside - IV - liposome, can produce a synergistic effect at the levels of cell proliferation, functional activation, anti - apoptosis, and microenvironment regulation through its unique physical and chemical properties and biological activities, promote the proliferation and activation of NK cells, resist NK cell apoptosis, and extend the in vitro culture survival time. Through multi - target regulation (proliferation signal, apoptosis pathway, microenvironment factor) and the advantages of liposome delivery (sustained release, low toxicity), a triple improvement of "amplification efficiency - functional activity - in - vivo persistence" is achieved. Adding astragaloside - IV in the form of liposome can effectively solve the problem of poor water solubility of astragaloside - IV, thereby improving the bioavailability.

[0025] (4) In the culture method of the NK cells disclosed by the present invention, during the culture process, low - frequency pulsed electromagnetic field is used for auxiliary treatment, which can regulate the cell cycle and proliferation signal, enhance cell metabolism and energy supply, promote the release of cytotoxic molecules, regulate cytokine secretion and immune regulation function, optimize the culture matrix and cell adhesion, inhibit cell apoptosis and maintain phenotypic stability; the combined effect with cytokines can reduce the dosage of cytokines while improving the culture efficiency; at the same time, it can also promote the delivery efficiency of nano - hydroxyapatite to NK cells, enhance its regulatory effect on the signal pathway, and further optimize cell function. Detailed implementation mode

[0026] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments in the following description are only examples, and other obvious variants can be conceived by those skilled in the art.

[0027] Example 1

[0028] An NK cell culture medium, comprising a basal medium and additives. The components and concentrations of the additives are as follows: cytokines 200 IU / L, nutritional supplements 3 mmol / L, metabolic regulators 1 mmol / L, vitamin C 90 mg / L, plant polysaccharides 150 μg / mL, 1,3-dicaffeoylquinic acid 50 μmol / L, nano-hydroxyapatite 48 μg / mL, signal pathway regulators 14 μmol / L, heparin sodium 5 IU / L, astragaloside-IV-liposome 10 μg / mL.

[0029] The cytokines are a mixture of IL-2, IL-15, IL-21, and IL-33 in a mass ratio of 15:7:2:1; the nutritional supplements are a mixture of glutamine and sodium pyruvate in a molar ratio of 2:1; the metabolic regulator is ethyl pyruvate; the plant polysaccharides are a mixture of astragalus polysaccharide, ginseng polysaccharide, ganoderma lucidum polysaccharide, lentinan, poria cocos polysaccharide, wolfberry polysaccharide, and tea polysaccharide in a mass ratio of 1:1:0.8:1:0.5:1:0.3; the particle size of the nano-hydroxyapatite is 10 nm; the signal pathway regulators are a mixture of Y-27632 and AZD8055 in a molar ratio of 3000:1.

[0030] The preparation method of the astragaloside-IV-liposome includes the following steps: mixing soybean phospholipids and cholesterol to form a lipid raw material, and dissolving it together with astragaloside-IV in a mixed solvent; rotating and evaporating at 45°C and 150 rpm for 30 minutes to form a uniform lipid film, and then vacuum drying for 2 hours to remove the residual solvent; adding PBS buffer solution with a pH of 7.4 preheated to 45°C to the flask containing the lipid film, hydrating in a water bath at 45°C for 30 minutes, and then ultrasonically treating for 3 minutes to form a primary emulsion suspension; subjecting the primary emulsion suspension to high-pressure homogenization treatment at 15000 psi for 5 cycles to obtain astragaloside-IV-liposome; the molar ratio of soybean phospholipids to cholesterol is 7:3; the mass ratio of astragaloside-IV, lipid raw material, and mixed solvent is 1:9:40; the mixed solvent is a mixture of chloroform and methanol in a volume ratio of 2:1; the amount ratio of the lipid film to the PBS buffer solution is 100 mg:10 mL; the basal medium is IMDM medium.

[0031] A method for preparing the NK cell culture medium, comprising the following steps: mixing the basal medium and each component in proportion, and adjusting the pH to 7.2; sterilizing with a 0.22 μm filter membrane, aliquoting, and storing at -20 °C.

[0032] A method for culturing NK cells using the above NK cell culture medium, comprising the following steps: Step S1: Using a 3D suspension culture system, inoculate peripheral blood mononuclear cells at an inoculation density of cells / mL into the NK cell culture medium, and culture under the conditions of 37 °C, 5% CO2 and 95% humidity; Step S2: After counting every day, replace half of the culture medium according to the situation; at the same time, adjust the cell concentration according to the cell growth situation, and maintain it at cells / mL; Step S3: Collect the cells after culturing for 10 days.

[0033] During the culture process in Step S1, low-frequency pulsed electromagnetic field-assisted treatment is adopted. The frequency of the low-frequency pulsed electromagnetic field is 50 Hz, the intensity is 50 mT, the pulse width is 1 ms, the treatment time is 15 min each time, and 3 times a day.

[0034] Example 2

[0035] An NK cell culture medium, comprising a basal medium and additives. The components and concentrations of the additives are as follows: cytokines 250 IU / L, nutritional supplements 4 mmol / L, metabolic regulators 1.2 mmol / L, vitamin C 95 mg / L, plant polysaccharides 180 μg / mL, 1,3-dicaffeoylquinic acid 60 μmol / L, nano-hydroxyapatite 49 μg / mL, signal pathway regulators 14.5 μmol / L, heparin sodium 6 IU / L, astragaloside-IV-liposome 30 μg / mL.

[0036] The cytokines are a mixture of IL-2, IL-15, IL-21, and IL-33 in a mass ratio of 17:7.5:2.5:1; the nutritional supplements are a mixture of glutamine and sodium pyruvate in a molar ratio of 2.5:1.2; the metabolic regulator is ethyl pyruvate; the plant polysaccharides are a mixture of astragalus polysaccharides, ginseng polysaccharides, ganoderma lucidum polysaccharides, lentinan polysaccharides, poria cocos polysaccharides, wolfberry polysaccharides, and tea polysaccharides in a mass ratio of 1.5:1:0.9:1.3:0.6:1:0.35; the particle size of the nano-hydroxyapatite is 30 nm; the signal pathway regulators are a mixture of Y-27632 and AZD8055 in a molar ratio of 3000:1.

[0037] The preparation method of astragaloside-IV liposome comprises the following steps: mixing soybean phospholipid and cholesterol to form lipid raw materials, and dissolving them together with astragaloside-IV in a mixed solvent; performing rotary evaporation at 45 °C and 150 rpm for 30 minutes to form a uniform lipid film, and then drying it under vacuum for 2 hours to remove the residual solvent; adding PBS buffer solution with pH = 7.4 preheated to 45 °C into the flask containing the lipid film, hydrating it in a water bath at 45 °C for 30 minutes, and then performing ultrasonic treatment for 3.5 minutes to form a primary emulsion suspension; subjecting the primary emulsion suspension to high-pressure homogenization treatment at 15000 psi for 6 cycles to obtain astragaloside-IV liposome; the molar ratio of soybean phospholipid to cholesterol is 7:3; the mass ratio of astragaloside-IV, lipid raw materials, and mixed solvent is 1:9:43; the mixed solvent is composed of chloroform and methanol mixed at a volume ratio of 2:1; the amount ratio of the lipid film to PBS buffer solution is 100 mg:10 mL; the basal medium is IMDM medium.

[0038] A preparation method of the NK cell medium comprises the following steps: mixing the basal medium and each component in proportion, and adjusting the pH to 7.3; sterilizing with a 0.22 μm filter membrane, and storing it at -20 °C after aliquoting.

[0039] A culturing method of NK cells using the above NK cell medium comprises the following steps: Step S1: Using a 3D suspension culture system, inoculating peripheral blood mononuclear cells at an inoculation density into the NK cell medium, and culturing them under the conditions of 37 °C, 5% CO2 and 95% humidity; Step S2: Counting every day and replacing half of the medium according to the situation; at the same time, adjusting the cell concentration according to the cell growth situation, and maintaining it at cells / mL; Step S3: Collecting the cells after culturing for 10 days.

[0040] During the culturing process in Step S1, low-frequency pulsed electromagnetic field-assisted treatment is adopted. The frequency of the low-frequency pulsed electromagnetic field is 60 Hz, the intensity is 60 mT, the pulse width is 3 ms, the treatment time is 17 min each time, and it is performed 4 times a day.

[0041] Example 3

[0042] An NK cell culture medium, comprising a basal medium and additives, and the components and concentrations of the additives are as follows: cytokines 300 IU / L, nutritional supplements 4.5 mmol / L, metabolic regulators 1.5 mmol / L, vitamin C 100 mg / L, plant polysaccharides 230 μg / mL, 1,3-dicaffeoylquinic acid 75 μmol / L, nano-hydroxyapatite 50 μg / mL, signal pathway regulators 15 μmol / L, heparin sodium 7 IU / L, astragaloside-IV-liposome 40 μg / mL.

[0043] The cytokines are a mixture of IL-2, IL-15, IL-21, and IL-33 in a mass ratio of 17:8:3:1; the nutritional supplements are a mixture of glutamine and sodium pyruvate in a molar ratio of 3:1.5; the metabolic regulator is ethyl pyruvate; the plant polysaccharides are a mixture of astragalus polysaccharide, ginseng polysaccharide, ganoderma lucidum polysaccharide, lentinan, poria cocos polysaccharide, wolfberry polysaccharide, and tea polysaccharide in a mass ratio of 2:1:1:1.5:0.65:1:0.4; the particle size of the nano-hydroxyapatite is 50 nm; the signal pathway regulators are a mixture of Y-27632 and AZD8055 in a molar ratio of 3000:1.

[0044] The preparation method of the astragaloside-IV-liposome includes the following steps: mixing soybean phospholipids and cholesterol to form a lipid raw material, and dissolving it together with astragaloside-IV in a mixed solvent; rotating and evaporating at 45 °C and 150 rpm for 30 minutes to form a uniform lipid film, and then vacuum drying for 2 hours to remove the residual solvent; adding a PBS buffer solution with a pH of 7.4 preheated to 45 °C to the flask containing the lipid film, hydrating in a 45 °C water bath for 30 minutes, and then ultrasonic treating for 4 minutes to form a primary emulsion suspension; subjecting the primary emulsion suspension to high-pressure homogenization treatment at 15000 psi for 7 cycles to obtain astragaloside-IV-liposome; the molar ratio of soybean phospholipids to cholesterol is 7:3; the mass ratio of astragaloside-IV, lipid raw material, and mixed solvent is 1:9:45; the mixed solvent is a mixture of chloroform and methanol in a volume ratio of 2:1; the amount ratio of the lipid film to the PBS buffer solution is 100 mg:10 mL; the basal medium is IMDM medium.

[0045] A preparation method of the NK cell culture medium includes the following steps: mixing the basal medium with each component in proportion, adjusting the pH to 7.3; sterilizing with a 0.22 μm filter membrane, and storing at -20 °C after sub-packaging.

[0046] A method for culturing NK cells using the above NK cell culture medium includes the following steps: Step S1, using a 3D suspension culture system, inoculating peripheral blood mononuclear cells at an inoculation density Inoculate at Step S2: After counting every day, replace half of the culture medium according to the situation; at the same time, adjust the cell concentration according to the cell growth situation to maintain it at cells / mL; Step S3: Collect the cells after culturing for 10 days.

[0047] During the culturing process described in Step S1, low-frequency pulsed electromagnetic field-assisted treatment is adopted. The frequency of the low-frequency pulsed electromagnetic field is 75 Hz, the intensity is 80 mT, the pulse width is 7 ms, the treatment time is 20 min each time, and 4 times a day.

[0048] Example 4

[0049] An NK cell culture medium, comprising a basal medium and additives. The components and concentrations of the additives are as follows: cytokine 350 IU / L, nutritional supplement 5.5 mmol / L, metabolic regulator 1.8 mmol / L, vitamin C 105 mg / L, plant polysaccharide 280 μg / mL, 1,3-dicaffeoylquinic acid 90 μmol / L, nano-hydroxyapatite 51 μg / mL, signal pathway regulator 15.5 μmol / L, heparin sodium 9 IU / L, astragaloside-IV-liposome 70 μg / mL.

[0050] The cytokine is a mixture of IL-2, IL-15, IL-21, and IL-33 in a mass ratio of 19:8.5:3.5:1; the nutritional supplement is a mixture of glutamine and sodium pyruvate in a molar ratio of 3.5:1.8; the metabolic regulator is ethyl pyruvate; the plant polysaccharide is a mixture of astragalus polysaccharide, ginseng polysaccharide, ganoderma polysaccharide, lentinan, poria cocos polysaccharide, wolfberry polysaccharide, and tea polysaccharide in a mass ratio of 2.5:1:1.1:1.8:0.75:1:0.45; the particle size of the nano-hydroxyapatite is 70 nm; the signal pathway regulator is a mixture of Y-27632 and AZD8055 in a molar ratio of 3000:1.

[0051] The preparation method of astragaloside-IV liposome comprises the following steps: mixing soybean phospholipid and cholesterol to form a lipid raw material, and dissolving the lipid raw material together with astragaloside-IV in a mixed solvent; performing rotary evaporation at 45 °C and 150 rpm for 30 minutes to form a uniform lipid film, and then performing vacuum drying for 2 hours to remove the residual solvent; adding PBS buffer solution with pH = 7.4 preheated to 45 °C into the flask containing the lipid film, performing water bath hydration at 45 °C for 30 minutes, and then performing ultrasonic treatment for 4.5 minutes to form a primary emulsion suspension; subjecting the primary emulsion suspension to high-pressure homogenization treatment at 15000 psi for 7 cycles to obtain astragaloside-IV liposome; the molar ratio of the soybean phospholipid to the cholesterol is 7:3; the mass ratio of the astragaloside-IV, the lipid raw material, and the mixed solvent is 1:9:48; the mixed solvent is a mixture of chloroform and methanol in a volume ratio of 2:1; the quantity ratio of the lipid film to the PBS buffer solution is 100 mg:10 mL; the basal medium is IMDM medium.

[0052] A preparation method of the NK cell medium comprises the following steps: mixing the basal medium and each component in proportion, and adjusting the pH to 7.4; performing sterilization with a 0.22-μm filter membrane, and storing in aliquots at -20 °C after subpackaging.

[0053] A method for culturing NK cells using the above NK cell medium comprises the following steps: Step S1: Using a 3D suspension culture system, inoculating peripheral blood mononuclear cells at an inoculation density of cells / mL into the NK cell medium, and culturing under the conditions of 37 °C, 5% CO2 and 95% humidity; Step S2: After counting every day, perform semi-quantitative medium replacement according to the situation; at the same time, adjust the cell concentration according to the cell growth situation and maintain it at cells / mL; Step S3: Collect the cells after culturing for 10 days.

[0054] During the culturing process in Step S1, low-frequency pulsed electromagnetic field-assisted treatment is adopted. The frequency of the low-frequency pulsed electromagnetic field is 90 Hz, the intensity is 90 mT, the pulse width is 9 ms, the treatment time is 23 min each time, and it is performed 5 times a day.

[0055] Example 5

[0056] An NK cell culture medium, comprising a basal medium and additives, and the components and concentrations of the additives are as follows: cytokine 400 IU / L, nutritional supplement 6 mmol / L, metabolic regulator 2 mmol / L, vitamin C 110 mg / L, plant polysaccharide 300 μg / mL, 1,3-dicaffeoylquinic acid 100 μmol / L, nano-hydroxyapatite 52 μg / mL, signal pathway regulator 16 μmol / L, heparin sodium 10 IU / L, astragaloside-IV-liposome 80 μg / mL.

[0057] The cytokine is a mixture of IL-2, IL-15, IL-21, and IL-33 in a mass ratio of 20:9:4:1; the nutritional supplement is a mixture of glutamine and sodium pyruvate in a molar ratio of 4:2; the metabolic regulator is ethyl pyruvate; the plant polysaccharide is a mixture of astragalus polysaccharide, ginseng polysaccharide, ganoderma lucidum polysaccharide, lentinan, poria cocos polysaccharide, wolfberry polysaccharide, and tea polysaccharide in a mass ratio of 3:1:1.2:2:0.8:1:0.5; the particle size of the nano-hydroxyapatite is 80 nm; the signal pathway regulator is a mixture of Y-27632 and AZD8055 in a molar ratio of 3000:1.

[0058] The preparation method of the astragaloside-IV-liposome comprises the following steps: mixing soybean phospholipid and cholesterol to form a lipid raw material, and dissolving it together with astragaloside-IV in a mixed solvent; rotating and evaporating at 45 °C and 150 rpm for 30 minutes to form a uniform lipid film, and then vacuum drying for 2 hours to remove the residual solvent; adding PBS buffer solution with a pH of 7.4 preheated to 45 °C to the flask containing the lipid film, hydrating in a water bath at 45 °C for 30 minutes, and then ultrasonically treating for 5 minutes to form a primary emulsion suspension; subjecting the primary emulsion suspension to high-pressure homogenization treatment at 15000 psi for 8 cycles to obtain astragaloside-IV-liposome; the molar ratio of soybean phospholipid to cholesterol is 7:3; the mass ratio of astragaloside-IV, lipid raw material, and mixed solvent is 1:9:50; the mixed solvent is a mixture of chloroform and methanol in a volume ratio of 2:1; the amount ratio of the lipid film to the PBS buffer solution is 100 mg:10 mL; the basal medium is IMDM medium.

[0059] A preparation method of the NK cell culture medium comprises the following steps: mixing the basal medium and each component in proportion, and adjusting the pH to 7.4; sterilizing with a 0.22 μm filter membrane, and storing in aliquots at -20 °C.

[0060] A method for culturing NK cells using the above NK cell culture medium comprises the following steps: Step S1, using a 3D suspension culture system, inoculating peripheral blood mononuclear cells at an inoculation density Inoculate at cells / mL into NK cell culture medium and culture under the conditions of 37 °C, 5% CO2 and 95% humidity; Step S2: After counting every day, replace half of the culture medium according to the situation; at the same time, adjust the cell concentration according to the cell growth situation and maintain it at cells / mL; Step S3: Collect the cells after culturing for 10 days.

[0061] During the culturing process described in Step S1, low-frequency pulsed electromagnetic field-assisted treatment is adopted. The frequency of the low-frequency pulsed electromagnetic field is 100 Hz, the intensity is 100 mT, the pulse width is 10 ms, the treatment time is 25 min each time, and it is 5 times a day.

[0062] Comparative Example 1 A NK cell culture medium and a method for culturing NK cells are basically the same as those in Example 1, except that nano-hydroxyapatite is not added, and an equal amount of astragalus polysaccharide is used instead of plant polysaccharide.

[0063] Comparative Example 2 A NK cell culture medium and a method for culturing NK cells are basically the same as those in Example 1, except that astragaloside-IV-liposome is not added, and low-frequency pulsed electromagnetic field-assisted treatment is not adopted during the culturing process.

[0064] In order to further illustrate the beneficial technical effects of the NK cell culture medium and the method for culturing NK cells involved in each embodiment of the present invention, relevant performance tests are carried out on the NK cell culture medium and the method for culturing NK cells involved in Examples 1-5 and Comparative Examples 1-2. The test results are shown in Table 1, and the test methods are as follows: Use the trypan blue staining method for cell counting and calculate the cell amplification multiple; use the lactate dehydrogenase release method to measure the killing effect of NK cells on K562 tumor cells, and the effector-to-target ratio is 10:1; use flow cytometry (CD56 +, CD3 - labeling) to detect the purity of NK cells.

[0065] Table 1 Performance test results of NK cell culture medium and method for culturing NK cells Project Example 1 Example 2 Example 3 Example 4 Example 5 Comparative Example 1 Comparative Example 2 Killing activity (%) 92.7 94.3 95.2 97.4 98.0 83.2 81.5 Cell expansion multiple 975 980 987 997 1000 947 933 Purity (%) 95.5 96.2 97.3 98.0 98.5 89.3 85.7 As can be seen from Table 1, compared with the comparative examples, the NK cell culture medium and the method for culturing NK cells involved in the embodiments of the present invention have more significant effects; the combined use of nano-hydroxyapatite, plant polysaccharide, astragaloside-IV-liposome and the adoption of low-frequency pulsed electromagnetic field-assisted treatment are beneficial to improving the above performance.

[0066] The above embodiments are only used to illustrate the technical concept and features of the present invention, and the purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly. It is not intended to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A NK cell culture medium, characterized in that, It includes a basal medium and additives. The components and concentrations of the additives are as follows: cytokines 200 - 400 IU / L, nutritional supplements 3 - 6 mmol / L, metabolic regulators 1 - 2 mmol / L, vitamin C 90 - 110 mg / L, plant polysaccharides 150 - 300 μg / mL, 1,3 - dicaffeoylquinic acid 50 - 100 μmol / L, nano-hydroxyapatite 48 - 52 μg / mL, signal pathway regulators 14 - 16 μmol / L, heparin sodium 5 - 10 IU / L, astragaloside-IV-liposome 10–80 μg / mL.

2. The NK cell culture medium according to claim 1, wherein The cytokines are composed of IL-2, IL-15, IL-21, and IL-33 mixed in a mass ratio of (15 - 20):(7 - 9):(2 - 4):1; the nutritional supplements are composed of glutamine and sodium pyruvate mixed in a molar ratio of (2 - 4):(1 - 2); the metabolic regulator is ethyl pyruvate.

3. The NK cell culture medium according to claim 1, wherein The plant polysaccharides are composed of astragalus polysaccharide, ginseng polysaccharide, ganoderma polysaccharide, lentinan, poria cocos polysaccharide, wolfberry polysaccharide, and tea polysaccharide mixed in a mass ratio of (1 - 3):1:(0.8 - 1.2):(1 - 2):(0.5 - 0.8):1:(0.3 - 0.5).

4. The NK cell culture medium according to claim 1, wherein The particle size of the nano-hydroxyapatite is 10 - 80 nm; the signal pathway regulators are composed of Y-27632 and AZD8055 mixed in a molar ratio of 3000:

1.

5. The NK cell culture medium according to claim 1, wherein, The preparation method of the astragaloside-IV-liposome includes the following steps: mixing soybean phospholipid and cholesterol to form a lipid raw material, and dissolving it together with astragaloside-IV in a mixed solvent; performing rotary evaporation at 45 °C and 150 rpm for 30 minutes to form a uniform lipid film, and then vacuum drying for 2 hours to remove the residual solvent; adding PBS buffer solution with a pH of 7.4 preheated to 45 °C to the flask containing the lipid film, hydrating in a water bath at 45 °C for 30 minutes, and then performing ultrasonic treatment for 3 - 5 minutes to form a primary emulsion suspension; subjecting the primary emulsion suspension to high-pressure homogenization treatment at 15000 psi for 5 - 8 cycles to obtain astragaloside-IV-liposome.

6. The NK cell culture medium according to claim 5, characterized in that, The molar ratio of the soybean phospholipid to cholesterol is 7:3; the mass ratio of astragaloside-IV, lipid raw material, and mixed solvent is 1:9:(40 - 50).

7. The NK cell culture medium according to claim 5, characterized in that, The mixed solvent is composed of chloroform and methanol mixed in a volume ratio of 2:1; the mass ratio of the lipid film to the PBS buffer solution is 100 mg:10 mL.

8. The NK cell culture medium according to claim 1, wherein, The basal medium is IMDM medium.

9. A method for preparing the NK cell culture medium according to any one of claims 1-8, characterized in that, It includes the following steps: mixing the basal medium with each component in proportion, adjusting the pH to 7.2 - 7.4; sterilizing with a 0.22 μm filter membrane, and storing at -20 °C after aliquoting.

10. A method for culturing NK cells using the NK cell culture medium according to any one of claims 1-8, characterized in that, It includes the following steps: Step S1: Using a 3D suspension culture system, peripheral blood mononuclear cells are inoculated into the NK cell medium at an inoculation density of cells / mL, and cultured under the conditions of 37 °C, 5% CO2 and 95% humidity; during the culture process described in step S1, low-frequency pulsed electromagnetic field assistance treatment is adopted, the frequency of the low-frequency pulsed electromagnetic field is 50-100 Hz, the intensity is 50-100 mT, the pulse width is 1-10 ms, the treatment time is 15-25 min each time, and 3-5 times a day; Step S2: After counting every day, replace half of the culture medium according to the situation; at the same time, adjust the cell concentration according to the cell growth situation and maintain it at cells / mL; Step S3: Collect cells after culturing for 10 - 14 days.