NK cell culture medium and NK cell culture method

Abstract

The present invention relates to a NK cell culture medium and a NK cell culture method, relating to the field of cell culture technology, comprising a basal culture medium and additives, wherein the components and concentrations of the additives are: 200-400 IU / L of cytokines, 3-6 mmol / L of nutritional supplements, 1-2 mmol / L of metabolic regulators, 90-110 mg / L of vitamin C, 150-300 μg / mL of plant polysaccharides, 50-100 μmol / L of 1,3-dicaffeoylquinic acid, 48-52 μg / mL of nano-hydroxyapatite, 14-16 μmol / L of signaling pathway regulators, 5-10 IU / L of heparin sodium, and 10-80 μg / mL of astragaloside IV liposomes. The culture medium and culture method can efficiently expand NK cells and maintain their activity and functional stability.

Description

Technical Field

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

[0002] Natural killer (NK) cells, a vital component of the immune system, play a key role in tumor immunosurveillance and antiviral resistance. Their ability to directly kill tumor cells without prior sensitization makes them highly promising immunotherapy cells. However, NK cells face challenges in vitro with low proliferation efficiency and difficulty maintaining activity. Currently, traditional NK cell culture media typically utilize animal-derived ingredients such as fetal bovine serum. However, these ingredients carry significant batch-to-batch variability, the potential for pathogen carriage, and safety concerns such as immunogenicity in clinical applications. Existing serum-free culture media also suffer from technical deficiencies, including suboptimal culture results and the need to improve NK cell expansion efficiency and activity maintenance.

[0003] To address the above-mentioned issues, a Chinese invention patent with authorization publication number CN114196630B discloses a NK cell culture medium and a method for culturing NK cells. IL-2, IL-15, and IL-21 in the NK cell culture medium of the invention synergistically act on mononuclear cells derived from umbilical cord blood to obtain a large number of NK cells with high purity, high cytotoxic activity, and high cytotoxic activity. The NK cell culture method of the invention has the advantages of high induction efficiency and rapid expansion rate. In the process of culturing NK cells using the culture medium of the invention, there is no need to introduce tumor cells or add animal serum, thus avoiding safety hazards and having the advantage of high safety. The NK cell culture medium of the invention has few added factors, low cost, and the culture method is simple and efficient to operate, which can meet the clinical demand for NK cells. However, the NK cell expansion efficiency and activity maintenance of the culture medium and culture method still need to be further improved.

[0004] It can be seen that developing a culture medium and culture method that can efficiently expand NK cells, maintain their activity and functional stability, and has safe ingredients and controllable costs has important practical significance. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a NK cell culture medium and NK cell culture method that can efficiently expand NK cells, maintain their activity and functional stability, and has safe ingredients and controllable costs.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is a NK cell culture medium, comprising a basal culture medium and additives, wherein the components and concentrations of the additives are: 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, and astragaloside-IV-liposomes 10–80 μg / mL.

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

[0008] Preferably, the nutritional supplement is 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 polysaccharide is a mixture of astragalus polysaccharide, ginseng polysaccharide, ganoderma lucidum polysaccharide, lentinan polysaccharide, poria 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).

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

[0012] Preferably, the signal pathway regulator is a mixture of Y-27632 and AZD8055 at a molar ratio of 3000:1.

[0013] Preferably, the preparation method of the astragaloside-IV-liposome comprises the following steps: mixing soybean lecithin and cholesterol to form a lipid raw material, dissolving the raw material and astragaloside-IV in a mixed solvent; rotary evaporation at 45°C and 150 rpm for 30 minutes to form a uniform lipid film, followed by 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 45°C water bath for 30 minutes, and then ultrasonically treating for 3-5 minutes to form a colostrum suspension; and subjecting the colostrum suspension to a 15,000 psi high-pressure homogenization treatment, and cycling for 5-8 times to obtain astragaloside-IV-liposome.

[0014] Preferably, the molar ratio of soybean lecithin 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 chloroform and methanol mixed in a volume ratio of 2:1.

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

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

[0019] Another object of the present invention is to provide a method for preparing the NK cell culture medium, comprising the following steps: mixing the basal culture 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 packaging.

[0020] Another object of the present invention is to provide a method for culturing NK cells using the above-mentioned NK cell culture medium, comprising the following steps:

[0021] Step S1: Using a 3D suspension culture system, peripheral blood mononuclear cells were seeded at a density of cells / mL were inoculated into NK cell culture medium and cultured at 37°C, 5% CO2, and 95% humidity;

[0022] 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 cells / mL;

[0023] Step S3: Collect cells after culturing for 10-14 days.

[0024] Preferably, low-frequency pulsed electromagnetic field auxiliary treatment is used in the culture process in step S1, 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 minutes each time, and 3-5 times a day.

[0025] Due to the application of the above technical solution, the present invention has the following beneficial effects:

[0026] (1) The NK cell culture medium disclosed in 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, easy large-scale industrial production, and high promotion and application value.

[0027] (2) The NK cell culture medium disclosed in the present invention comprises a basal culture medium and additives, wherein the components and concentrations of the additives are as follows: 200-400 IU / L of cytokines, 3-6 mmol / L of nutritional supplements, 1-2 mmol / L of metabolic regulators, 90-110 mg / L of vitamin C, 150-300 μg / mL of plant polysaccharides, 50-100 μmol / L of 1,3-dicaffeoylquinic acid, 48-52 μg / mL of nano-hydroxyapatite, 14-16 μmol / L of signaling pathway regulators, 5-10 IU / L of sodium heparin, and 10–80 μg / mL of astragaloside IV-liposomes. Through the mutual cooperation and joint action of the components, the prepared culture medium can efficiently amplify NK cells, maintain their activity and functional stability, and has safe ingredients and controllable costs. The synergistic combination of multiple components such as cytokines, signal pathway regulators, and nanohydroxyapatite forms a three-dimensional technology system of "signal regulation-metabolic optimization-microenvironment improvement", breaking through the limitations of single-component optimization of existing culture media, and significantly improving the NK cell expansion effect and killing rate. The plant polysaccharide is a mixture of astragalus polysaccharide, ginseng polysaccharide, ganoderma lucidum polysaccharide, shiitake polysaccharide, poria 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 formula selection, the addition of plant polysaccharides can effectively promote NK cell proliferation and activation, enhance the cytotoxicity of NK cells, regulate the immune microenvironment, and inhibit NK cell exhaustion.

[0028] (3) The NK cell culture medium disclosed in the present invention, with the addition of astragaloside IV-liposomes, can produce synergistic effects in terms of cell proliferation, functional activation, anti-apoptosis and microenvironmental regulation through its unique physicochemical properties and biological activity, promoting NK cell proliferation and activation, resisting NK cell apoptosis, and prolonging in vitro culture survival time. Through multi-target regulation (proliferation signals, apoptosis pathways, microenvironmental factors) and the advantages of liposome delivery (sustained release, low toxicity), a triple improvement in "amplification efficiency-functional activity-in vivo persistence" is achieved. The addition of astragaloside IV in the form of liposomes can effectively solve the problem of poor water solubility of astragaloside IV, thereby improving bioavailability.

[0029] (4) The NK cell culture method disclosed in the present invention adopts low-frequency pulsed electromagnetic field auxiliary treatment during the culture process, which can regulate cell cycle and proliferation signals, enhance cell metabolism and energy supply, promote the release of cytotoxic molecules, regulate cytokine secretion and immune regulation function, optimize culture matrix and cell adhesion, inhibit cell apoptosis and maintain phenotypic stability; the combined effect with cytokines can reduce the amount of cytokines used while improving culture efficiency; at the same time, it can also promote the delivery efficiency of nanohydroxyapatite to NK cells, enhance its regulatory effect on signal pathways, and further optimize cell function. DETAILED DESCRIPTION

[0030] The following description is intended to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are merely examples, and those skilled in the art may conceive of other obvious variations.

[0031] Example 1

[0032] A NK cell culture medium comprises a basal culture medium and additives, wherein the components and concentrations of the additives are as follows: 200 IU / L of cytokines, 3 mmol / L of nutritional supplements, 1 mmol / L of metabolic regulators, 90 mg / L of vitamin C, 150 μg / mL of plant polysaccharides, 50 μmol / L of 1,3-dicaffeoylquinic acid, 48 μg / mL of nano-hydroxyapatite, 14 μmol / L of signaling pathway regulators, 5 IU / L of heparin sodium, and 10 μg / mL of astragaloside-IV-liposomes.

[0033] The cytokines are IL-2, IL-15, IL-21, and IL-33 mixed in a mass ratio of 15:7:2:1; the nutritional supplement is glutamine and sodium pyruvate mixed in a molar ratio of 2:1; the metabolic regulator is ethyl pyruvate; the plant polysaccharides are astragalus polysaccharide, ginseng polysaccharide, ganoderma lucidum polysaccharide, lentinan polysaccharide, poria polysaccharide, wolfberry polysaccharide, and tea polysaccharide mixed 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; and the signal pathway regulator is Y-27632 and AZD8055 mixed in a molar ratio of 3000:1.

[0034] The preparation method of astragaloside-IV-liposomes comprises the following steps: mixing soybean lecithin and cholesterol to form a lipid raw material, and dissolving the lipid raw material and astragaloside-IV in a mixed solvent; rotary evaporation at 45°C and 150 rpm for 30 minutes to form a uniform lipid film, followed by vacuum drying for 2 hours to remove residual solvent; adding PBS buffer preheated to 45°C and pH=7.4 to a flask containing the lipid film, hydrating in a 45°C water bath for 30 minutes, and then ultrasonically treating for 3 minutes to form a colostrum suspension; subjecting the colostrum suspension to 15,000 psi high-pressure homogenization for 5 cycles to obtain astragaloside-IV-liposomes; the molar ratio of soybean lecithin 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 molar ratio of the lipid film to the PBS buffer is 100 mg:10 mL; and the basal culture medium is IMDM culture medium.

[0035] A method for preparing the NK cell culture medium comprises the following steps: mixing the basal culture medium with each component in proportion, adjusting the pH to 7.2; sterilizing with a 0.22 μm filter membrane, and storing at -20°C after packaging.

[0036] A method for culturing NK cells using the above-mentioned NK cell culture medium comprises the following steps:

[0037] Step S1: Using a 3D suspension culture system, peripheral blood mononuclear cells were seeded at a density of cells / mL were inoculated into NK cell culture medium and cultured at 37°C, 5% CO2, and 95% humidity;

[0038] 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 cells / mL;

[0039] Step S3: Collect cells after culturing for 10 days.

[0040] During the culture process in step S1, low-frequency pulsed electromagnetic field auxiliary treatment is adopted, wherein 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 minutes each time, and it is done 3 times a day.

[0041] Example 2

[0042] A NK cell culture medium comprises a basal culture medium and additives, wherein the components and concentrations of the additives are as follows: 250 IU / L of cytokines, 4 mmol / L of nutritional supplements, 1.2 mmol / L of metabolic regulators, 95 mg / L of vitamin C, 180 μg / mL of plant polysaccharides, 60 μmol / L of 1,3-dicaffeoylquinic acid, 49 μg / mL of nano-hydroxyapatite, 14.5 μmol / L of signaling pathway regulators, 6 IU / L of heparin sodium, and 30 μg / mL of astragaloside-IV-liposomes.

[0043] The cytokines are IL-2, IL-15, IL-21, and IL-33 mixed in a mass ratio of 17:7.5:2.5:1; the nutritional supplement is glutamine and sodium pyruvate mixed in a molar ratio of 2.5:1.2; the metabolic regulator is ethyl pyruvate; the plant polysaccharides are astragalus polysaccharide, ginseng polysaccharide, ganoderma lucidum polysaccharide, lentinan polysaccharide, poria polysaccharide, wolfberry polysaccharide, and tea polysaccharide mixed 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; and the signal pathway regulator is Y-27632 and AZD8055 mixed in a molar ratio of 3000:1.

[0044] The preparation method of astragaloside-IV-liposomes comprises the following steps: mixing soybean lecithin and cholesterol to form a lipid raw material, and dissolving the lipid raw material and astragaloside-IV in a mixed solvent; rotary evaporation at 45°C and 150 rpm for 30 minutes to form a uniform lipid film, followed by vacuum drying for 2 hours to remove residual solvent; adding PBS buffer preheated to 45°C and pH=7.4 to a flask containing the lipid film, hydrating in a 45°C water bath for 30 minutes, and then ultrasonically treating for 3.5 minutes to form a colostrum suspension; subjecting the colostrum suspension to 15,000 psi high-pressure homogenization for 6 cycles to obtain astragaloside-IV-liposomes; the molar ratio of soybean lecithin to cholesterol is 7:3; the mass ratio of astragaloside-IV, lipid raw material, and mixed solvent is 1:9:43; 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 PBS buffer is 100 mg:10 mL; and the basal culture medium is IMDM culture medium.

[0045] A method for preparing the NK cell culture medium comprises the following steps: mixing the basal culture 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 packaging.

[0046] A method for culturing NK cells using the above-mentioned NK cell culture medium comprises the following steps:

[0047] Step S1: Using a 3D suspension culture system, peripheral blood mononuclear cells were seeded at a density of Inoculate in NK cell culture medium and culture at 37°C, 5% CO2, and 95% humidity;

[0048] 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 cells / mL;

[0049] Step S3: Collect cells after culturing for 10 days.

[0050] During the culture process in step S1, low-frequency pulsed electromagnetic field auxiliary 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 minutes each time, and it is 4 times a day.

[0051] Example 3

[0052] A NK cell culture medium comprises a basal culture medium and additives, wherein the components and concentrations of the additives are as follows: 300 IU / L of cytokines, 4.5 mmol / L of nutritional supplements, 1.5 mmol / L of metabolic regulators, 100 mg / L of vitamin C, 230 μg / mL of plant polysaccharides, 75 μmol / L of 1,3-dicaffeoylquinic acid, 50 μg / mL of nano-hydroxyapatite, 15 μmol / L of signaling pathway regulators, 7 IU / L of heparin sodium, and 40 μg / mL of astragaloside-IV-liposomes.

[0053] The cytokines are IL-2, IL-15, IL-21, and IL-33 mixed in a mass ratio of 17:8:3:1; the nutritional supplement is glutamine and sodium pyruvate mixed in a molar ratio of 3:1.5; the metabolic regulator is ethyl pyruvate; the plant polysaccharides are astragalus polysaccharide, ginseng polysaccharide, ganoderma lucidum polysaccharide, lentinan polysaccharide, poria polysaccharide, wolfberry polysaccharide, and tea polysaccharide mixed 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; and the signal pathway regulator is Y-27632 and AZD8055 mixed in a molar ratio of 3000:1.

[0054] The preparation method of astragaloside-IV-liposomes comprises the following steps: mixing soybean lecithin and cholesterol to form a lipid raw material, and dissolving the lipid raw material and astragaloside-IV in a mixed solvent; rotary evaporation at 45°C and 150 rpm for 30 minutes to form a uniform lipid film, followed by vacuum drying for 2 hours to remove residual solvent; adding PBS buffer solution with a pH of 7.4 preheated to 45°C to a flask containing the lipid film, hydrating in a 45°C water bath for 30 minutes, and then ultrasonically treating for 4 minutes to form a colostrum suspension; subjecting the colostrum suspension to 15,000 psi high-pressure homogenization for 7 cycles to obtain astragaloside-IV-liposomes; the molar ratio of soybean lecithin 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 PBS buffer solution is 100 mg:10 mL; and the basal culture medium is IMDM culture medium.

[0055] A method for preparing the NK cell culture medium comprises the following steps: mixing the basal culture 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 packaging.

[0056] A method for culturing NK cells using the above-mentioned NK cell culture medium comprises the following steps:

[0057] Step S1: Using a 3D suspension culture system, peripheral blood mononuclear cells were seeded at a density of cells / mL were inoculated into NK cell culture medium and cultured at 37°C, 5% CO2, and 95% humidity;

[0058] 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 cells / mL;

[0059] Step S3: Collect cells after culturing for 10 days.

[0060] During the culture process in step S1, low-frequency pulsed electromagnetic field auxiliary treatment is adopted, wherein 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 minutes each time, and it is 4 times a day.

[0061] Example 4

[0062] A NK cell culture medium comprises a basal culture medium and additives, wherein the components and concentrations of the additives are as follows: 350 IU / L of cytokines, 5.5 mmol / L of nutritional supplements, 1.8 mmol / L of metabolic regulators, 105 mg / L of vitamin C, 280 μg / mL of plant polysaccharides, 90 μmol / L of 1,3-dicaffeoylquinic acid, 51 μg / mL of nano-hydroxyapatite, 15.5 μmol / L of signaling pathway regulators, 9 IU / L of heparin sodium, and 70 μg / mL of astragaloside-IV-liposomes.

[0063] The cytokines are IL-2, IL-15, IL-21, and IL-33 mixed in a mass ratio of 19:8.5:3.5:1; the nutritional supplement is glutamine and sodium pyruvate mixed in a molar ratio of 3.5:1.8; the metabolic regulator is ethyl pyruvate; the plant polysaccharides are astragalus polysaccharide, ginseng polysaccharide, ganoderma lucidum polysaccharide, lentinan polysaccharide, poria polysaccharide, wolfberry polysaccharide, and tea polysaccharide mixed 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; and the signal pathway regulator is Y-27632 and AZD8055 mixed in a molar ratio of 3000:1.

[0064] The preparation method of astragaloside-IV-liposomes comprises the following steps: mixing soybean lecithin and cholesterol to form a lipid raw material, and dissolving the lipid raw material and astragaloside-IV in a mixed solvent; rotary evaporation at 45°C and 150 rpm for 30 minutes to form a uniform lipid film, followed by vacuum drying for 2 hours to remove 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 45°C water bath for 30 minutes, and then ultrasonically treating for 4.5 minutes to form a colostrum suspension; subjecting the colostrum suspension to 15,000 psi high-pressure homogenization for 7 cycles to obtain astragaloside-IV-liposomes; the molar ratio of soybean lecithin to cholesterol is 7:3; the mass ratio of astragaloside-IV, lipid raw material, and mixed solvent is 1:9:48; the mixed solvent is a mixture of chloroform and methanol in a volume ratio of 2:1; the molar ratio of the lipid film to the PBS buffer solution is 100 mg:10 mL; and the basal culture medium is IMDM culture medium.

[0065] A method for preparing the NK cell culture medium comprises the following steps: mixing the basal culture medium with each component in proportion, adjusting the pH to 7.4; sterilizing with a 0.22 μm filter membrane, and storing at -20°C after packaging.

[0066] A method for culturing NK cells using the above-mentioned NK cell culture medium comprises the following steps:

[0067] Step S1: Using a 3D suspension culture system, peripheral blood mononuclear cells were seeded at a density of cells / mL were inoculated into NK cell culture medium and cultured at 37°C, 5% CO2, and 95% humidity;

[0068] 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 cells / mL;

[0069] Step S3: Collect cells after culturing for 10 days.

[0070] During the culture process in step S1, low-frequency pulsed electromagnetic field auxiliary 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 minutes each time, and it is 5 times a day.

[0071] Example 5

[0072] A NK cell culture medium comprises a basal culture medium and additives, wherein the components and concentrations of the additives are as follows: 400 IU / L of cytokines, 6 mmol / L of nutritional supplements, 2 mmol / L of metabolic regulators, 110 mg / L of vitamin C, 300 μg / mL of plant polysaccharides, 100 μmol / L of 1,3-dicaffeoylquinic acid, 52 μg / mL of nano-hydroxyapatite, 16 μmol / L of signaling pathway regulators, 10 IU / L of heparin sodium, and 80 μg / mL of astragaloside-IV-liposomes.

[0073] The cytokines are IL-2, IL-15, IL-21, and IL-33 mixed in a mass ratio of 20:9:4:1; the nutritional supplement is glutamine and sodium pyruvate mixed in a molar ratio of 4:2; the metabolic regulator is ethyl pyruvate; the plant polysaccharides are astragalus polysaccharide, ginseng polysaccharide, ganoderma lucidum polysaccharide, lentinan polysaccharide, poria polysaccharide, wolfberry polysaccharide, and tea polysaccharide mixed 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; and the signal pathway regulator is Y-27632 and AZD8055 mixed in a molar ratio of 3000:1.

[0074] The preparation method of astragaloside-IV-liposomes comprises the following steps: mixing soybean lecithin and cholesterol to form a lipid raw material, and dissolving the lipid raw material and astragaloside-IV in a mixed solvent; rotary evaporation at 45°C and 150 rpm for 30 minutes to form a uniform lipid film, followed by vacuum drying for 2 hours to remove residual solvent; adding PBS buffer solution with a pH of 7.4 preheated to 45°C to a flask containing the lipid film, hydrating in a 45°C water bath for 30 minutes, and then ultrasonically treating for 5 minutes to form a colostrum suspension; subjecting the colostrum suspension to 15,000 psi high-pressure homogenization for 8 cycles to obtain astragaloside-IV-liposomes; the molar ratio of soybean lecithin 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 PBS buffer solution is 100 mg:10 mL; and the basal culture medium is IMDM culture medium.

[0075] A method for preparing the NK cell culture medium comprises the following steps: mixing the basal culture medium with each component in proportion, adjusting the pH to 7.4; sterilizing with a 0.22 μm filter membrane, and storing at -20°C after packaging.

[0076] A method for culturing NK cells using the above-mentioned NK cell culture medium comprises the following steps:

[0077] Step S1: Using a 3D suspension culture system, peripheral blood mononuclear cells were seeded at a density of cells / mL were inoculated into NK cell culture medium and cultured at 37°C, 5% CO2, and 95% humidity;

[0078] 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 cells / mL;

[0079] Step S3: Collect cells after culturing for 10 days.

[0080] During the culture process in step S1, low-frequency pulsed electromagnetic field auxiliary treatment is adopted, wherein 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 minutes each time, and 5 times a day.

[0081] Comparative Example 1

[0082] A NK cell culture medium and a NK cell culture method 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.

[0083] Comparative Example 2

[0084] A NK cell culture medium and a NK cell culture method are basically the same as those in Example 1, except that astragaloside IV-liposome is not added and low-frequency pulsed electromagnetic field auxiliary treatment is not used during the culture process.

[0085] In order to further illustrate the beneficial technical effects of the NK cell culture medium and the NK cell culture method involved in each embodiment of the present invention, relevant performance tests were performed on the NK cell culture medium and the NK cell culture method involved in Examples 1-5 and Comparative Examples 1-2. The test results are shown in Table 1. The testing method is as follows: cells are counted by trypan blue staining to calculate the cell proliferation multiple; the lactate dehydrogenase release method is used to determine the killing effect of NK cells on K562 tumor cells, with an effector-target ratio of 10:1; and the purity of NK cells is detected by flow cytometry (CD56+, CD3- markers).

[0086] Table 1 Performance test results of NK cell culture medium and NK cell culture method

[0087] 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 times 975 980 987 997 1000 947 933 purity(%) 95.5 96.2 97.3 98.0 98.5 89.3 85.7

[0088] As can be seen from Table 1, the NK cell culture medium and NK cell culture method involved in the embodiments of the present invention are more effective than those in the comparative examples; the combined use of nano-hydroxyapatite, plant polysaccharides, and astragaloside-IV-liposomes and the use of low-frequency pulsed electromagnetic field-assisted treatment are beneficial to improving the above-mentioned properties.

[0089] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A NK cell culture medium, characterized in that The invention comprises a basal culture medium and additives, wherein 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, signaling pathway regulators 14-16 μmol / L, heparin sodium 5-10 IU / L, and astragaloside-IV-liposomes 10–80 μg / mL; The cytokines are IL-2, IL-15, IL-21, and IL-33 mixed in a mass ratio of (15-20):(7-9):(2-4):1; the nutritional supplement is glutamine and sodium pyruvate mixed in a molar ratio of (2-4):(1-2); the metabolic regulator is ethyl pyruvate; the plant polysaccharides are astragalus polysaccharide, ginseng polysaccharide, ganoderma lucidum polysaccharide, lentinan, poria 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); the signal pathway regulator is Y-27632 and AZD8055 mixed in a molar ratio of 3000:1; the basal culture medium is IMDM culture medium; The preparation method of astragaloside-IV-liposomes comprises the following steps: mixing soybean lecithin and cholesterol to form a lipid raw material, dissolving the mixture with astragaloside-IV in a mixed solvent; rotary evaporating at 45°C and 150 rpm for 30 minutes to form a uniform lipid film, followed by vacuum drying for 2 hours to remove residual solvent; adding PBS buffer solution with a pH of 7.4 preheated to 45°C to the flask containing the lipid film, hydrating the mixture in a 45°C water bath for 30 minutes, and then ultrasonically treating the mixture for 3-5 minutes to form a colostrum suspension; subjecting the colostrum suspension to a 15,000 psi high-pressure homogenization treatment for 5-8 cycles to obtain astragaloside-IV-liposomes; the molar ratio of soybean lecithin to cholesterol is 7:3; and the mass ratio of astragaloside-IV, lipid raw material, and mixed solvent is 1:9:(40-50). The mixed solvent is chloroform and methanol mixed in a volume ratio of 2:1; the amount ratio of the lipid film and PBS buffer is 100 mg:10 mL.

2. The NK cell culture medium according to claim 1, characterized in that The particle size of the nano-hydroxyapatite is 10-80 nm.

3. A method for preparing the NK cell culture medium according to claim 1 or 2, characterized in that: The method comprises the following steps: mixing the basic culture 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 packaging.

4. A method for culturing NK cells using the NK cell culture medium according to claim 1 or 2, characterized in that: The steps include: Step S1: Using a 3D suspension culture system, peripheral blood mononuclear cells were seeded at a density of cells / mL were inoculated into NK cell culture medium and cultured at 37°C, 5% CO2, and 95% humidity. During the culture process, low-frequency pulsed electromagnetic field (LPEM) was used as an auxiliary treatment. The LPEM had a frequency of 50-100 Hz, an intensity of 50-100 mT, a pulse width of 1-10 ms, and a treatment time of 15-25 minutes each time, 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 to maintain cells / mL; Step S3: Collect cells after culturing for 10-14 days.