CN114891740B - Culture method and application of NK cells with high proliferation capacity and high cytotoxicity - Google Patents

Culture method and application of NK cells with high proliferation capacity and high cytotoxicity Download PDF

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CN114891740B
CN114891740B CN202210302864.3A CN202210302864A CN114891740B CN 114891740 B CN114891740 B CN 114891740B CN 202210302864 A CN202210302864 A CN 202210302864A CN 114891740 B CN114891740 B CN 114891740B
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杨桂花
杨海霞
赵进军
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Hezhe Technology Co ltd
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Abstract

The application relates to the technical field of cell culture, and in particular discloses a culture method of NK cells with high proliferation capacity and high cytotoxicity and application thereof, comprising the following steps: s1, coating an anti-human CD16 monoclonal antibody with the concentration of 0.1-2.0 mg/mL in a cell culture flask, wherein the coating condition is 25 ℃, and the incubation time is 1-5 h; s2, taking a cell culture bottle subjected to coating treatment, adding an NK cell culture medium, and then adding mononuclear cells obtained by centrifugal separation from peripheral blood into the cell culture bottle, and adjusting the cell density to be 1X 106/mL-2X 106/mL; s3, continuously culturing the cell culture flask in a incubator with CO2 with the concentration of 2% at 37 ℃ for 9-15 days, and supplementing IL-2 with the concentration of 2-100 mug/mL every 3 days. The NK cells with high purity, high amplification efficiency and strong cytotoxicity can be obtained by adopting the preparation method of the application.

Description

Culture method and application of NK cells with high proliferation capacity and high cytotoxicity
Technical Field
The application relates to the technical field of cell culture, in particular to a culture method of NK cells with high proliferation capacity and high cytotoxicity and application thereof.
Background
NK cells, also called natural killer cells (natural killer cell), are a third class of lymphocytes other than T, B cells, a cell subset with unique functions, are one of lymphocytes in human immune cells, are a key subset of innate immune cells, are considered to be one of the main effector cells of the body for controlling the occurrence, development and metastasis of tumors, and are also the first natural defense line of the body for resisting tumors and infections.
The main mechanisms of NK cell anti-tumor immunity include: 1) Releasing cytoplasmic granules comprising perforin and granzyme to cause apoptosis of target cells, thereby clearing mutant tumor cells; 2) Death receptors mediate target apoptosis, NK cells express at least 3 TNF superfamily death ligands: fas ligand (Fas ligand, fasL), TNF and TNF-related apoptosis-inducing ligand (TNF related apoptosis inducing ligand, TRAIL) kill tumor cells by binding to their corresponding death receptors on their surfaces; 3) Cytokine mediated killing such as INF-gamma, TNF, granulocyte-macrophage colony stimulating factor, IL-5, IL-10, IL-13, etc.; 4) Antibody-dependent cell-mediated cytotoxicity (ADCC) is mediated primarily through fcγriii (CD 16) with low affinity at its membrane surface.
NK cells play an important role in both innate and adaptive immunity and are considered as important effector cells for tumor immunotherapy. NK cells are rare subpopulations of lymphocytes, accounting for only about 10% -20% of peripheral blood lymphocytes. NK cell content in peripheral blood of normal human body can not meet the clinical treatment requirement. Therefore, obtaining NK cells with high purity, high amplification efficiency and strong cytotoxicity has become a research hotspot.
Disclosure of Invention
In order to obtain NK cells with high purity, high amplification efficiency and strong cytotoxicity, the application provides a culture method of NK cells with high proliferation capacity and high cytotoxicity and application thereof.
In a first aspect, the present application provides a method for culturing NK cells with high proliferation capacity and high cytotoxicity, which adopts the following technical scheme:
a method for culturing NK cells with high proliferation capacity and high cytotoxicity, comprising the steps of:
s1, coating an anti-human CD16 monoclonal antibody with the concentration of 0.1-2.0 mg/mL in a cell culture flask, wherein the coating condition is 25 ℃, and the incubation time is 1-5 h;
s2, taking a cell culture bottle subjected to coating treatment, adding an NK cell culture medium, and then adding mononuclear cells obtained by centrifugal separation from peripheral blood into the cell culture bottle to adjust the cell density to be 1 multiplied by 10 6 /mL～2×10 6 /mL; the NK cell culture medium comprises a basic culture solution and an activating culture solution, wherein the adding amount of the activating culture solution is 3-5% of that of the basic culture solution; the activation culture solution comprises the following components in concentration: 1.2 to 1.5g/L of human serum albumin, 1.5 to 2.0g/L of human serum albumin, 120 to 160mg/L of vitamin C, 220 to 240mg/L of vitamin E, 120 to 150mg/L of coenzyme A, 0.5 to 5mg/L of recombinant human insulin, 280 to 330mg/L of L-glutamine, 420 to 530ug/mL of dimercaptoethanol, 20 to 30ug/mL of indomethacin, and 0.1 to 10 mu g/mL of Na + 50-100 ng/mL of monoclonal antibody CD2, 50-100 ng/mL of NKp46 antibody, 0.01-0.06 KE/mL of OK432 and 2-100 mug/mL of cytokine IL-2;
s3, placing the cell culture flask at 37 ℃ and CO with the concentration of 2% 2 The incubator is continuously cultured for 9 to 15 days, and IL-2 with the concentration of 2 to 100 mug/mL is added every 3 days.
By adopting the method to culture NK cells, NK cells with high purity, high amplification efficiency and strong cytotoxicity can be obtained. The reason is that the anti-human CD16 monoclonal antibody is selected to be coated in a cell culture flask, and the anti-human CD16 monoclonal antibody is combined with CD16 molecules in NK cells, so that a CD16 co-stimulation signal path is activated, and the activation and proliferation of the ANK cells can be promoted. The human serum albumin and the human serum albumin in the NK cell culture medium can provide basic culture components for the culture of NK cells; vitamin C and dimercaptoethanol can prevent oxidation of cells, increase toughness of cell wall, and Na + In the process of cell growth, the ion maintains the balance of the osmotic pressure inside and outside the cell, and can prevent the cell wall from cracking so as to promote the cell growth; vitamin E is a bioactive substance for cell growth, and can regulate and control during cell growth and metabolismThe preparation is carried out; the coenzyme A metabolism pathway can induce oxidative phosphorylation and enhance the anti-tumor effect of NK cells; recombinant human insulin can promote NK cell self-regulation and enhance cytotoxicity; l-glutamine is an encoded amino acid in protein synthesis, and can provide nutrients for the culture of NK cells; indomethacin can play an anti-inflammatory and anti-toxic role, has strong capability of inducing cytotoxicity (ADCC), and can inhibit the expression of Survivin factors in tumor cells, thereby effectively assisting in improving the killing activity of NK cells; monoclonal antibody CD2 and multifunctional antibody targeting NKp46 can activate anti-tumor immunity of NK cells; OK-432 can enhance the cytotoxicity of NK cells, promote secretion of IFN-r, IL-2, TNF-alpha and the like, and regulate the anti-tumor immune response of organisms; in the application, monoclonal antibodies CD2 and NKp46 and OK-432 are taken as a biological effector and added into a culture medium at the initial stage of cell culture, so that NK cells can be activated; in addition, the IL-2 can promote the growth of NK precursor cells and NK mature cells, increase the expression level of cytolytic enzymes secreted by NK cells and enhance NK cytotoxicity; the monoclonal antibody CD2 and NKp46 antibody and OK-432 and IL-2 are simultaneously present in a culture medium at the initial stage of cell culture, so that the expression rate of NK cells can be effectively improved, the amplification factor of the NK cells is greatly improved, and the in-vitro tumor killing activity is enhanced.
Preferably, the activation culture solution comprises the following components in each concentration: 1.3g/L human serum albumin, 1.8g/L human serum albumin, 130-140 mg/L vitamin C, 230-235 mg/L vitamin E, 130-140 mg/L coenzyme A, 3mg/L recombinant human insulin, 320 mg/L-glutamine, 450-480 ug/mL dimercaptoethanol, 26ug/mL indomethacin, 5 ug/mL Na + 60-70 ng/mL of monoclonal antibody CD2, 60-70 ng/mL of NKp46 antibody, 0.06KE/mL of OK432 and 2-50 mug/mL of cytokine IL-2.
Preferably, the activation culture solution comprises the following components in each concentration: 1.3g/L human serum albumin, 1.8g/L human serum albumin, 135mg/L vitamin C, 230mg/L vitamin E, 135mg/L coenzyme A, 3mg/L recombinant human insulin, 320 mg/L-glutamine, 460ug/mL dimercaptoethanol, 26ug/mL indomethacin, 5 ug/mL Na + Monoclonal antibody of 65ng/mLCD2, 65ng/mL NKp46 antibody, 0.06KE/mL OK432, 50 μg/mL cytokine IL-2.
By adopting the technical scheme, the combined factors with the concentrations are adopted in the activating culture solution, so that the expression rate of NK cells can be effectively improved, the amplification factor and cytotoxicity of the NK cells are greatly improved, and the in-vitro tumor killing activity is effectively enhanced.
Preferably, in the step S2, before the mononuclear cells are added into a cell culture flask, the mononuclear cells are pretreated for 10 to 15 hours with a culture solution consisting of the cytokine IL-12 with the concentration of 15 to 20ng/mL, the cytokine IL-15 with the concentration of 1 to 10ng/mL and the cytokine IL-18 with the concentration of 30 to 50ng/mL, then washed for 2 to 3 times by using PBS buffer solution, and then placed into the cell culture flask for culture.
By adopting the technical scheme, IL-12 achieves the effects of inhibiting tumor angiogenesis and mediating tumor apoptosis through tumor necrosis factor-related apoptosis-inducing ligand (tumor necrosis factor related apoptosis inducing ligand, TRAIL) and apoptosis factor ligand (fas ligand, fasL) by promoting NK cells to release interferon gamma (IFN-gamma) and tumor necrosis factor alpha (tumor necrosis factor alpha, TNF-alpha); IL-18 can enhance the effect of IL-12 by up-regulating the expression of NK cell surface IL-12receptor (IL-12R); IL-15 can sustain NK cell differentiation and expansion, and resist IL-2 mediated NK cell apoptosis.
Preferably, in the step S2, before the mononuclear cells are added into a cell culture flask, the mononuclear cells are pretreated for 13 hours with a culture solution composed of cytokine IL-12 with a concentration of 15ng/mL, cytokine IL-15 with a concentration of 5ng/mL and cytokine IL-18 with a concentration of 50ng/mL, then washed for 2 times by using PBS buffer, and then placed into the cell culture flask for culturing.
By adopting the technical scheme, the pretreatment is carried out before the NK cell culture by adopting a mode of combining the cytokines IL-12, IL-15 and IL-18, so that the NK cell can be efficiently amplified and the killing activity of the NK cell can be obviously activated.
Preferably, the basal medium is selected from Corning KBM 581 lymphocyte serum-free medium.
By adopting the technical scheme, the basic culture solution is rich in nutrition and meets the requirement of NK cell culture.
In a second aspect, the present application provides NK cells prepared by the culture method of NK cells having high proliferation potency and high cytotoxicity.
In a third aspect, the present application provides the use of high proliferative capacity and high cytotoxic NK cells in medicine for the treatment of tumors, infections or diseases of the immune system.
In summary, the application has the following beneficial effects:
1. by adopting the method to culture NK cells, NK cells with high purity, high amplification efficiency and strong cytotoxicity can be obtained.
2. The pretreatment is carried out before the NK cell culture by adopting a mode of combining the cytokines IL-12, IL-15 and IL-18, so that the NK cells can be efficiently amplified and the killing activity of the NK cells can be obviously activated.
Drawings
FIG. 1 is a bar graph showing NK cell expansion for different days in the method provided in examples and comparative examples of the present application.
Detailed Description
The application is described in further detail below with reference to the drawings and examples.
The instruments, materials, reagents, etc. used in the present application are commercially available unless otherwise specified.
Examples
Example 1
A method for culturing NK cells with high proliferation capacity and high cytotoxicity, comprising the steps of:
s1, coating an anti-human CD16 monoclonal antibody with the concentration of 0.1mg/mL in a cell culture flask, wherein the coating condition is 25 ℃, and the incubation time is 1h;
collecting human peripheral blood, transferring the collected peripheral blood into a 50mL centrifuge tube, centrifuging at 20 ℃ for 2000rmp for 10min, and collecting plasma for later use; separating and collecting peripheral blood mononuclear cells by using lymphocyte separating medium (corning), and cleaning the collected mononuclear cells with PBS for three times and counting for later use;
wherein, the method for separating and collecting peripheral blood mononuclear cells by using lymphocyte separation liquid comprises the following steps: 1. the blood sample after plasma aspiration was prepared according to 1:1, adding PBS according to the proportion, and uniformly mixing; 2. slowly adding the diluted blood sample on the lymphocyte separation liquid level, wherein the ratio of the diluted blood sample to the lymphocyte separation liquid is 1:1, a step of; 3. centrifuging at 2000rpm for 15min, and slowly increasing and decreasing at 20deg.C. 4. After centrifugation, mononuclear cell layer cells were aspirated.
S2, taking a cell culture bottle subjected to coating treatment, adding an NK cell culture medium, and then adding mononuclear cells obtained by centrifugal separation from peripheral blood into the cell culture bottle to adjust the cell density to be 1 multiplied by 10 6 /mL; the NK cell culture medium comprises a basic culture medium and an activating culture medium, wherein the adding amount of the activating culture medium is 4% of that of the basic culture medium, and the basic culture medium is selected from Corning KBM 581 lymphocyte serum-free culture medium; the activation culture solution comprises the following components in concentration: 1.2g/L human serum albumin, 1.5g/L human serum albumin, 120mg/L vitamin C, 220mg/L vitamin E, 120mg/L coenzyme A, 0.5mg/L recombinant human insulin, 280 mg/L-glutamine, 420ug/mL dimercaptoethanol, 20ug/mL indomethacin, 0.1 ug/mL Na + 50ng/mL of monoclonal antibody CD2, 50ng/mL of NKp46 antibody, 0.01KE/mL of OK432, 2 μg/mL of cytokine IL-2.
S3, placing the cell culture flask at 37 ℃ and CO with the concentration of 2% 2 Is continuously cultured for 14 days, and IL-2 with the concentration of 2 mug/mL is supplemented every 3 days.
S4, collecting cells, washing for 3 times by using PBS, and performing cell quality inspection, so that a large number of amplified NK cells with high purity, high proliferation capacity and high cytotoxicity are obtained after the cells are qualified, and are used for tumor cell treatment or cryopreservation.
Example 2
Example 2 differs from example 1 in that the activation medium comprises the following components in the respective concentrations: 1.3g/L human serum albumin, 1.8g/L human serum albumin, 130mg/L vitamin C,230mg/L vitamin E, 130mg/L coenzyme A, 3mg/L recombinant human insulin, 320 mg/L-glutamine, 450ug/mL dimercaptoethanol, 26ug/mL indomethacin, 5 ug/mL Na + 60ng/mL of monoclonal antibody CD2, 60ng/mL of NKp46 antibody, 0.06KE/mL of OK432, 50 μg/mL of cytokine IL-2.
Example 3
Example 3 differs from example 1 in that the activation medium comprises the following components in the respective concentrations: 1.3g/L human serum albumin, 1.8g/L human serum albumin, 135mg/L vitamin C, 230mg/L vitamin E, 135mg/L coenzyme A, 3mg/L recombinant human insulin, 320 mg/L-glutamine, 460ug/mL dimercaptoethanol, 26ug/mL indomethacin, 5 ug/mL Na + 65ng/mL of monoclonal antibody CD2, 65ng/mL of NKp46 antibody, 0.06KE/mL of OK432, 50 μg/mL of cytokine IL-2.
Example 4
Example 4 differs from example 1 in that the activation medium comprises the following components in the respective concentrations: 1.3g/L human serum albumin, 1.8g/L human serum albumin, 135mg/L vitamin C, 230mg/L vitamin E, 135mg/L coenzyme A, 3mg/L recombinant human insulin, 320 mg/L-glutamine, 460ug/mL dimercaptoethanol, 26ug/mL indomethacin, 5 ug/mL Na + 65ng/mL of monoclonal antibody CD2, 100ng/mL of NKp46 antibody, 0.06KE/mL of OK432, 50 μg/mL of cytokine IL-2.
Example 5
Example 5 differs from example 1 in that the activation medium comprises the following components in the respective concentrations: 1.3g/L human serum albumin, 1.8g/L human serum albumin, 140mg/L vitamin C, 235mg/L vitamin E, 140mg/L coenzyme A, 3mg/L recombinant human insulin, 320 mg/L-glutamine, 480ug/mL dimercaptoethanol, 26ug/mL indomethacin, 5 ug/mL Na + 70ng/mL of monoclonal antibody CD2, 70ng/mL of NKp46 antibody, 0.06KE/mL of OK432, 50 μg/mL of cytokine IL-2.
Example 6
Example 6 differs from example 1 in that the activation medium comprises the following components in the respective concentrations: 1.5g/L human serum albumin, 2.0g/L human serumAlbumin, vitamin C160 mg/L, vitamin E240 mg/L, coenzyme A150 mg/L, recombinant human insulin 5mg/L, L-glutamine 330mg/L, dimercaptoethanol 530ug/mL, indomethacin 30ug/mL, na 10 μg/mL + 100ng/mL of monoclonal antibody CD2, 100ng/mL of NKp46 antibody, 0.06KE/mL of OK432, 100 μg/mL of cytokine IL-2.
Example 7
Example 7 differs from example 3 in that in step S2, the mononuclear cells were pretreated with a culture solution composed of cytokine IL-12 at a concentration of 15ng/mL, cytokine IL-15 at a concentration of 1g/mL and cytokine IL-18 at a concentration of 30ng/mL for 10 hours before being added to the cell culture flask, washed with PBS buffer for 2 times, and then placed in the cell culture flask for culturing.
Example 8
Example 8 differs from example 3 in that in step S2, the mononuclear cells were pretreated with a culture medium comprising cytokine IL-12 at a concentration of 18ng/mL, cytokine IL-15 at a concentration of 5ng/mL and cytokine IL-18 at a concentration of 40ng/mL for 13 hours before being added to the cell culture flask, washed 2 times with PBS buffer, and then placed in the cell culture flask for culturing.
Example 9
Example 9 differs from example 3 in that in step S2, the mononuclear cells were pretreated with a medium composed of cytokine IL-12 at a concentration of 15ng/mL, cytokine IL-15 at a concentration of 5ng/mL and cytokine IL-18 at a concentration of 50ng/mL for 13 hours before being added to the cell culture flask, washed 2 times with PBS buffer, and then placed in the cell culture flask for culturing.
Example 10
Example 10 differs from example 3 in that in step S2, the mononuclear cells were pretreated with a medium comprising cytokine IL-12 at a concentration of 20ng/mL, cytokine IL-15 at a concentration of 10ng/mL and cytokine IL-18 at a concentration of 50ng/mL for 15 hours before being added to the cell culture flask, washed 3 times with PBS buffer, and then placed in the cell culture flask for culturing.
Comparative example
Comparative example 1
Comparative example 1 differs from example 1 in that in step S2, monoclonal antibody CD2 was not added to the activation medium.
Comparative example 2
Comparative example 2 differs from example 1 in that no targeted NKp46 antibody was added to the activation medium in step S2.
Comparative example 3
Comparative example 3 differs from example 1 in that in step S2, the targeting OK-432 was not added to the activation broth.
Comparative example 4
Comparative example 4 was different from example 1 in that in step S2, the concentration of monoclonal antibody CD2 added to the activation medium was 10ng/mL and the concentration of NKp46 antibody was 10ng/mL.
Comparative example 5
Comparative example 5 was different from example 1 in that in step S2, the concentration of monoclonal antibody CD2 added to the activation medium was 150ng/mL and the concentration of NKp46 antibody was 150ng/mL.
Performance test
1. NK cell expansion fold detection
Cultured cells were taken from examples 1 to 10 and comparative examples 1 to 5 on days 0, 7 and 14, respectively, counted after staining with trypan blue, and the total number of cells on the day of counting was divided by the number of cells before culturing (i.e., day 0), the number being the fold of expansion of the cells, and the results are shown in Table 1.
Table 1 comparison of amplification factors of examples and comparative examples
As can be seen from Table 1, the NK cell expansion factors of examples 1 to 10 of the present application are higher than those of comparative examples 1 to 5, wherein the NK cell numbers of the culture of example 9 reached 4000 to 8000 times after 7 days and the NK cell numbers of the culture of comparative examples 1 to 5 reached about 5000 times after 14 days.
It can be seen from the combination of examples 3 and 4 that NK cells can be effectively expanded when the concentration ratio of monoclonal antibody CD2 and NKp46 antibodies is 1:1.
As can be seen from the combination of example 1 and comparative examples 1-3, the monoclonal antibodies CD2 and NKp46 and OK-432 in the present application are added to the culture medium as a bioeffective agent at the initial stage of cell culture, and can activate NK cells, thereby greatly increasing the expansion factor of NK cells. It can be seen from the combination of example 1 and comparative examples 4 to 5 that the addition of monoclonal antibodies CD2 and NKp46 at a final concentration less than or greater than the final concentration defined in the present application affects the expansion of NK cells.
It can be seen from the combination of examples 3 and 7-10 that pretreatment of NK cells prior to culture by using a combination of cytokine IL-12, cytokine IL-15 and cytokine IL-18 can efficiently amplify NK cells. The NK cells in the example 9 have obvious expansion condition (burrs are arranged around the colony to generate and gradually increase) from the 7 th day of culture, the NK cell colony is smaller at the 14 th day, and the NK cells are rapidly increased after being dispersed, so that a large amount of expansion is realized, the cells are dispersed, and the activity is good.
2. NK cell purity assay
NK cells cultured for 14 days in example 3, example 9 and comparative example 1 were taken for flow assay to count the purity of NK cells. The detection result showed that the NK cells obtained in example 3 had a purity of 90.8% after 14 days of culture. The NK cells obtained in example 9 were cultured for 14 days and the purity of NK cells was 95.2%. The NK cells obtained in comparative example 1 were cultured for 14 days, and the purity of NK cells was 82.5%.
3. NK cell killing detection the tumor cells were subject to K562 tumor cells, and the NK cells obtained in inventive example 3, example 9 and comparative 1 were used to detect the killing activity against tumor cells. Killing experiments were performed with K562 as target cells, with effective target ratios of 20:1, 10:1, 5:1, and 1:1, respectively, and the results are shown in Table 2.
TABLE 2 killing Activity of NK cells obtained in example 9 and comparative example 1 against K562 tumor cells
It can be seen from Table 2 that pretreatment of NK cells prior to culturing by combination of cytokines IL-12, IL-15 and IL-18 can efficiently amplify NK cells and remarkably activate NK cell killing activity.
In conclusion, the NK cells are cultured by adopting the culture method provided by the application, so that the NK cells with high purity, high amplification efficiency and strong cytotoxicity can be obtained.
It is to be understood that the above embodiments are merely illustrative of the application of the principles of the present application, but not in limitation thereof. Various modifications and improvements may be made by those skilled in the art without departing from the spirit and substance of the application, and are also considered to be within the scope of the application.

Claims (5)

1. A method for culturing NK cells with high proliferation capacity and high cytotoxicity, comprising the steps of:
s1, coating an anti-human CD16 monoclonal antibody with the concentration of 0.1-2.0 mg/mL in a cell culture flask, wherein the coating condition is 25 ℃, and the incubation time is 1-5 h;
s2, taking a cell culture bottle subjected to coating treatment and adding an NK cell culture medium, then pretreating mononuclear cells obtained by centrifugal separation from peripheral blood for 10-15 hours by using a culture solution consisting of cytokine IL12 with the concentration of 15-20 ng/mL, cytokine IL15 with the concentration of 1-10 ng/mL and cytokine IL18 with the concentration of 30-50 ng/mL, then washing for 2-3 times by using PBS buffer solution, and then placing the culture solution into the cell culture bottle for culturing, adding the culture solution into the cell culture bottle, and regulating the cell density to be 1 multiplied by 10 6 /mL～2×10 6 /mL; wherein NK cell cultureThe culture medium comprises basic culture solution and activating culture solution, and the adding amount of the activating culture solution is 3-5% of that of the basic culture solution; the activation culture solution comprises the following components in concentration: 1.2-1.5 g/L human serum albumin, 1.5-2.0 g/L human serum albumin, 120-160 mg/L vitamin C, 220-240 mg/L vitamin E, 120-150 mg/L coenzyme A, 0.5-5 mg/L recombinant human insulin, 280-330 mg/L-glutamine, 420-530 ug/mL dimercaptoethanol, 20-30 ug/mL indomethacin, 0.1-10 ug/mL Na + 50-100 ng/mL of monoclonal antibody CD2, 50-100 ng/mL of NKp46 antibody, 0.01-0.06 KE/mL of OK432 and 2-100 mug/mL of cytokine IL2;
s3, placing the cell culture flask at 37 ℃ and CO with the concentration of 2% 2 The incubator is continuously cultured for 9 to 15 days, and IL2 with the concentration of 2 to 100 mug/mL is added every 3 days.
2. The method for culturing high proliferation potency and high cytotoxicity NK cells according to claim 1, wherein: the activation culture solution comprises the following components in concentration: 1.3g/L human serum albumin, 1.8g/L human serum albumin, 130-140 mg/L vitamin C, 230-235 mg/L vitamin E, 130-140 mg/L coenzyme A, 3mg/L recombinant human insulin, 320 mg/L-glutamine, 450-480 ug/mL dimercaptoethanol, 26ug/mL indomethacin, 5 ug/mL Na + 60-70 ng/mL of monoclonal antibody CD2, 60-70 ng/mL of NKp46 antibody, 0.06KE/mL of OK432 and 2-50 mug/mL of cytokine IL2.
3. The method for culturing high proliferation potency and high cytotoxicity NK cells according to claim 2, wherein: the activation culture solution comprises the following components in concentration: 1.3g/L human serum albumin, 1.8g/L human serum albumin, 135mg/L vitamin C, 230mg/L vitamin E, 135mg/L coenzyme A, 3mg/L recombinant human insulin, 320 mg/L-glutamine, 460ug/mL dimercaptoethanol, 26ug/mL indomethacin, 5 ug/mL Na + 65ng/mL of monoclonal antibody CD2, 65ng/mL of NKp46 antibody, 0.06KE/mL of OK432, 50 μg/mL of cytokine IL2.
4. The method for culturing high proliferation potency and high cytotoxicity NK cells according to claim 3, wherein: in the step S2, before the mononuclear cells are added into a cell culture bottle, the mononuclear cells are pretreated for 13 hours by a culture solution consisting of the cytokine IL12 with the concentration of 15ng/mL, the cytokine IL15 with the concentration of 5ng/mL and the cytokine IL18 with the concentration of 50ng/mL, then washed for 2 times by a PBS buffer solution, and then placed into the cell culture bottle for culture.
5. A method of culturing high proliferative and highly cytotoxic NK cells according to any one of claims 1-3, wherein: the basal culture solution is selected from Corning KBM 581 lymphocyte serum-free culture solution.
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