Method for efficiently culturing and amplifying NK (Natural Killer) cells in vitro
By combining specific antibody coating and cytokine culture medium, NK cells were efficiently expanded, solving the problem of unstable NK cell expansion in existing technologies. This resulted in high proliferation rate and high positivity rate, with significant cytotoxicity.
Patent Information
- Application Number
- CN202511358039.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-23
- Publication Date
- 2025-11-14
AI Technical Summary
In existing technologies, in vitro expansion methods for NK cells have high safety risks, low expansion fold and purity, and are unstable. In particular, the activity of seed mononuclear cells after cryopreservation is impaired, leading to difficulties in culture.
One method involves the following steps: peripheral blood extraction and separation, plasma inactivation, cell density adjustment, lymphocyte separation, cell cryopreservation, cell coating, cell revival, and culture in serum-free culture medium containing specific cytokines and proteins. Specifically, this includes coating culture flasks with antibodies against CD16, CD244, CD226, and NKG2D, and culturing NK cells in culture medium containing MG53 protein, IL-2, IL-12, and IL-18.
It achieves efficient expansion of NK cells with high proliferation rate and high NK cell positivity rate, and has high cytotoxicity to K562 cells with good safety, making it suitable for cryopreserved mononuclear cell seeding.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of in vitro cell culture technology, and particularly relates to a method for efficiently culturing and expanding NK cells in vitro. Background Technology
[0002] NK cells, also known as natural killer cells, are an important type of lymphocyte in the human body's innate immune system. Their phenotype is generally CD3. - CD16 + CD56 + NK cells have the advantage of recognizing and killing tumor cells without requiring antigen sensitization or MHC restriction, playing an important role in the body's anti-tumor and early antiviral immune responses. Therefore, NK cells show great promise for the treatment of cancer patients. However, the low proportion and small number of NK cells in the body limit their application, leading to the development of in vitro NK cell expansion technology.
[0003] Currently, there are two main NK cell culture processes: feeder cell process and non-feeder cell process. The feeder cell process has a significant effect on the activation and expansion of NK cells; however, because feeder cells are irradiated tumor cells, there is a risk of tumor cell residue, so it is mostly used in scientific research. The non-feeder cell process mainly uses a combination of cytokines to activate mononuclear cells to expand and culture NK cells. It has better safety and high clinical application value, but the expansion fold and purity are not high and are unstable, especially for cryopreserved mononuclear cell seeds, where cell culture becomes more difficult due to impaired cell viability. Therefore, it is necessary to find a safe, high-expansion method for in vitro NK cell culture suitable for cryopreserved mononuclear cell seeds. Summary of the Invention
[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for efficiently culturing and expanding NK cells in vitro.
[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for efficiently culturing and expanding NK cells in vitro includes the following steps: S1: Peripheral blood is drawn and placed in a sterile heparin sodium blood collection tube. The separation process is completed within 2-4 hours. The specific separation process is as follows: the sterile heparin sodium blood collection tube containing peripheral blood is placed in a centrifuge and centrifuged to obtain the upper plasma layer and the lower blood layer. S2: Inactivate the upper plasma obtained in step S1 at 56℃ for 30 min, then centrifuge, discard the platelets and complement components at the bottom, and the resulting supernatant is the inactivated autologous plasma, which is stored aseptically at -80℃ for later use. S3: Adjust the cell density of the lower layer of blood obtained in step S1 with physiological saline; place the lymphocyte separation solution in a centrifuge tube, and then slowly add the lower layer of blood with adjusted cell density to the upper layer of the lymphocyte separation solution. Then place the centrifuge tube in a centrifuge and centrifuge; after centrifugation, aspirate the mononuclear cell layer, transfer it to another centrifuge tube, add physiological saline to resuspend and mix well, centrifuge, discard the supernatant, and repeat the washing 2-3 times. S4: The mononuclear cells washed in step S3 are processed at a rate of 1×10⁻⁶. 7 Resuspend the cells at a density of cells / ml in the cryopreservation solution, aliquot them into cryovials, place the cryovials in a programmed cooling box, and incubate them overnight at -80°C. The next day, remove the cryovials and transfer them to a liquid nitrogen tank for long-term storage. S5: Evenly cover the bottom of the untreated culture flask with PBS buffer containing CD16 antibody, CD244 antibody, CD226 antibody and NKG2D antibody. Then place the culture flask in a biosafety cabinet and let it stand at room temperature for 1-2 hours. After that, aspirate the coating solution and wash the culture flask twice with PBS buffer. S6: When the water bath temperature rises to 37°C, quickly place the cryovial containing the mononuclear cell suspension from step S4 into it, gently shake it left and right to thaw it quickly, transfer the thawed cell suspension to α-MEM medium containing human serum albumin and MG53 protein, centrifuge, discard the supernatant, and the resulting cell pellet is the revived mononuclear cells. S7: Transfer the resuscitated mononuclear cells from step S6 at a rate of 1×10⁻⁶. 6 ~2×10 6 The cells were seeded at a density of MG53 protein / ml in the culture flasks coated in step S5, and serum-free cell culture medium containing MG53 protein, inactivated autologous plasma obtained in step S2, IL-2, IL-12 and IL-18 was added. The cells were then incubated statically at 37°C in a 5% CO2 incubator. S8: Cell samples were taken on days 3, 5, 7, 9, and 11 of culture for cell counting. Based on the counting results, serum-free cell culture medium containing MG53 protein, inactivated autologous plasma obtained in step S2, IL-2, IL-12, and IL-18 was added to the culture flasks to adjust the cell density to 0.8 × 10⁻⁶. 6 ~1.2×10 6 Cells / ml; continue static incubation at 37℃ and 5% CO2 until day 14 of incubation; S9: Collect cells cultured to day 14 to obtain NK cells.
[0006] In step S5, the coating concentration of the CD16 antibody is 5 μg / ml, the coating concentration of the CD244 antibody is 10 μg / ml, the coating concentration of the CD226 antibody is 20 μg / ml, and the coating concentration of the NKG2D antibody is 5 μg / ml.
[0007] In step S6, the α-MEM culture medium contains 1 wt% human serum albumin and 0-1 mM MG53 protein.
[0008] In step S7, the serum-free cell culture medium contains 0-1 mM MG53 protein, 10 wt% inactivated autologous plasma, 500-1000 U / ml IL-2, 5-15 ng / ml IL-12 and 5-15 ng / ml IL-18.
[0009] In step S8, the serum-free cell culture medium contains 0-1 mM MG53 protein, 10 wt% inactivated autologous plasma, 500-1000 U / ml IL-2, 5-15 ng / ml IL-12 and 5-15 ng / ml IL-18. Attached Figure Description
[0010] Figure 1 : NK cell growth morphology diagram.
[0011] Figure 2 NK cell growth curve.
[0012] Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 Figure: Results of flow cytometry analysis of NK cell positivity rate.
[0013] Figure 8 Figure: Results of NK cell cytotoxicity assay. Detailed Implementation
[0014] To make the content of this invention easier to understand, the technical solution of this invention will be further described below in conjunction with specific embodiments, but this invention is not limited thereto.
[0015] All reagents and raw materials used in this invention are commercially available. Specifically, the lymphocyte separation medium was purchased from Tianjin Haoyang (catalog number LTS10770125); the cell cryopreservation medium was purchased from Youkang Biotechnology (catalog number NC1001.1); the CD16 antibody was purchased from Nearshore Protein (catalog number GMP-A091); the CD244 antibody was purchased from Nearshore Protein (catalog number C07C); the CD226 antibody was purchased from Nearshore Protein (catalog number CS90); the NKG2D antibody was purchased from Nearshore Protein (catalog number GMP-A075); the serum-free cell culture medium was purchased from Zhuhai Beso (catalog number 01400P10); and the MG53 protein... The following antibodies were purchased: white protein (abcam, catalog number ab132761), IL-2 (Nearshore Protein, catalog number GMP-CD66), IL-12 (Nearshore Protein, catalog number GMP-CI58), IL-18 (Nearshore Protein, catalog number GMP-CH29), CD3-FITC antibody (BD, catalog number 555339), CD16-PE antibody (BD, catalog number 555407), CD56-APC antibody (BD, catalog number 555518), Lactate dehydrogenase cytotoxicity assay kit (LDH Cytotoxicity Assay Kit) (Beyotime Biotechnology, catalog number C0016), and α-MEM medium (Gibco, catalog number C12571500CP).
[0016] Example 1 (Group A): A method for efficiently culturing and expanding NK cells in vitro includes the following steps: S1: Peripheral blood collection and separation processing: The healthy adult volunteers tested negative for multiple tests, including hepatitis B surface antigen, hepatitis C antibody, human immunodeficiency virus antibody, syphilis antibody, and cytomegalovirus IgM antibody. 50 ml of peripheral blood was drawn from each volunteer and placed in sterile heparin sodium blood collection tubes (10 ml per tube). The separation process was completed within 3 hours. The specific separation process was as follows: the sterile heparin sodium blood collection tubes containing peripheral blood were placed in a centrifuge and centrifuged at 3000 rpm for 10 min to obtain the upper plasma layer and the lower blood layer.
[0017] S2: Inactivation of plasma: The upper plasma obtained in step S1 was inactivated at 56°C for 30 min, then centrifuged at 3000g for 10 min. The platelets and complement components at the bottom were discarded. The resulting supernatant was the inactivated autologous plasma, which was stored aseptically at -80°C for later use.
[0018] S3: Isolation of mononuclear cells: The lower layer blood obtained in step S1 was adjusted to a cell density of 5 × 10⁻⁶ cells using physiological saline. 6Cells / ml. Place an appropriate amount of lymphocyte separation medium in a 50ml centrifuge tube. Then, slowly add the adjusted cell density lower layer of blood to the upper layer of the lymphocyte separation medium (the volume ratio of lymphocyte separation medium to the adjusted cell density lower layer of blood is 1:2; when adding the sample, use the tip of a pipette to gently draw several fine lines along the tube wall at the interface of the liquid surface to facilitate smooth spread of the blood). Then, place the centrifuge tube in a centrifuge, set the centrifugation force to 800g, and adjust the acceleration and deceleration settings to 0. Centrifuge for 30 minutes. After centrifugation, aspirate the mononuclear cell layer and transfer it to another 50ml centrifuge tube. Add 30ml of physiological saline to resuspend and mix well. Centrifuge at 300g for 10 minutes, discard the supernatant, and repeat the washing process three times.
[0019] S4: Cryopreservation of mononuclear cells: The mononuclear cells washed in step S3 were then processed at a rate of 1×10⁻⁶. 7 Resuspend the cells at a density of cells / ml in cryopreservation solution, aliquot 1.5ml into 2ml cryovials, place the cryovials in a programmed cooling box, and incubate overnight at -80°C. The next day, remove the cryovials and transfer them to a liquid nitrogen tank for long-term storage.
[0020] S5: Coating of the culture flask: The bottom of an untreated T75 culture flask was evenly covered with 10 ml of 1×PBS buffer (pH = 7.2–7.4) containing 5 μg / ml CD16 antibody, 10 μg / ml CD244 antibody, 20 μg / ml CD226 antibody and 5 μg / ml NKG2D antibody. The culture flask was then placed in a biosafety cabinet and incubated at room temperature for 2 hours. After that, the coating solution was aspirated and the culture flask was washed twice with 1×PBS buffer (pH = 7.2–7.4).
[0021] S6: Resuscitation of mononuclear cells: Once the water bath temperature reaches 37°C, quickly place the cryovial containing 1.5 ml of mononuclear cell suspension from step S4 into the water bath and gently shake it from side to side to thaw it quickly. Transfer the thawed cell suspension to 20 ml of α-MEM medium containing 1 wt% human serum albumin, centrifuge at 300 g for 5 min, discard the supernatant, and the resulting cell pellet is the revived mononuclear cells.
[0022] S7: Cell Repair and Activation The resuscitated mononuclear cells from step S6 were 1×10 6 The cells were seeded at a density of cells / ml in the culture flasks coated in step S5. 20 ml of serum-free cell culture medium containing 10 wt% of the inactivated autologous plasma obtained in step S2, 1000 U / ml IL-2, 10 ng / ml IL-12 and 10 ng / ml IL-18 was added, and the cells were incubated statically in a 37°C, 5% CO2 incubator.
[0023] S8: Cell Expansion Cells were sampled on days 3, 5, 7, 9, and 11 of culture for cell counting. Based on the counting results, serum-free cell culture medium containing 10 wt% of the inactivated autologous plasma obtained in step S2, 1000 U / ml IL-2, 10 ng / ml IL-12, and 10 ng / ml IL-18 was added to the culture flasks to adjust the cell density to 1 × 10⁻⁶ cells / ml. 6 Cells / ml; continue static incubation at 37℃, 5% CO2 until day 14.
[0024] S9: Cellular Harvest: Cells cultured to day 14 were collected to obtain NK cells.
[0025] Example 2 (Group B): A method for efficiently culturing and expanding NK cells in vitro includes the following steps: S1: Peripheral blood collection and separation processing: The same as step S1 in Example 1.
[0026] S2: Inactivation of plasma: The same as step S2 in Example 1.
[0027] S3: Isolation of mononuclear cells: The same as step S3 in Example 1.
[0028] S4: Cryopreservation of mononuclear cells: The same as step S4 in Example 1.
[0029] S5: Coating of the culture flask: The same as step S5 in Example 1.
[0030] S6: Resuscitation of mononuclear cells: Once the water bath temperature reaches 37°C, quickly place the cryovial containing 1.5 ml of mononuclear cell suspension from step S4 into the water bath and gently shake it from side to side to thaw it rapidly. Transfer the thawed cell suspension to 20 ml of α-MEM medium containing 1 wt% human serum albumin and 1 mM MG53 protein, centrifuge at 300 g for 5 min, discard the supernatant, and the resulting cell pellet is the revived mononuclear cells.
[0031] S7: Cell Repair and Activation The same as step S7 in Example 1.
[0032] S8: Cell Expansion The same as step S8 in Example 1.
[0033] S9: Cellular Harvest: The same as step S9 in Example 1.
[0034] Example 3 (Group C): A method for efficiently culturing and expanding NK cells in vitro, the specific steps of which are as follows: S1: Peripheral blood collection and separation processing: The same as step S1 in Example 1.
[0035] S2: Inactivation of plasma: The same as step S2 in Example 1.
[0036] S3: Isolation of mononuclear cells: The same as step S3 in Example 1.
[0037] S4: Cryopreservation of mononuclear cells: The same as step S4 in Example 1.
[0038] S5: Coating of the culture flask: The same as step S5 in Example 1.
[0039] S6: Resuscitation of mononuclear cells: The same as step S6 in Example 1.
[0040] S7: Cell Repair and Activation The resuscitated mononuclear cells from step S6 were 1×10 6 The cells were seeded at a density of cells / ml in the culture flasks coated in step S5. 20 ml of serum-free cell culture medium containing 0.5 mM MG53 protein, 10 wt% of the inactivated autologous plasma obtained in step S2, 1000 U / ml IL-2, 10 ng / ml IL-12 and 10 ng / ml IL-18 was added, and the cells were incubated statically in a 37°C, 5% CO2 incubator.
[0041] S8: Cell Expansion The same as step S8 in Example 1.
[0042] S9: Cellular Harvest: The same as step S9 in Example 1.
[0043] Example 4 (Group D): A method for efficiently culturing and expanding NK cells in vitro, the specific steps of which are as follows: S1: Peripheral blood collection and separation processing: The same as step S1 in Example 1.
[0044] S2: Inactivation of plasma: The same as step S2 in Example 1.
[0045] S3: Isolation of mononuclear cells: The same as step S3 in Example 1.
[0046] S4: Cryopreservation of mononuclear cells: The same as step S4 in Example 1.
[0047] S5: Coating of the culture flask: The same as step S5 in Example 1.
[0048] S6: Resuscitation of mononuclear cells: The same as step S6 in Example 1.
[0049] S7: Cell Repair and Activation The same as step S7 in Example 1.
[0050] S8: Cell Expansion Cells were sampled on days 3, 5, 7, 9, and 11 of culture for cell counting. Based on the counting results, serum-free cell culture medium containing 0.5 mM MG53 protein, 10 wt% of inactivated autologous plasma obtained in step S2, 1000 U / ml IL-2, 10 ng / ml IL-12, and 10 ng / ml IL-18 was added to the culture flasks to adjust the cell density to 1 × 10⁻⁶ cells / ml. 6 Cells / ml; continue static incubation at 37℃, 5% CO2 until day 14.
[0051] S9: Cellular Harvest: The same as step S9 in Example 1.
[0052] Example 5 (Group E): A method for efficiently culturing and expanding NK cells in vitro, the specific steps of which are as follows: S1: Peripheral blood collection and separation processing: The same as step S1 in Example 1.
[0053] S2: Inactivation of plasma: The same as step S2 in Example 1.
[0054] S3: Isolation of mononuclear cells: The same as step S3 in Example 1.
[0055] S4: Cryopreservation of mononuclear cells: The same as step S4 in Example 1.
[0056] S5: Coating of the culture flask: The same as step S5 in Example 1.
[0057] S6: Resuscitation of mononuclear cells: Once the water bath temperature reaches 37°C, quickly place the cryovial containing 1.5 ml of mononuclear cell suspension from step S4 into the water bath and gently shake it from side to side to thaw it rapidly. Transfer the thawed cell suspension to 20 ml of α-MEM medium containing 1 wt% human serum albumin and 1 mM MG53 protein, centrifuge at 300 g for 5 min, discard the supernatant, and the resulting cell pellet is the revived mononuclear cells.
[0058] S7: Cell Repair and Activation The resuscitated mononuclear cells from step S6 were 1×10 6 The cells were seeded at a density of cells / ml in the culture flasks coated in step S5. 20 ml of serum-free cell culture medium containing 0.5 mM MG53 protein, 10 wt% of the inactivated autologous plasma obtained in step S2, 1000 U / ml IL-2, 10 ng / ml IL-12 and 10 ng / ml IL-18 was added, and the cells were incubated statically in a 37°C, 5% CO2 incubator.
[0059] S8: Cell Expansion Cells were sampled on days 3, 5, 7, 9, and 11 of culture for cell counting. Based on the counting results, serum-free cell culture medium containing 0.5 mM MG53 protein, 10 wt% of inactivated autologous plasma obtained in step S2, 1000 U / ml IL-2, 10 ng / ml IL-12, and 10 ng / ml IL-18 was added to the culture flasks to adjust the cell density to 1 × 10⁻⁶ cells / ml. 6 Cells / ml; continue static incubation at 37℃, 5% CO2 until day 14.
[0060] S9: Cellular Harvest: The same as step S9 in Example 1.
[0061] NK cell quality testing: like Figure 1 As shown, all five groups of cells exhibited good growth, with group E having the most and largest cell colonies; conversely, group A had the fewest and smallest cell colonies. Figure 2 As shown, group E had the highest cell proliferation rate, followed by groups C, B, and D, while group A had the lowest proliferation rate.
[0062] The following steps were performed for flow cytometry analysis of the NK cell positivity rates in Examples 1-5: Cells were washed twice with PBS containing 0.1% BSA, and then resuspended in 300 μL of PBS; 5 μL of LCD3-FITC antibody, 5 μL of CD16-PE antibody, and 5 μL of CD56-APC antibody were added to the cell suspension, and the cells were incubated at room temperature in the dark for 30 min; after incubation, 2 mL of PBS was added to wash the cells, followed by centrifugation at 200 g for 5 min; the supernatant was discarded, and the cells were remixed with 300 μL of PBS, and finally quantitative analysis was performed by flow cytometry. Flow cytometry analysis was performed on a BD C6Plus instrument, and data graphs were generated simultaneously. Figures 3 to 7 As shown, there are five groups of CD3: A, B, C, D, and E. - CD16 + CD56 + The NK cell positivity rates were 67.56%, 83.78%, 85.82%, 80.39%, and 95.23%, respectively; among them, group E had the highest NK cell positivity rate, while group A had the lowest.
[0063] The cytotoxic activity of NK cells against K562 cells in Examples 1-5 was detected using the LDH Cytotoxicity Assay Kit. The specific procedures are as follows: (1) Take a 96-well plate and set up the seeding system according to the following 6 groups, where the effector cells are the five groups of NK cells A, B, C, D and E corresponding to Examples 1 to 5, and the target cells are K562 cells: effector cell group: 50 μL effector cell suspension + 50 μL complete culture medium, target cell spontaneous release group: 50 μL target cell suspension + 50 μL complete culture medium, target cell maximum release group: 50 μL target cell suspension + 50 μL complete culture medium + 10 μL 10× lysis buffer, experimental group: 50 μL target cell suspension + 50 μL effector cell suspension (set up 5 subgroups according to effector-target ratio of 10:1, 5:1, 2:1, 1:1 and 0.5:1, and adjust the cell concentration of each group according to the corresponding ratio), volume correction control group: 100 μL complete culture medium + 10 μL 10× lysis buffer, background control group: 100 μL complete culture medium. Note: Except for the experimental group, the target cell concentrations of the other groups are the same as those of the experimental group, and the effector cell concentrations are matched according to the requirements of the corresponding groups.
[0064] (2) Place the inoculated 96-well plate into a multi-well plate centrifuge and centrifuge at 250g for 4 min; then transfer it to a 37℃, 5% CO2 incubator for 6 h.
[0065] (3) 45 min before the end of incubation, add 10 μL of 10× lysis buffer to each well of the “target cell maximum release group”; after centrifugation at 250g for 4 min, carefully aspirate 50 μL of supernatant from each well and transfer it to the corresponding well of another clean 96-well plate to avoid aspirating cell precipitate.
[0066] (4) Add 50 μL of substrate mixture diluted again according to the kit instructions to each well of the 96-well plate after transferring the supernatant, and incubate at room temperature in the dark for 30 min.
[0067] (5) Add 50 μL of stop solution to each well, mix gently, zero the instrument with the “background control group”, and measure the absorbance of each well at a wavelength of 450 nm; calculate the killing activity of each group of effector cells against target cells according to the formula provided in the kit.
[0068] like Figure 8 As shown, under all effector-target ratios (10:1 to 0.5:1), the killing activity of NK cells in group E against K562 cells was the highest (exhibiting high cytotoxicity), followed by groups C, B, and D, while group A had the lowest killing activity.
[0069] Conclusion: The method in Example 5 was used to culture and expand cryopreserved mononuclear cells, resulting in NK cells with high proliferation rate, high NK cell positivity rate, and high cytotoxicity against K562 cells.
[0070] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.
Claims
1. A method for efficiently culturing and expanding NK cells in vitro, characterized in that: Includes the following steps: S1: Peripheral blood is drawn and placed in a sterile heparin sodium blood collection tube. The separation process is completed within 2-4 hours. The specific separation process is as follows: the sterile heparin sodium blood collection tube containing peripheral blood is placed in a centrifuge and centrifuged to obtain the upper plasma and lower blood layers respectively. S2: Inactivate the upper plasma obtained in step S1 at 56℃ for 30 min, then centrifuge, discard the platelets and complement components at the bottom, and the resulting supernatant is the inactivated autologous plasma, which is stored aseptically at -80℃ for later use. S3: Adjust the cell density of the lower layer of blood obtained in step S1 with physiological saline; take the lymphocyte separation fluid and place it in a centrifuge tube, then slowly add the lower layer of blood with adjusted cell density to the upper layer of the lymphocyte separation fluid, and then place the centrifuge tube in a centrifuge; after centrifugation, aspirate the mononuclear cell layer, transfer it to another centrifuge tube, add physiological saline to resuspend and mix well, centrifuge, discard the supernatant, and repeat the washing 2-3 times; S4: The mononuclear cells washed in step S3 are processed at a rate of 1×10⁻⁶. 7 Resuspend the cells at a density of cells / ml in the cryopreservation solution, aliquot them into cryovials, place the cryovials in a programmed cooling box, and incubate them overnight at -80°C. The next day, remove the cryovials and transfer them to a liquid nitrogen tank for long-term storage. S5: Evenly cover the bottom of the untreated culture flask with PBS buffer containing CD16 antibody, CD244 antibody, CD226 antibody and NKG2D antibody. Then place the culture flask in a biosafety cabinet and let it stand at room temperature for 1-2 hours. After that, aspirate the coating solution and wash the culture flask twice with PBS buffer. S6: When the water bath temperature rises to 37°C, quickly place the cryovial containing the mononuclear cell suspension from step S4 into it, gently shake it left and right to thaw it quickly, transfer the thawed cell suspension to α-MEM medium containing human serum albumin and MG53 protein, centrifuge, discard the supernatant, and the resulting cell pellet is the revived mononuclear cells. S7: Transfer the resuscitated mononuclear cells from step S6 at a rate of 1×10⁻⁶. 6 ~2×10 6 The cells were seeded at a density of cells / ml in the culture flasks coated in step S5, and serum-free cell culture medium containing MG53 protein, inactivated autologous plasma obtained in step S2, IL-2, IL-12 and IL-18 was added. The cells were then incubated statically in a 37°C, 5% CO2 incubator. S8: Cell samples were taken on days 3, 5, 7, 9, and 11 of culture for cell counting. Based on the counting results, serum-free cell culture medium containing MG53 protein, inactivated autologous plasma obtained in step S2, IL-2, IL-12, and IL-18 was added to the culture flasks to adjust the cell density to 0.8 × 10⁻⁶. 6 ~1.2×10 6 Cells / ml; continue static incubation at 37℃ and 5% CO2 until day 14 of incubation; S9: Collect cells cultured to day 14 to obtain NK cells.
2. The method according to claim 1, characterized in that: In step S5, the coating concentration of the CD16 antibody is 5 μg / ml, the coating concentration of the CD244 antibody is 10 μg / ml, the coating concentration of the CD226 antibody is 20 μg / ml, and the coating concentration of the NKG2D antibody is 5 μg / ml.
3. The method according to claim 1, characterized in that: In step S6, the α-MEM culture medium contains 1 wt% human serum albumin and 0-1 mM MG53 protein.
4. The method according to claim 1, characterized in that: In step S7, the serum-free cell culture medium contains 0-1 mM MMG53 protein, 10 wt% inactivated autologous plasma, 500-1000 U / ml IL-2, 5-15 ng / ml IL-12 and 5-15 ng / ml IL-18.
5. The method according to claim 1, characterized in that: In step S8, the serum-free cell culture medium contains 0-1 mM MMG53 protein, 10 wt% inactivated autologous plasma, 500-1000 U / ml IL-2, 5-15 ng / ml IL-12 and 5-15 ng / ml IL-18.