In-vitro amplification culture method of NK (Natural Killer) cells

Abstract

The invention belongs to the technical field of immune cell culture, and particularly relates to an in-vitro amplification culture method of NK cells. The amplification method comprises the steps of CD16 pretreatment, plasma inactivation, lymphocyte separation and multi-stage fluid infusion. The preparation method comprises the following steps: pretreating CD16 in a T75 culture flask, promoting attachment and activation of NK cells, simulating a natural activation state, improving proliferation efficiency, treating whole blood and inactivating plasma to eliminate immunosuppressive factors, reserving nutritional ingredients, supporting amplification of the NK cells, enriching the NK cells through a lymphocyte separating medium, removing impurities and improving purity, mixing an RPMI-1640 culture medium with a solution A, and culturing for 12-24 hours at a high temperature; oK432 and IL-15 are added, the microenvironment is optimized, growth of the NK cells is remarkably promoted, liquid supplementing operation and bottling and bagging are carried out for multiple times, sufficient space and nutrition are provided, the cells are gradually transferred into a serum-free culture medium, and the cell safety and clinical application potential are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of immune cell culture, and specifically relates to an in vitro expansion and culture method of NK cells. Background Art

[0002] Natural Killer Cells (NK Cells) are an important effector cell in the innate immune system. They are named for their ability to directly recognize and kill virus-infected cells and tumor cells without antigen presentation. NK cells play a key role in immune surveillance and elimination of abnormal cells. Unlike T cells and B cells, NK cells do not rely on specific antigen receptors, have natural cytotoxicity, and can regulate immune responses by secreting cytokines (such as IFN-γ). With the development of immunotherapy, the potential application of NK cells in tumor immunotherapy has received widespread attention. Studies have shown that NK cells have important application prospects in the fields of anti-tumor, anti-viral infection, and transplant immunity. However, the content of NK cells in peripheral blood is low, which is difficult to meet the needs of clinical treatment. Therefore, developing an effective in vitro expansion method to obtain sufficient number and functional activity of NK cells is one of the current research hotspots. Due to the low proportion of NK cells in the body, accounting for only 5-15% of peripheral blood mononuclear cells (PBMCs), the direct application of unexpanded NK cells is not sufficient in number to produce significant therapeutic effects. Therefore, in vitro expansion technology is regarded as an important means to increase the number and activity of NK cells. Through in vitro culture and expansion, a large number of high-purity and high-activity NK cells can be obtained, thereby providing sufficient cell resources for clinical applications. However, traditional NK cell expansion methods have problems such as low efficiency and unstable cell activity. Therefore, it is extremely necessary to develop a more efficient and controllable NK cell in vitro expansion method. Summary of the invention

[0003] In view of the defects of the prior art, the object of the present invention is to provide an in vitro expansion and culture method of NK cells.

[0004] The technical effect of the present invention is achieved by the following technical solution: A method for in vitro proliferation and culture of NK cells, comprising the following steps: S1: Use a pipette to take 6 mL of PBS buffer and add it to a T75 culture flask, add 10 μg / mL CD16 to prepare CD16 working solution, tighten the bottle cap, slowly turn it upside down to allow CD16 to fully disperse at the bottom of the bottle; seal it with a sealing film, put it in a bag, and then place it at 4°C overnight; S2: Draw 50 mL of whole blood into a sterile sodium heparin blood collection tube and mix by inverting several times; S3: at room temperature, centrifuge the whole blood treated in step S2. After centrifugation, transfer the upper plasma into a centrifuge tube using a pipette, and then put it into a water bath and inactivate it at 56°C for 30 minutes; S4: placing the plasma inactivated in step S3 in a 4°C refrigerator for 20-30 minutes, then taking out the plasma from the refrigerator, centrifuging it at 3000 rpm for 5 minutes, taking the supernatant for later use, and obtaining pretreated plasma; S5: resuspend and mix the lower sediment after centrifugation in step S4 with an equal volume of physiological saline; put 5 mL of lymphocyte separation solution into a centrifuge tube and slowly add 5 mL of the resuspended and mixed blood cells, and then centrifuge at room temperature; S6: After completing the centrifugation in step S5, aspirate the white film layer in the middle of the centrifuge solution, put the white film layer into a new centrifuge tube, resuspend and mix with physiological saline to 50 mL, centrifuge at 2000 rpm for 5 min, discard the supernatant, repeat the resuspending and centrifugation once to obtain a cell pellet; S7: Mix RPMI-1640 medium and nutrient supplement solution in a volume ratio of 9:1 to prepare a complete culture solution; S8: adding the cell pellet prepared in step S6 to 9 mL of complete culture medium prepared in step S7, resuspending and mixing, and then adding 133 μL of OK432, 100 μL of IL-15 and 1 mL of pretreated plasma prepared in step S4 to obtain a cell suspension; S9: adding the cell suspension prepared in step S8 to the T75 culture flask prepared in step S1, and placing in an incubator at 37°C, 5% CO2 for culture; S10: First fluid replenishment operation: On the second day after completing the culture operation in step S9, take 9 mL of the complete culture medium prepared in step S7, add it to a 50 mL centrifuge tube, mix OK432, IL-15 and the pretreated plasma prepared in step S4 in a ratio of 20 μL:15 μL:1.5 mL, and then slowly add it to the culture bottle to ensure that the final volume of the liquid in the culture bottle is 25 mL, and continue to culture in a 37°C, 5% CO2 incubator; S11: Second fluid replenishment operation. On the 2nd to 3rd day after the first fluid replenishment operation of step S10 is completed, the complete culture solution prepared in step S7 and the pretreated plasma prepared in step S4 are added to a 50 mL centrifuge tube in a ratio of 32 mL:3 mL, and then fully mixed. Then, the mixture is slowly added to the culture bottle to ensure that the final volume of the liquid in the culture bottle is 60 mL, and the culture is continued in a 37°C, 5% CO2 incubator; S12: The third rehydration operation, on the second day after the second rehydration operation of step S11, the cell suspension in the culture flask is transferred to a T175 culture flask, 10 mL of the pretreated plasma prepared in step S4 is added, and then the complete culture medium prepared in step S7 is added to ensure that the final volume of the liquid in the culture flask is 150 mL; S13: The fourth rehydration operation, on the second day after the third rehydration operation of step S12 is completed, the cell suspension in the culture flask is evenly distributed to two T225 culture flasks, 6-7 mL of the pretreated plasma prepared in step S4 is added to each culture flask, and then the complete culture medium prepared in step S7 is used to replenish the final volume of the liquid in each culture flask to 200 mL, a total of 400 mL; S14: Mix the nutrient supplement solution and OptiVitro T cell serum-free medium at a volume ratio of 0.8:9.2 to prepare a complete serum-free culture medium; S15: The fifth rehydration operation. On the second day after the fourth rehydration operation in step S13 is completed, the cell suspension in the culture flask is equally divided into four T225 culture flasks, recorded as T225-1, T225-2, T225-3 and T225-4, and the complete serum-free culture medium prepared in step S14 is added to each culture flask to ensure that the final volume of the liquid in each culture flask is 200 mL; S16: Sixth rehydration: On the second day after the fifth rehydration operation in step S15, disinfect the connection port of the culture bag and connect the connection port to the syringe port to prepare for bagging; take out T225-1 and T225-2 in step S15, tap the culture bottle to resuspend the cells, then add them to the culture bag, add the complete serum-free culture solution prepared in step S14, and rinse the culture bottle to ensure that the final volume of the culture bag liquid is 800 mL; after bagging, bag T225-3 and T225-4 in step S15 in the same way; S17: Seventh rehydration: On the second day after the sixth rehydration operation of step S16 is completed, the complete serum-free culture medium prepared in step S14 is added to each culture bag until the final volume of the liquid is 1050 mL, and the total volume of the two bags is 2100 mL; S18: On the 2nd to 3rd day after the seventh rehydration operation of step S17 is completed, the cells in the two culture bags are collected into two 500mL centrifuge bottles, recorded as centrifuge bottle-1 and centrifuge bottle-2, 50mL of normal saline is added to centrifuge bottle-1, and the cells are transferred to centrifuge bottle-2, and then normal saline is added to the final volume of the centrifuge bottle liquid to 500mL, the precipitate is collected by centrifugation, 10-20mL of normal saline and 2-3mL of nutrient preservation solution are added, the mixture is bagged, and the temperature is gradually reduced at a rate of 1°C / min until it reaches -80°C, and then stored in a frozen state.

[0005] Preferably, in step S1, the mass concentration of CD16 in the CD16 working solution is 1 μg / mL; Preferably, in step S3, the specific parameters of the centrifugal treatment are 1800 rpm, 9 up and 7 down, and time 15 min; Preferably, in step S5, the specific parameters of the centrifugation treatment are 2000 rpm, 9 up and 0 down, and time 20 min; the lymphocyte separation fluid is prepared by Ficoll density gradient centrifugation using PBS buffer as solvent; Preferably, in step S6, the middle buffy coat layer is a cell layer at the junction of the two liquid surfaces.

[0006] Preferably, in step S7, the specific preparation steps of the nutrient supplement solution are as follows: A1: Dissolve sodium alginate in PBS buffer, heat in a water bath to control the temperature at 37°C, stir and dissolve evenly, and cool naturally to room temperature to obtain a 0.2wt% sodium alginate solution; dissolve hydroxypropyl methylcellulose in PBS buffer, continue to stir and dissolve evenly, and obtain a 0.1wt% hydroxypropyl methylcellulose solution; A2: Mix equal volumes of the sodium alginate solution and hydroxypropyl methylcellulose solution prepared in step A1, heat in a water bath to control the temperature at 35°C, add insulin, transferrin and selenoprotein, stir to dissolve evenly, then add glutamine and glutathione, stir to dissolve evenly, filter through a 0.22 μm filter membrane to obtain a nutrient supplement solution.

[0007] Preferably, in step A2, the amount of insulin added is 5 μg / mL; the amount of transferrin added is 30-50 μg / mL; the amount of selenoprotein added is 0.2 mg / mL; the amount of glutamine added is 0.5-1 mg / mL; and the amount of glutathione added is 0.04 mg / mL.

[0008] Preferably, in step S8, the total volume of the cell suspension is 10 mL, and the cell density is 5×10 6 The concentrations of OK432 and IL-15 added were both 1 μg / mL; Preferably, in step S10, the concentration of OK432 and IL-15 added is 1 μg / mL; Preferably, in step S18, the collection operation is centrifugation at 2000-3000 rpm for 5-8 min, discarding the supernatant, and repeating the centrifugation operation twice.

[0009] Preferably, the specific steps for preparing the nutrient preservation solution in step S18 are as follows: B1: Add β-cyclodextrin to 20 times the mass volume of deionized water, and continue stirring until it is evenly dissolved to obtain a β-cyclodextrin solution. Add dimethyl sulfoxide to the β-cyclodextrin solution, pre-treat with 60W ultrasound for 15 minutes, and continue stirring at 300-500rpm at room temperature for 4-6 hours, and then dialyze in a 3kDa dialysis bag for 48 hours to obtain a β-cyclodextrin-DMSO inclusion complex; B2: Evaporate and concentrate the β-cyclodextrin-DMSO inclusion complex prepared in step B1 to 1 / 4 of the original volume at 35°C, dialyze in a 2 kDa dialysis bag for 24 hours, and then control the temperature at 4°C in an ice bath, add nicotinamide and NAD+ to the dialyzed liquid, stir and disperse evenly, then add IL-2 and gentamicin, stir and disperse evenly, and obtain a nutrient preservation solution.

[0010] Preferably, in step B1, the ratio of the amount of dimethyl sulfoxide to β-cyclodextrin is 1 mL:2 g; in step B2, the amount of nicotinamide added is 10-50 ng / mL, the amount of NAD+ added is 10-50 ng / mL, the amount of gentamicin added is 1000 U / mL, and the amount of IL-2 added is 6 ng / mL.

[0011] The beneficial effects of the present invention are as follows: The present invention effectively promotes the attachment and activation of NK cells by pre-treating CD16 in a T75 culture bottle. CD16 binding antibodies can simulate the natural activation state of NK cells, thereby promoting their proliferation. After processing whole blood and pre-treating plasma, potential immunosuppressive factors in the plasma are removed, while retaining nutrients and proteins that contribute to the growth of NK cells; the inactivated plasma is used for subsequent culture medium supplementation, which can provide necessary growth factors for NK cells and support cell expansion and maintenance. Lymphocytes are separated and resuspended by lymphocyte separation fluid, effectively enriching NK cells, removing most of the unnecessary cell components, ensuring that NK cells are mainly amplified in the subsequent culture process, thereby improving the culture efficiency and purity of NK cells. OK432 and IL-15 are added to the complete culture medium prepared by mixing RPMI-1640 culture medium with nutrient supplement solution to form an optimized microenvironment, which significantly supports the growth and expansion of NK cells; OK432 and IL-15, as known potent factors for NK cell activation and proliferation, can greatly increase the expansion rate of NK cells. The present invention gradually increases the volume of the culture medium (from 25 mL to 1050 mL) through multiple fluid replenishment operations, which not only adapts to the growth of the number of cells, but also effectively maintains the sufficiency of nutrients and the dilution of wastes in the culture environment, avoiding the restriction of cell proliferation due to malnutrition or waste accumulation; the bottle division and bagging operations provide sufficient space for the large-scale expansion of NK cells, preventing growth inhibition caused by excessive cell density; in addition, the gradual transition to serum-free culture medium reduces the influence of exogenous proteins, improves the safety and controllability of cell culture, and makes it suitable for the subsequent preparation of clinical-grade cell preparations.

[0012] The present invention increases the viscosity of the culture medium by using hydroxypropyl methylcellulose, and provides a more uniform and stable culture environment. Hydroxypropyl methylcellulose can effectively prevent the sedimentation of cells during the culture process, maintain the uniformity of the culture medium, and hydroxypropyl methylcellulose will not cross-link with sodium alginate. In the absence of calcium ions, the two are combined to appropriately thicken the culture medium to ensure that NK cells are evenly distributed and maintain good growth during the amplification process. In the initial culture stage, the growth factors and nutritional supplements in the nutrient supplement solution can support the proliferation of NK cells, and provide necessary antioxidant protection, reducing the damage of oxidative stress to cells. During the rehydration process, the ingredients of the nutrient supplement solution ensure that as the volume of the culture medium increases, the nutrient concentration in the cell environment can remain stable, avoiding malnutrition caused by the dilution effect, and the addition of sodium alginate and hydroxypropyl methylcellulose helps to stabilize the growth state of cells in the dynamically changing culture medium. In addition, the combination of the nutrient supplement solution and the serum-free culture medium improves the amplification efficiency of the cells while maintaining the activity and function of the cells. The present invention modifies the traditional cryoprotectant dimethyl sulfoxide (DMSO) and forms an inclusion complex with cyclodextrin, thereby significantly reducing the toxicity of dimethyl sulfoxide and enhancing its osmotic protection effect, effectively preventing the formation of ice crystals in cells and protecting cell structures. At the same time, nicotinamide, NAD+ and IL-2 are combined with NK cell expansion to produce excellent synergistic effects, effectively achieving the reduction of cell damage during cryopreservation, reducing oxidative stress, ensuring that cells can quickly recover their functions after thawing and recovery, and maintaining efficient proliferation and activation capabilities. The application of this protective solution ensures that NK cells can quickly recover their proliferation ability and activity after cryopreservation, thereby providing high-quality NK cell preparations for subsequent cell applications. This protection method forms an effective closed loop with the overall process of cell culture and expansion, so that a high level of cell quality can be maintained during the cell preservation stage after the expansion is completed. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 This is a flow cytometry test result diagram of NK cells after amplification and culture in Example 2 of the present invention; Figure 2 It is a growth amplification multiple diagram of NK cells amplified and cultured in Examples 1 to 3 of the present invention and Comparative Examples 2 to 3; Figure 3 This is a toxicity test diagram of NK cells after expansion and culture in Example 2 of the present invention and Comparative Examples 2 to 3; Figure 4 This is a freezing and rehydration test diagram of NK cells after amplification and culture in Example 2 of the present invention and Comparative Example 1; Figure 5 It is a graph showing the results of IFN-γ release in the biological activity test of NK cells after expansion and culture in Example 2 of the present invention and Comparative Examples 2 to 3. DETAILED DESCRIPTION

[0015] The technical scheme of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. It should be noted that, unless otherwise specified, all raw materials involved in the present invention are purchased through conventional commercial channels, and OptiVitro T cell serum-free medium is purchased from Suzhou Yikesai Biotechnology Co., Ltd.

[0016] Example 1: A method for in vitro expansion and culture of NK cells, comprising the following steps: S1: Use a pipette to take 6 mL of PBS buffer and add it to a T75 culture flask, add 0.6 mL of 10 ug / mL CD16 to prepare CD16 working solution, tighten the bottle cap, slowly turn it upside down to allow 1 ug / mL CD16 to fully disperse at the bottom of the bottle; seal it with a sealing film, put it in a bag, and then place it at 4°C overnight; S2: Draw 50 mL of whole blood into a sterile sodium heparin blood collection tube and mix by inverting several times; S3: at room temperature, the whole blood treated in step S2 was centrifuged, the parameters were set to 1800 rpm / (757 g), 9 up and 7 down, and the time was 15 min. After the centrifugation, the upper plasma was transferred into a centrifuge tube with a pipette, and then placed in a water bath and inactivated at 56°C for 30 min. S4: placing the plasma inactivated in step S3 in a 4°C refrigerator for 20 min, then taking the plasma out of the refrigerator, centrifuging at 3000 rpm / (1057 g) for 5 min, taking the supernatant for later use, and obtaining pretreated plasma; S5: The blood cell precipitate in the supernatant of step S4 is resuspended and mixed with an equal volume of physiological saline; 5 mL of lymphocyte separation solution is placed in a centrifuge tube and 5 mL of the resuspended and mixed blood cells is slowly added, and then centrifuged at room temperature, with parameters set to 2000 rpm / (939 g), 9 for rise and 0 for fall, and time for 20 min; S6: After completing the centrifugation in step S5, aspirate the white film layer in the middle of the centrifuge solution, put the white film layer into a new centrifuge tube, resuspend and mix with physiological saline to 50 mL, centrifuge at 2000 rpm for 5 min, discard the supernatant, repeat the above resuspending, volume fixing and centrifugation once to obtain a cell pellet; S7: Mix RPMI-1640 medium and nutrient supplement solution in a volume ratio of 9:1 to prepare a complete culture solution; S8: adding the cell pellet prepared in step S6 to 9 mL of complete culture medium prepared in step S7, resuspending and mixing, and then adding 133 μL of 1 μg / mL OK432, 100 μL of 1 μg / mL IL-15 and 1 mL of pretreated plasma prepared in step S4 to obtain a cell suspension; S9: adding the cell suspension prepared in step S8 to the T75 culture flask prepared in step S1, and placing in an incubator at 37°C, 5% CO2 for culture; S10: First fluid replenishment operation: On the second day after completing the culture operation in step S9, take 9 mL of the complete culture medium prepared in step S7, add it to a 50 mL centrifuge tube, mix the pretreated plasma, OK432 and IL-15 prepared in step S4 in a ratio of 1.5 mL: 20 μL: 15 μL, and then slowly add it to the culture bottle to ensure that the final volume of the liquid in the culture bottle is 25 mL, and continue to culture in a 37°C, 5% CO2 incubator; S11: Second fluid replenishment operation. On the second day after the first fluid replenishment operation of step S10 is completed, the complete culture solution prepared in step S7 and the pretreated plasma prepared in step S4 are added to a 50 mL centrifuge tube in a ratio of 32 mL:3 mL, and then fully mixed. Then, the mixture is slowly added to the culture bottle to ensure that the final volume of the liquid in the culture bottle is 60 mL, and the culture is continued in a 37°C, 5% CO2 incubator; S12: The third rehydration operation, on the second day after the second rehydration operation of step S11, the cell suspension in the culture flask is transferred to a T175 culture flask, 10 mL of the pretreated plasma prepared in step S4 is added, and then the complete culture medium prepared in step S7 is added to ensure that the final volume of the liquid in the culture flask is 150 mL; S13: the fourth rehydration operation, on the second day after the third rehydration operation of step S12 is completed, the cell suspension in the culture flask is evenly distributed to two T225 culture flasks, 6 mL of the pretreated plasma prepared in step S4 is added to each culture flask, and then the complete culture medium prepared in step S7 is used to replenish the final volume of the liquid in each culture flask to 200 mL, a total of 400 mL; S14: Mix the nutrient supplement solution and OptiVitro T cell serum-free medium at a volume ratio of 0.8:9.2 to prepare a complete serum-free culture medium; S15: The fifth rehydration operation. On the second day after the fourth rehydration operation in step S13 is completed, the cell suspension in the culture flask is equally divided into four T225 culture flasks, recorded as T225-1, T225-2, T225-3 and T225-4, and the complete serum-free culture medium prepared in step S14 is added to each culture flask to ensure that the final volume of the liquid in each culture flask is 200 mL; S16: Sixth rehydration: On the second day after the fifth rehydration operation in step S15, disinfect the connection port of the culture bag and connect the connection port to the syringe port to prepare for bagging; take out T225-1 and T225-2 in step S15, tap the culture bottle to resuspend the cells, and then put them into the same culture bag, add the complete serum-free culture solution prepared in step S14, and rinse the culture bottle to ensure that the final volume of the culture bag liquid is 800 mL; after bagging, bag T225-3 and T225-4 in step S15 in the same way; S17: Seventh rehydration: On the second day after the sixth rehydration operation of step S16 is completed, the complete serum-free culture medium prepared in step S14 is added to each culture bag until the final volume of the liquid is 1050 mL, and the total volume of the two bags is 2100 mL; S18: On the second day after the seventh rehydration operation of step S17 is completed, the cells in the two culture bags are collected into two 500 mL centrifuge bottles, recorded as centrifuge bottle-1 and centrifuge bottle-2, 50 mL of normal saline is added to centrifuge bottle-1, and the cells are transferred to centrifuge bottle-2, and then normal saline is added to the final volume of the centrifuge bottle liquid to 500 mL, centrifuged at 2000 rpm for 8 minutes, the supernatant is discarded, and the centrifugation operation is repeated twice, and 10 mL of normal saline and 2 mL of nutrient preservation solution are added, mixed and bagged, and the temperature is gradually reduced at a rate of 1°C / min until it reaches -80°C, and frozen for storage; In step S7, the specific preparation steps of the nutrient supplement solution are as follows: A1: Dissolve sodium alginate in PBS buffer, heat in a water bath to control the temperature at 37°C, stir and dissolve evenly, and cool naturally to room temperature to obtain a 0.2wt% sodium alginate solution; dissolve hydroxypropyl methylcellulose in PBS buffer, continue to stir and dissolve evenly, and obtain a 0.1wt% hydroxypropyl methylcellulose solution; A2: Add 100 mL of the sodium alginate solution prepared in step A1 to 100 mL of hydroxypropyl methylcellulose solution, heat in a water bath to control the temperature at 35°C, add 1 mg of insulin, 6 mg of transferrin and 40 mg of selenoprotein, stir to dissolve evenly, then add 100 mg of glutamine and 8 mg of glutathione, stir to dissolve evenly, filter with a 0.22 μm filter membrane to obtain a nutrient supplement solution; In step S18, the specific steps of preparing the nutrient preservation solution are as follows: B1: 1 g of β-cyclodextrin was added to 20 mL of deionized water and stirred continuously until it was uniformly dissolved to obtain a β-cyclodextrin solution. 0.5 mL of dimethyl sulfoxide was added to the β-cyclodextrin solution. After 60 W ultrasonic pretreatment for 15 min, the solution was stirred continuously at 300 rpm at room temperature for 6 h, and then dialyzed in a 3 kDa dialysis bag for 48 h to obtain a β-cyclodextrin-DMSO inclusion complex. B2: The β-cyclodextrin-DMSO inclusion complex prepared in step B1 was evaporated and concentrated to 1 / 4 of the original volume at 35°C, dialyzed in a 2 kDa dialysis bag for 24 h, and then the temperature was controlled at 4°C in an ice bath. 50 ng of nicotinamide and 50 ng of NAD+ were added to the dialyzed liquid, stirred and dispersed evenly, and then 5000 U of gentamicin and 30 ng of IL-2 were added, stirred and dispersed evenly to obtain a nutrient preservation solution.

[0017] Example 2: A method for in vitro expansion and culture of NK cells, comprising the following steps: S1: Use a pipette to take 6 mL of PBS buffer and add it to a T75 culture flask, add 0.6 mL of 10 ug / mL CD16 to prepare CD16 working solution, tighten the bottle cap, slowly turn it upside down to allow 1 ug / mL CD16 to fully disperse at the bottom of the bottle; seal it with a sealing film, put it in a bag, and then place it at 4°C overnight; S2: Draw 50 mL of whole blood into a sterile sodium heparin blood collection tube and mix by inverting several times; S3: at room temperature, the whole blood treated in step S2 was centrifuged, the parameters were set to 1800 rpm / (757 g), 9 up and 7 down, and the time was 15 min. After the centrifugation, the upper plasma was transferred into a centrifuge tube with a pipette, and then placed in a water bath and inactivated at 56°C for 30 min. S4: placing the plasma inactivated in step S3 in a 4°C refrigerator for 28 minutes, then taking out the plasma from the refrigerator, centrifuging at 3000 rpm / (1057 g) for 5 minutes, taking the supernatant for later use, and obtaining pretreated plasma; S5: The blood cell precipitate in the supernatant of step S4 is resuspended and mixed with an equal volume of physiological saline; 5 mL of lymphocyte separation solution is placed in a centrifuge tube and 5 mL of the resuspended and mixed blood cells is slowly added, and then centrifuged at room temperature, with parameters set to 2000 rpm / (939 g), 9 for rise and 0 for fall, and time for 20 min; S6: After completing the centrifugation in step S5, aspirate the white film layer in the middle of the centrifuge solution, put the white film layer into a new centrifuge tube, resuspend and mix with physiological saline to 50 mL, centrifuge at 2000 rpm for 5 min, discard the supernatant, repeat the above resuspending, volume fixing and centrifugation once to obtain a cell pellet; S7: Mix RPMI-1640 medium and nutrient supplement solution in a volume ratio of 9:1 to prepare a complete culture solution; S8: adding the cell pellet prepared in step S6 to 9 mL of complete culture medium prepared in step S7, resuspending and mixing, and then adding 133 μL of 1 μg / mL OK432, 100 μL of 1 μg / mL IL-15 and 1 mL of pretreated plasma prepared in step S4 to obtain a cell suspension; S9: adding the cell suspension prepared in step S8 to the T75 culture flask prepared in step S1, and placing in an incubator at 37°C, 5% CO2 for culture; S10: First fluid replenishment operation: On the second day after completing the culture operation in step S9, take 9 mL of the complete culture medium prepared in step S7, add it to a 50 mL centrifuge tube, mix the pretreated plasma, OK432 and IL-15 prepared in step S4 in a ratio of 1.5 mL: 20 μL: 15 μL, and then slowly add it to the culture bottle to ensure that the final volume of the liquid in the culture bottle is 25 mL, and continue to culture in a 37°C, 5% CO2 incubator; S11: Second fluid replenishment operation: on the third day after the first fluid replenishment operation of step S10, the complete culture medium prepared in step S7 and the pretreated plasma prepared in step S4 are added to a 50 mL centrifuge tube in a ratio of 32 mL:3 mL, and then fully mixed. Then, the mixture is slowly added to the culture bottle to ensure that the final volume of the liquid in the culture bottle is 60 mL, and the culture is continued in a 37°C, 5% CO2 incubator; S12: The third rehydration operation, on the second day after the second rehydration operation of step S11, the cell suspension in the culture flask is transferred to a T175 culture flask, 10 mL of the pretreated plasma prepared in step S4 is added, and then the complete culture medium prepared in step S7 is added to ensure that the final volume of the liquid in the culture flask is 150 mL; S13: The fourth rehydration operation, on the second day after the third rehydration operation of step S12 is completed, the cell suspension in the culture flask is evenly distributed to two T225 culture flasks, 7 mL of the pretreated plasma prepared in step S4 is added to each culture flask, and then the complete culture medium prepared in step S7 is used to replenish the final volume of the liquid in each culture flask to 200 mL, a total of 400 mL; S14: Mix the nutrient supplement solution and OptiVitro T cell serum-free medium at a volume ratio of 0.8:9.2 to prepare a complete serum-free culture medium; S15: The fifth rehydration operation. On the second day after the fourth rehydration operation in step S13 is completed, the cell suspension in the culture flask is equally divided into four T225 culture flasks, recorded as T225-1, T225-2, T225-3 and T225-4, and the complete serum-free culture medium prepared in step S14 is added to each culture flask to ensure that the final volume of the liquid in each culture flask is 200 mL; S16: Sixth rehydration: On the second day after the fifth rehydration operation in step S15, disinfect the connection port of the culture bag and connect the connection port to the syringe port to prepare for bagging; take out T225-1 and T225-2 in step S15, tap the culture bottle to resuspend the cells, and then put them into the same culture bag, add the complete serum-free culture solution prepared in step S14, and rinse the culture bottle to ensure that the final volume of the culture bag liquid is 800 mL; after bagging, bag T225-3 and T225-4 in step S15 in the same way; S17: Seventh rehydration: On the second day after the sixth rehydration operation of step S16 is completed, the complete serum-free culture medium prepared in step S14 is added to each culture bag until the final volume of the liquid is 1050 mL, and the total volume of the two bags is 2100 mL; S18: On the second day after the seventh rehydration operation of step S17 is completed, the cells in the two culture bags are collected into two 500 mL centrifuge bottles, recorded as centrifuge bottle-1 and centrifuge bottle-2, 50 mL of normal saline is added to centrifuge bottle-1, and the cells are transferred to centrifuge bottle-2, and then normal saline is added to the final volume of the centrifuge bottle liquid to 500 mL, centrifuged at 2500 rpm for 8 minutes, the supernatant is discarded, and the centrifugation operation is repeated twice, and 15 mL of normal saline and 3 mL of nutrient preservation solution are added, mixed and bagged, and the temperature is gradually reduced at a rate of 1°C / min until it reaches -80°C, and frozen for storage; In step S7, the specific preparation steps of the nutrient supplement solution are as follows: A1: Dissolve sodium alginate in PBS buffer, heat in a water bath to control the temperature at 37°C, stir and dissolve evenly, and cool naturally to room temperature to obtain a 0.2wt% sodium alginate solution; dissolve hydroxypropyl methylcellulose in PBS buffer, continue to stir and dissolve evenly, and obtain a 0.1wt% hydroxypropyl methylcellulose solution; A2: Add 100 mL of the sodium alginate solution prepared in step A1 to 100 mL of hydroxypropyl methylcellulose solution, heat in a water bath to control the temperature at 35°C, add 1 mg of insulin, 10 mg of transferrin and 40 mg of selenoprotein, stir to dissolve evenly, then add 150 mg of glutamine and 8 mg of glutathione, stir to dissolve evenly, filter with a 0.22 μm filter membrane to obtain a nutrient supplement solution; In step S18, the specific steps of preparing the nutrient preservation solution are as follows: B1: 1 g of β-cyclodextrin was added to 20 mL of deionized water and stirred continuously until it was uniformly dissolved to obtain a β-cyclodextrin solution. 0.5 mL of dimethyl sulfoxide was added to the β-cyclodextrin solution. After 60 W ultrasonic pretreatment for 15 min, the solution was stirred continuously at 500 rpm for 4 h at room temperature, and then dialyzed in a 3 kDa dialysis bag for 48 h to obtain a β-cyclodextrin-DMSO inclusion complex. B2: The β-cyclodextrin-DMSO inclusion complex prepared in step B1 was evaporated and concentrated to 1 / 4 of the original volume at 35°C, dialyzed in a 2 kDa dialysis bag for 24 h, and then the temperature was controlled at 4°C in an ice bath. 250 ng nicotinamide and 250 ng NAD+ were added to the dialyzed liquid, stirred and dispersed evenly, and then 5000 U gentamicin and 30 ng IL-2 were added, stirred and dispersed evenly to obtain a nutrient preservation solution.

[0018] Example 3: A method for in vitro expansion and culture of NK cells, comprising the following steps: S1: Use a pipette to take 6 mL of PBS buffer and add it to a T75 culture flask, add 0.6 mL of 10 ug / mL CD16 to prepare CD16 working solution, tighten the bottle cap, slowly turn it upside down to allow 1 ug / mL CD16 to fully disperse at the bottom of the bottle; seal it with a sealing film, put it in a bag, and then place it at 4°C overnight; S2: Draw 50 mL of whole blood into a sterile sodium heparin blood collection tube and mix by inverting several times; S3: at room temperature, the whole blood treated in step S2 was centrifuged, the parameters were set to 1800 rpm / (757 g), 9 up and 7 down, and the time was 15 min. After the centrifugation, the upper plasma was transferred into a centrifuge tube with a pipette, and then placed in a water bath and inactivated at 56°C for 30 min. S4: placing the plasma inactivated in step S3 in a 4°C refrigerator for 30 minutes, then taking out the plasma from the refrigerator, centrifuging it at 3000 rpm / (1057 g) for 5 minutes, taking the supernatant for later use, and obtaining pretreated plasma; S5: The blood cell precipitate in the supernatant of step S4 is resuspended and mixed with an equal volume of physiological saline; 5 mL of lymphocyte separation solution is placed in a centrifuge tube and 5 mL of the resuspended and mixed blood cells is slowly added, and then centrifuged at room temperature, with parameters set to 2000 rpm / (939 g), 9 for rise and 0 for fall, and time for 20 min; S6: After completing the centrifugation in step S5, aspirate the white film layer in the middle of the centrifuge solution, put the white film layer into a new centrifuge tube, resuspend and mix with physiological saline to 50 mL, centrifuge at 2000 rpm for 5 min, discard the supernatant, repeat the above resuspending, volume fixing and centrifugation once to obtain a cell pellet; S7: Mix RPMI-1640 medium and nutrient supplement solution in a volume ratio of 9:1 to prepare a complete culture solution; S8: adding the cell pellet prepared in step S6 to 9 mL of complete culture medium prepared in step S7, resuspending and mixing, and then adding 133 μL of 1 μg / mL OK432, 100 μL of 1 μg / mL IL-15 and 1 mL of pretreated plasma prepared in step S4 to obtain a cell suspension; S9: adding the cell suspension prepared in step S8 to the T75 culture flask prepared in step S1, and placing in an incubator at 37°C, 5% CO2 for culture; S10: First fluid replenishment operation: On the second day after completing the culture operation in step S9, take 9 mL of the complete culture medium prepared in step S7, add it to a 50 mL centrifuge tube, mix the pretreated plasma, OK432 and IL-15 prepared in step S4 in a ratio of 1.5 mL: 20 μL: 15 μL, and then slowly add it to the culture bottle to ensure that the final volume of the liquid in the culture bottle is 25 mL, and continue to culture in a 37°C, 5% CO2 incubator; S11: Second fluid replenishment operation. On the second day after the first fluid replenishment operation of step S10 is completed, the complete culture solution prepared in step S7 and the pretreated plasma prepared in step S4 are added to a 50 mL centrifuge tube in a ratio of 32 mL:3 mL, and then fully mixed. Then, the mixture is slowly added to the culture bottle to ensure that the final volume of the liquid in the culture bottle is 60 mL, and the culture is continued in a 37°C, 5% CO2 incubator; S12: The third rehydration operation, on the second day after the second rehydration operation of step S11, the cell suspension in the culture flask is transferred to a T175 culture flask, 10 mL of the pretreated plasma prepared in step S4 is added, and then the complete culture medium prepared in step S7 is added to ensure that the final volume of the liquid in the culture flask is 150 mL; S13: The fourth rehydration operation, on the second day after the third rehydration operation of step S12 is completed, the cell suspension in the culture flask is evenly distributed to two T225 culture flasks, 6.5 mL of the pretreated plasma prepared in step S4 is added to each culture flask, and then the complete culture medium prepared in step S7 is used to replenish the final volume of the liquid in each culture flask to 200 mL, a total of 400 mL; S14: Mix the nutrient supplement solution and OptiVitro T cell serum-free medium at a volume ratio of 0.8:9.2 to prepare a complete serum-free culture medium; S15: The fifth rehydration operation. On the second day after the fourth rehydration operation in step S13 is completed, the cell suspension in the culture flask is equally divided into four T225 culture flasks, recorded as T225-1, T225-2, T225-3 and T225-4, and the complete serum-free culture medium prepared in step S14 is added to each culture flask to ensure that the final volume of the liquid in each culture flask is 200 mL; S16: Sixth rehydration: On the second day after the fifth rehydration operation in step S15, disinfect the connection port of the culture bag and connect the connection port to the syringe port to prepare for bagging; take out T225-1 and T225-2 in step S15, tap the culture bottle to resuspend the cells, and then put them into the same culture bag, add the complete serum-free culture solution prepared in step S14, and rinse the culture bottle to ensure that the final volume of the culture bag liquid is 800 mL; after bagging, bag T225-3 and T225-4 in step S15 in the same way; S17: Seventh rehydration: On the second day after the sixth rehydration operation of step S16 is completed, the complete serum-free culture medium prepared in step S14 is added to each culture bag until the final volume of the liquid is 1050 mL, and the total volume of the two bags is 2100 mL; S18: On the second day after the seventh rehydration operation of step S17 is completed, the cells in the two culture bags are collected into two 500 mL centrifuge bottles, recorded as centrifuge bottle-1 and centrifuge bottle-2, 50 mL of normal saline is added to centrifuge bottle-1, and the cells are transferred to centrifuge bottle-2, and then normal saline is added to the final volume of the centrifuge bottle liquid to 500 mL, centrifuged at 3000 rpm for 5 minutes, the supernatant is discarded, and the centrifugation operation is repeated twice, and then 20 mL of normal saline and 3 mL of nutrient preservation solution are added, mixed and bagged, and the temperature is gradually reduced at a rate of 1°C / min until it reaches -80°C, and frozen for storage; In step S7, the specific preparation steps of the nutrient supplement solution are as follows: A1: Dissolve sodium alginate in PBS buffer, heat in a water bath to control the temperature at 37°C, stir and dissolve evenly, and cool naturally to room temperature to obtain a 0.2wt% sodium alginate solution; dissolve hydroxypropyl methylcellulose in PBS buffer, continue to stir and dissolve evenly, and obtain a 0.1wt% hydroxypropyl methylcellulose solution; A2: Add 100 mL of the sodium alginate solution prepared in step A1 to 100 mL of hydroxypropyl methylcellulose solution, heat in a water bath to control the temperature at 35°C, add 1 mg of insulin, 10 mg of transferrin and 40 mg of selenoprotein, stir to dissolve evenly, then add 200 mg of glutamine and 8 mg of glutathione, stir to dissolve evenly, filter with a 0.22 μm filter membrane to obtain a nutrient supplement solution; In step S18, the specific steps of preparing the nutrient preservation solution are as follows: B1: Add 1 g of β-cyclodextrin to 20 mL of deionized water and continue stirring until it is uniformly dissolved to obtain a β-cyclodextrin solution. Add 0.5 mL of dimethyl sulfoxide to the β-cyclodextrin solution, pre-treat with 60 W ultrasound for 15 min, continue stirring at 400 rpm at room temperature for 5 h, and then dialyze in a 3 kDa dialysis bag for 48 h to obtain a β-cyclodextrin-DMSO inclusion complex. B2: The β-cyclodextrin-DMSO inclusion complex prepared in step B1 was evaporated and concentrated to 1 / 4 of the original volume at 35°C, dialyzed in a 2 kDa dialysis bag for 24 h, and then placed in an ice bath to control the temperature at 4°C. 150 ng of nicotinamide and 150 ng of NAD+ were added to the dialyzed liquid, stirred and dispersed evenly, and then 5000 U of gentamicin and 30 ng of IL-2 were added, stirred and dispersed evenly to obtain a nutrient preservation solution.

[0019] Comparative Example 1: The operation of Comparative Example 1 is basically the same as that of Example 2, except that conventional dimethyl sulfoxide protective solution is used to replace the nutrient preservation solution in Comparative Example 1.

[0020] Comparative Example 2: The operation of Comparative Example 2 is basically the same as that of Example 2, except that in Comparative Example 2, only RPMI-1640 culture medium is used for multiple fluid replenishment culture operations.

[0021] Comparative Example 3: The operation of Comparative Example 3 is basically the same as that of Example 2, except that in Comparative Example 3, the nutrient supplement solution is used for only one rehydration, and no subsequent multiple rehydration operations are performed. The specific operation steps are as follows: S1: Use a pipette to take 6 mL of PBS buffer and add it to a T75 culture flask, add 10 ug / mL of CD16, tighten the bottle cap, slowly turn it upside down to allow CD16 to fully disperse at the bottom of the bottle; seal it with a sealing film, put it in a bag, and then place it at 4°C overnight; S2: Draw 50 mL of whole blood into a sterile sodium heparin blood collection tube and mix by inverting several times; S3: at room temperature, centrifuge the whole blood treated in step S2. After centrifugation, transfer the upper plasma into a centrifuge tube using a pipette, and then put it into a water bath and inactivate it at 56°C for 30 minutes; S4: placing the plasma inactivated in step S3 in a 4°C refrigerator for 28 minutes, taking out the plasma from the refrigerator, centrifuging at 3000 rpm / (1057 g) for 5 minutes, taking the supernatant for later use, and obtaining pretreated plasma; S5: The blood cell precipitate in the supernatant of step S4 is resuspended and mixed with an equal volume of physiological saline; the lymphocyte separation solution is placed in a centrifuge tube and the resuspended and mixed blood cells are slowly added, and then centrifuged at room temperature; S6: After completing the centrifugation in step S5, aspirate the white film layer in the middle of the centrifuge solution, put the white film layer into a new centrifuge tube, resuspend and mix with physiological saline to 50 mL, centrifuge at 2000 rpm for 5 min, discard the supernatant, repeat the above resuspending, volume fixing and centrifugation once to obtain a cell pellet; S7: Mix RPMI-1640 medium and nutrient supplement solution in a volume ratio of 9:1 to prepare a complete culture solution; S8: adding the cell pellet prepared in step S6 to 9 mL of complete culture medium prepared in step S7, resuspending and mixing, and then adding 133 μL of OK432, 100 μL of IL-15 and 1 mL of pretreated plasma prepared in step S4 to obtain a cell suspension; S9: adding the cell suspension prepared in step S8 to the T75 culture flask prepared in step S1, and placing in an incubator at 37°C, 5% CO2 for culture; S10: On the second day after completing the culture operation in step S9, take 9 mL of the complete culture medium prepared in step S7, add it to a 50 mL centrifuge tube, and add 1.5 mL of the pretreated plasma prepared in step S4, 20 μL of OK432 and 15 μL of IL15, mix thoroughly according to the above dosage ratio, and slowly add it to the culture bottle to ensure that the final volume of the liquid in the culture bottle is 25 mL, and place it in a 37°C, 5% CO2 incubator for continued culture; S11: On the 7th day after completing the culture operation of step S10, collect the cells in the culture flask into a 500mL centrifuge bottle, add physiological saline to the final volume of the centrifuge bottle liquid to 500mL, centrifuge at 2500rpm for 8min, discard the supernatant, repeat the above centrifugation operation and wash twice, add 15mL of physiological saline and 3mL of nutrient preservation solution, mix and bag, gradually reduce the temperature at a rate of 1℃ / min until it reaches -80℃, and store in a frozen state.

[0022] Performance Test: Flow cytometry test: Anti-CD3-FITC, anti-CD16-PE, and anti-CD56-APC were used to perform flow cytometry on the NK cells amplified in Example 2. The results are as follows: Figure 1 shown.

[0023] Depend on Figure 1 The results show that the expansion and culture method of the present invention can achieve a high proportion of NK cell purity. In the flow cytometry test, 93.42% of the cells are CD3-CD16+CD56+ NK cells, showing a very high purity. This shows that the method shows excellent effect in the expansion and purification process of NK cells, and successfully obtains high-purity NK cell samples.

[0024] Growth and expansion test: The NK cells expanded in Examples 1 to 3 and Comparative Examples 2 to 3 were counted every three days using an automatic cell counter, i.e., on the 1st day, 4th day, 7th day, 10th day, 13th day and 16th day after the start of the expansion culture. It should be noted that since the culture time of Comparative Example 3 was only 9 days (less than 10 days), the expansion rate on the 9th day was additionally recorded, and then the expansion rate was calculated (expansion rate = number of cells at a specific time / number of starting cells). The expansion rate results are as follows: Figure 2 shown.

[0025] Depend on Figure 2 It can be seen from the results that the expansion method of the present invention shows an excellent expansion rate, especially in the middle and late stages of culture, thanks to the optimization of the multi-stage fluid replenishment operation, NK cells can continue to grow; by comparing the results of Comparative Example 2 and Example 2, it is found that in the late stage of culture (10 days later), the difference in the expansion rate between the two is significant. Comparative Example 2, which lacks nutrient supplement solution, is limited by the limitations of the culture medium components, resulting in significantly limited expansion effect; Comparison of Comparative Example 3 with Example 2 further shows that NK cells show a trend of stagnation or even decline in expansion in the late stage of culture, and the staged fluid replenishment operation has a significant positive effect on cell expansion.

[0026] Cytotoxicity test: K562 cells were adjusted to 1×10 6The density of NK cells after expansion and culture in Example 2 and Comparative Examples 2-3 was adjusted to 1×10 6 cells / mL, K562 cells were mixed with NK cells obtained by different amplification methods at a ratio of 5:1 and 1:1, and recorded as test groups 1, 2, and 3 in sequence; Example 2 (test group 1) and comparative examples 2-3 (test groups 2-3) were dispensed into 96-well plates, and 6 replicate wells were set for each test group; two control groups were set at the same time: a target cell spontaneous release control (containing only target cells, without NK cells) and a target cell maximum release control (after adding target cells, 1% Triton X-100 was added to achieve complete cell lysis); the 96-well plate was placed at 37°C, 5% Incubate in a CO2 incubator for 4 hours. After the incubation, take 100 μL of the supernatant and transfer it to a new 96-well plate. Use the LDH detection kit to add the detection reagent and measure the absorbance at a wavelength of 490 nm. Calculate the cytotoxicity rate of NK cells to target cells according to the formula: Cytotoxicity rate (%) = (LDH release in the experimental group - spontaneous release of target cells) / (maximum release of target cells - spontaneous release of target cells) × 100%. The results are shown in Figure 3 shown.

[0027] Depend on Figure 3 The results show that due to the use of a multi-stage rehydration operation, the cell growth environment is optimized, and the activity and functionality of NK cells during the expansion process are ensured. The NK cells of Example 2 can still show a high cytotoxicity rate of 78.64% at a ratio of 5:1; from the results of Comparative Example 2, it can be seen that the lack of the components in the nutrient supplement solution combined with the RPMI-1640 culture medium, the lack of stable nutrition and antioxidant protection, the proliferation and activity of NK cells are significantly affected, and at a ratio of 1:1, only 72.15% cytotoxicity rate is shown; from the results of Comparative Example 3, it can be seen that a one-time rehydration and a shorter culture time lead to insufficient nutrient supply and waste management, resulting in a decline in the cell state in the later stage of culture, and a significantly reduced toxicity rate. At a ratio of 1:1, only 61.46% cytotoxicity rate is shown.

[0028] Rehydration test: The NK cells after cryopreservation in Example 2 and Comparative Example 1 were taken out and rapidly thawed (rapidly thawed in a 37°C water bath to avoid ice crystal formation). After recovery, an equal amount of culture medium was added to dilute the cells, and the cryoprotectant, i.e., the nutrient preservation solution, was gradually removed. The survival rate of the recovered cells was detected by trypan blue staining, and the survival rate was recorded and compared with that of the control group (the cell survival rate of Example 2 before freezing). The results are as follows: Figure 4 shown.

[0029] Depend on Figure 4The results show that the nutrient preservation solution prepared by the present invention can effectively protect the cell activity after expansion culture, and the survival rate after thawing and recovery can reach 93.27%, which is maintained at a high level, slightly lower than the 97.82% survival rate of unfrozen cells; from the results of Comparative Example 2, it can be seen that due to the lack of oxidative stress protection during the thawing process and the lack of glutathione and IL-2, oxidative stress may be aggravated, cell activity recovery is slow, and the survival rate after thawing and recovery is significantly reduced to only 71.59%.

[0030] Cell activity test (IFN-γ release test): The reagents used were basically the same as those in the above cytotoxicity test; K562 cells were adjusted to 1×10 6 The density of NK cells after expansion and culture in Example 2 and Comparative Examples 2-3 was adjusted to 1×10 6 cells / mL, K562 cells were mixed with NK cells obtained by different amplification methods at a ratio of 5:1 and 1:1, and recorded as test groups 1, 2, and 3 in sequence; Example 2 (test group 1) and comparative examples 2 to 3 (test groups 2 to 3) were respectively divided into 96-well plates, and each test group was set up with 6 replicate wells. At the same time, two control groups were set up: a negative control (containing only NK cells, no target cells) and a positive control (adding NK cells and target cells and adding IL-2 stimulation), and the 96-well plate was placed in an incubator at 37°C and 5% CO2 for 4 hours. After the incubation, 100 μL of the supernatant was transferred to a new 96-well plate, and the detection antibody and substrate were added using the IFN-γ ELISA kit. The plates were incubated and washed according to the operating procedures in the manual. Finally, the absorbance was measured at a wavelength of 450 nm, and the activity of NK cells was evaluated based on the peak IFN-γ secretion amount. The results are shown in the figure. Figure 5 shown.

[0031] Depend on Figure 5 The results show that the expansion and culture method of the present invention can effectively achieve the expansion and activation of NK cells, and Example 2 shows an extremely high IFN-γ release level that is better than the positive control group; from the results of Comparative Example 2 and Example 2, it can be seen that the multi-stage fluid replenishment operation ensures the continuous supply of nutrients and dilution of waste in the culture medium in Example 2, while in Comparative Example 2, due to the lack of supplementation of nutrient supplement solution, the nutrients in the culture medium become insufficient during the culture process, resulting in the inhibition of NK cell proliferation and a significant impact on the secretion level of IFN-γ; from the results of Comparative Example 3 and Example 2, it can be seen that although Comparative Example 3 used nutrient supplement solution, only one fluid replenishment operation was performed, and it failed to continuously provide nutrients and dilute waste through multiple fluid replenishment operations as in Example 2, resulting in limited cell proliferation and significantly affecting the functional expression of NK cells.

[0032] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for in vitro expansion and culture of NK cells, characterized in that: The following steps are involved: S1: Use a pipette to take PBS buffer and add it to a T75 culture flask, add CD16 to prepare CD16 working solution, tighten the bottle cap, slowly turn it upside down to allow CD16 to fully disperse at the bottom of the bottle; seal it with a sealing film, put it in a bag, and then place it at 4°C overnight; S2: Draw whole blood into a sterile sodium heparin blood collection tube and mix by inverting several times; S3: at room temperature, centrifuge the whole blood treated in step S2. After centrifugation, transfer the upper plasma into a centrifuge tube using a pipette, and then place the tube in a water bath for inactivation. S4: placing the plasma inactivated in step S3 in a 4°C refrigerator for 20-30 minutes, then taking out the plasma from the refrigerator, centrifuging it, and taking the supernatant for later use to obtain pretreated plasma; S5: The blood cell precipitate in the supernatant of step S4 is resuspended and mixed with an equal volume of physiological saline; the lymphocyte separation solution is placed in a centrifuge tube and slowly added to the resuspended blood cells, and then centrifuged at room temperature; S6: After completing the centrifugation in step S5, aspirate the white film layer in the middle of the centrifuge solution, put the white film layer into a new centrifuge tube, resuspend and mix with physiological saline to a fixed volume, centrifuge, discard the supernatant, repeat the resuspending, fixing volume, and centrifugation operations once to obtain a cell pellet; S7: Mix RPMI-1640 medium and nutrient supplement solution in a volume ratio of 9:1 to prepare a complete culture solution; S8: adding the cell precipitate prepared in step S6 to the complete culture medium prepared in step S7, resuspending and mixing, and then adding OK432, IL-15 and the pretreated plasma prepared in step S4 to obtain a cell suspension; S9: adding the cell suspension prepared in step S8 to the T75 culture flask prepared in step S1, and placing in an incubator at 37°C, 5% CO2 for culture; S10: First fluid replenishment operation: On the second day after completing the culture operation in step S9, take the complete culture medium prepared in step S7, add it to a 50 mL centrifuge tube, fully mix OK432, IL-15 and the pretreated plasma prepared in step S4 according to the proportion, and slowly add it to the culture bottle to ensure that the final volume of the liquid in the culture bottle is 25 mL, and continue to culture in a 37°C, 5% CO2 incubator; S11: Second fluid replenishment operation: On the 2nd to 3rd day after the first fluid replenishment operation of step S10 is completed, the complete culture solution prepared in step S7 and the pretreated plasma prepared in step S4 are added to a 50 mL centrifuge tube in proportion and fully mixed, and then slowly added to the culture bottle to ensure that the final volume of the liquid in the culture bottle is 60 mL, and the culture is continued in a 37°C, 5% CO2 incubator; S12: The third rehydration operation, on the second day after the second rehydration operation of step S11, the cell suspension in the culture flask is transferred to a T175 culture flask, the pretreated plasma prepared in step S4 is added, and then the complete culture medium prepared in step S7 is added to ensure that the final volume of the liquid in the culture flask is 150 mL; S13: the fourth rehydration operation, on the second day after the third rehydration operation of step S12 is completed, the cell suspension in the culture flask is evenly distributed to two T225 culture flasks, the pretreated plasma prepared in step S4 is added to each culture flask, and then the complete culture medium prepared in step S7 is used to replenish the final volume of the liquid in each culture flask to 200 mL, a total of 400 mL; S14: Mix the nutrient supplement solution and OptiVitro T cell serum-free medium at a volume ratio of 0.8:9.2 to prepare a complete serum-free culture medium; S15: The fifth rehydration operation. On the second day after the fourth rehydration operation in step S13 is completed, the cell suspension in the culture flask is equally divided into four T225 culture flasks, recorded as T225-1, T225-2, T225-3 and T225-4, and the complete serum-free culture medium prepared in step S14 is added to each culture flask to ensure that the final volume of the liquid in each culture flask is 200 mL; S16: Sixth rehydration: On the second day after the fifth rehydration operation in step S15, disinfect the connection port of the culture bag and connect the connection port to the syringe port to prepare for bagging; take out T225-1 and T225-2 in step S15, tap the culture bottle to resuspend the cells, and then put them into the same culture bag, add the complete serum-free culture solution prepared in step S14, and rinse the culture bottle to ensure that the final volume of the culture bag liquid is 800 mL; after bagging, bag T225-3 and T225-4 in step S15 in the same way; S17: Seventh rehydration: On the second day after the sixth rehydration operation of step S16 is completed, the complete serum-free culture medium prepared in step S14 is added to each culture bag until the final volume of the liquid is 1050 mL, and the total volume of the two bags is 2100 mL; S18: On the 2nd to 3rd day after the seventh rehydration operation of step S17, the cells in the two culture bags were collected into two 500mL centrifuge bottles, recorded as centrifuge bottle-1 and centrifuge bottle-2, 50mL of normal saline was added to centrifuge bottle-1, and the cells were transferred to centrifuge bottle-2, and then normal saline was added to the final volume of the centrifuge bottle liquid to 500mL, the precipitate was collected by centrifugation, normal saline and nutrient preservation solution were added, the mixture was bagged, and the temperature was gradually reduced at a rate of 1°C / min until it reached -80°C, and then frozen for storage.

2. The in vitro expansion and culture method of NK cells according to claim 1, characterized in that: In step S1, the mass concentration of CD16 in the CD16 working solution is 1 μg / mL.

3. The in vitro expansion and culture method of NK cells according to claim 2, characterized in that: In step S3, the specific parameters of the centrifugal treatment are 1800 rpm, 9 up and 7 down, and time 15 min.

4. The in vitro expansion and culture method of NK cells according to claim 3, characterized in that: In step S5, the specific parameters of the centrifugal treatment are 2000 rpm, 9 up and 0 down, and time 20 min.

5. The in vitro expansion and culture method of NK cells according to claim 4, characterized in that: In step S6, the middle white film layer is the cell layer at the junction of the two liquid surfaces.

6. The in vitro expansion and culture method of NK cells according to claim 5, characterized in that: In step S7, the specific preparation steps of the nutrient supplement solution are as follows: A1: Dissolve sodium alginate in PBS buffer, heat in a water bath to control the temperature, stir and dissolve evenly, and cool naturally to room temperature to obtain a sodium alginate solution; dissolve hydroxypropyl methylcellulose in PBS buffer, continue to stir and dissolve evenly to obtain a hydroxypropyl methylcellulose solution; A2: Mix equal volumes of the sodium alginate solution and hydroxypropyl methylcellulose solution prepared in step A1, heat in a water bath to control the temperature, add insulin, transferrin and selenoprotein, stir to dissolve evenly, then add glutamine and glutathione, stir to dissolve evenly, filter with a filter membrane to obtain a nutrient supplement solution; In step A2, the amount of insulin added is 5 μg / mL; the amount of transferrin added is 30-50 μg / mL; the amount of selenoprotein added is 0.2 mg / mL; the amount of glutamine added is 0.5-1 mg / mL; and the amount of glutathione added is 0.04 mg / mL.

7. The in vitro expansion and culture method of NK cells according to claim 6, characterized in that: The concentrations of OK432 and IL-15 added in step S8 are both 1 μg / mL; the concentrations of OK432 and IL-15 added in step S10 are both 1 μg / mL.

8. The in vitro expansion and culture method of NK cells according to claim 7, characterized in that: The collection operation in step S18 is to centrifuge at 2000-3000 rpm for 5-8 minutes, discard the supernatant, and repeat the centrifugation operation twice.

9. The in vitro expansion and culture method of NK cells according to claim 8, characterized in that: The specific steps for preparing the nutrient preservation solution in step S18 are as follows: B1: Add β-cyclodextrin to deionized water and continue stirring until it is uniformly dissolved to obtain a β-cyclodextrin solution. Add dimethyl sulfoxide to the β-cyclodextrin solution, and continue stirring at room temperature after ultrasonic pretreatment. Then, dialyze in a dialysis bag to obtain a β-cyclodextrin-DMSO inclusion complex. B2: The β-cyclodextrin-DMSO inclusion complex prepared in step B1 is evaporated and concentrated at 35°C, dialyzed in a dialysis bag, and then the temperature is controlled by ice bath, nicotinamide and NAD+ are added to the dialyzed liquid, stirred and dispersed evenly, and then IL-2 and gentamicin are added, stirred and dispersed evenly, to obtain a nutrient preservation solution; In step B1, the ratio of dimethyl sulfoxide to β-cyclodextrin is 1 mL:2 g; in step B2, the amount of nicotinamide added is 10-50 ng / mL, the amount of NAD+ added is 10-50 ng / mL, the amount of gentamicin added is 1000 U / mL, and the amount of IL-2 added is 6 ng / mL.

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