High-efficiency and low-toxicity natural killer cell cryopreservation method

Abstract

The invention relates to an efficient and low-toxicity natural killer cell cryopreservation method. The high-efficiency and low-toxicity natural killer cell cryopreservation method comprises the following steps: resuspending NK cells by using a cell cryopreservation solution, and performing cryopreservation on the resuspended NK cells, the cryopreserved NK cells are unfrozen and recovered, cell cryopreservation liquid is removed through centrifugation, and then a fresh culture medium containing plasma membrane repair molecules is added to culture the recovered NK cells. The used cell cryopreservation liquid has the characteristic of low toxicity, and the cytotoxicity of the cell cryopreservation liquid is obviously lower than that of a DMSO group under the condition of 4 DEG C or 37 DEG C. Starting from an ice crystal inhibition strategy and a cell membrane damage repair strategy, efficient cryopreservation and recovery of the NK cells are realized by using a cell cryopreservation solution containing various types of ice inhibition molecules and a fresh culture medium containing plasma membrane repair molecules.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for cryopreserving natural killer cells with high efficiency and low toxicity. Background Art

[0002] Immune cell therapy is a new cancer therapy following traditional therapies such as surgical resection and chemotherapy, and has broad application prospects in the treatment of various cancer diseases. Among them, natural killer cells (NK cells) have the advantages of wide sources, can independently complete the process of recognizing and killing tumor cells, and do not induce graft-versus-host disease, etc., and are a research hotspot in the current field of immune cell therapy. The long-term cell preservation technology can provide sufficient and quality-stable NK cell resources for the preparation of NK cell therapy products, which is crucial for supporting its clinical transformation.

[0003] Ultra-low temperature cryopreservation is a common means for long-term cell preservation. However, during the cryopreservation process, water inside and outside the cells will inevitably form ice crystals, causing mechanical damage, osmotic damage and oxidative damage to cell membranes and organelles, etc., ultimately affecting the survival and therapeutic efficacy of NK cells. Currently, dimethyl sulfoxide (DMSO) is the most commonly used cryoprotectant for NK cells, which can effectively reduce the damage caused by ice crystals to cells. Although cryopreserving NK cells with DMSO (5-20 wt%) can achieve a relatively high cryopreservation effect, a large number of studies have shown that the migration ability, tumor necrosis factor, related apoptosis-inducing ligand and interferon-γ expression levels of NK cells cryopreserved with DMSO decrease. In addition, DMSO is toxic and difficult to completely remove from cells. A number of research reports have shown that cell therapy products cryopreserved with DMSO will cause various adverse reactions after being infused into the human body, including cardiovascular diseases, renal failure, vomiting, etc. Recently, researchers have developed DMSO-free new NK cell cryopreservation technologies using nanoparticle delivery of trehalose or low-toxic cryoprotectants such as poly-L-lysine, dextran and ectoine, but currently all have the problem of low cryopreservation efficiency, which limits the subsequent therapeutic efficacy of NK cells. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for cryopreserving natural killer cells with high efficiency and low toxicity to solve the technical problems of low cryopreservation efficiency of the existing NK cell cryopreservation technology and the toxicity of the cryoprotectant DMSO used.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A method for cryopreserving natural killer cells with high efficiency and low toxicity, comprising the following steps:

[0007] S1: Resuspend NK cells with a cell cryopreservation solution and cryopreserve the resuspended NK cells.

[0008] S2: Thaw and recover the cryopreserved NK cells, centrifuge to remove the cell cryopreservation solution, and then add a fresh medium containing a plasma membrane repair molecule to culture the recovered NK cells.

[0009] The cell cryopreservation solution includes polymers, organic permeable small molecules, and sugars.

[0010] Furthermore, the plasma membrane repair molecule is one or more of poloxamer, carnosine, polyene phosphatidylcholine, MG53 protein, recombinant human MG53 protein, 16α-methyl-9,11-dehydroprednisolone, SNARE protein, S-100A11 protein, synaptotagmin, and ESCRT complex, and the concentration of the plasma membrane repair molecule is 0 - 500 μM.

[0011] Furthermore, the addition method of the fresh medium containing a plasma membrane repair molecule is: add it within 0 - 72 h after thawing and recovering the cryopreserved NK cells.

[0012] Furthermore, the cryopreservation density of the NK cells is 1×10 6 ~100×10 6 cells / mL.

[0013] Furthermore, the polymer is one or more of polyvinylpyrrolidone, dextran, polyvinyl alcohol, polyethylene glycol, polyproline, polyacrylamide, hyaluronic acid, hydroxyethyl starch, and sodium carboxymethylcellulose, and the polymer accounts for 0.5 - 10 w / v% of the cell cryopreservation solution; the organic permeable small molecule is one or more of glycerol, ethylene glycol, propylene glycol, glycerol, and urea, and the organic permeable small molecule accounts for 0.5 - 10 w / v% of the cell cryopreservation solution; the sugars include one or more of glucose, sorbose, trehalose, sucrose, fructose, xylitol, mannose, and lactose, and the sugars account for 0.5 - 10 w / v% of the cell cryopreservation solution.

[0014] Furthermore, the cell cryopreservation solution further includes a medium suitable for NK cells.

[0015] Furthermore, the components of the medium suitable for NK cells include α-MEM, inositol with a final concentration of 0.18 - 0.22 mM, β-mercaptoethanol with a final concentration of 0.09 - 0.11 mM, folic acid with a final concentration of 0.18 - 0.2 mM, recombinant human interleukin-2 with a final concentration of 100 - 100 U / mL, horse serum with a final concentration of 10 - 13%, fetal bovine serum with a final concentration of 10 - 13%, and double antibody with a final concentration of 1 - 2%.

[0016] Furthermore, for the cryopreservation, the temperature is first decreased to -75 to -80°C, stored at -75 to -80°C for 0 to 24 hours, and then stored in liquid nitrogen or a liquid nitrogen atmosphere; the rate of temperature decrease is -1 to 10°C / min.

[0017] Furthermore, for the resuscitation, the cryopreserved NK cells are thawed under the condition of a 37 to 45°C water bath.

[0018] Furthermore, before adding the fresh culture medium containing the plasma membrane repair molecule in S2, the centrifuged NK cells need to be resuspended.

[0019] Advantages of the present invention:

[0020] Starting from the strategies of inhibiting ice crystals and repairing cell membrane damage, the present invention uses a cell cryopreservation solution containing various types of ice-inhibiting molecules and a fresh culture medium containing plasma membrane repair molecules to achieve efficient cryopreservation and resuscitation of NK cells.

[0021] The present invention cryopreserves NK cells using a cell cryopreservation solution with good ice-inhibiting function, reducing the damage of NK cells during cryopreservation. The survival rate of NK cells after thawing is 83%, showing no significant difference compared with the survival rate of 82% in Comparative Example 2 using 10 wt% DMSO for cryopreservation. After thawing, the cell cryopreservation solution is removed by centrifugation, and then a fresh culture medium containing plasma membrane repair molecules is added for resuscitation culture to repair the cryopreservation damage of NK cells and further improve the activity and function of NK cells after resuscitation. The present invention improves the activity and function of NK cells after resuscitation by using a cell cryopreservation solution with good ice-inhibiting function for cryopreservation and a fresh culture medium containing plasma membrane repair molecules for resuscitation when cryopreserved NK cells are resuscitated.

[0022] From Figure 3 and Figure 4 it can be seen that the NK cells in Comparative Example 1 died after 12 hours of resuscitation, while adding a fresh culture medium containing plasma membrane repair molecules can significantly alleviate the phenomenon of delayed cell death, and the survival rate is comparable to that of Comparative Example 2. When the effector-to-target ratio is 1:1, the killing ability of the NK cells after resuscitation of the present invention against K562 cells shows no significant difference compared with that of Comparative Example 2. When the effector-to-target ratios are 5:1 and 10:1, the killing ability of the NK cells after resuscitation of the present invention against K562 cells is significantly better than that of Comparative Example 2. In addition, under normal culture (37°C) and low temperature (4°C) conditions, the survival rate of the NK cells in Comparative Example 2 significantly decreases with the prolongation of the incubation time. However, with the prolongation of the incubation time, the survival rate of the NK cells in the cell cryopreservation solution and the fresh culture medium containing plasma membrane repair molecules of the present invention is better than that of Comparative Example 2. The cell cryopreservation solution and the fresh culture medium containing plasma membrane repair molecules of the present invention have no obvious toxicity to NK cells, and the survival rates are all significantly better than that of Comparative Example 2. Description of the Drawings

[0023] Figure 1 Orthogonal experiment optimization of the content of polymers, organic penetrating small molecules and sugars in the cell cryopreservation solution. Among them, A is the content diagram of polymers, organic penetrating small molecules and sugars in Comparative Example 1 and Examples 4-11, and B is the cell survival rate diagram of NK cells after cryopreservation with the cell cryopreservation solution of Comparative Example 1 and Examples 4-11; **, p < 0.01 (n = 3);

[0024] Figure 2 Survival rate diagram of NK cells with different concentrations of plasma membrane repair molecules after cryopreservation with the cell cryopreservation solution of Example 1. Among them, A is the survival rate diagram of NK cells after resuscitation with different concentrations of plasma membrane repair molecules, and B is the growth state diagram of NK cells after resuscitation with different concentrations of plasma membrane repair molecules;

[0025] Figure 3 Survival rate diagram of NK cells after cryopreservation and resuscitation in fresh medium, Example 1, and Comparative Examples 1-2. Among them, A is the survival rate diagram of NK cells, and B is the growth state diagram of NK cells; *, p < 0.05 (n = 3), **, p < 0.01 (n = 3), ***, p < 0.001 (n = 3);

[0026] Figure 4 Killing ability diagram of NK cells against K562 cells 24 hours after cryopreservation, resuscitation in fresh medium, Example 1, and Comparative Example 2. Among them, A is the flow cytometry diagram, and B is the quantitative flow cytometry diagram;

[0027] Figure 5 Survival rate diagram of NK cells after co-incubation with fresh medium, DMSO, E3P2G2, and P188 at 37°C for different times; *, p < 0.05 (n = 3), **, p < 0.01 (n = 3), ***, p < 0.001 (n = 3);

[0028] Figure 6 Survival rate diagram of NK cells after co-incubation with fresh medium, DMSO, E3P2G2, and P188 at 4°C for different times; *, p < 0.05 (n = 3), **, p < 0.01 (n = 3), ***, p < 0.001 (n = 3). Detailed implementation mode

[0029] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0030] The components of the medium applicable to NK cells include α-MEM, inositol with a final concentration of 0.2 mM, β-mercaptoethanol with a final concentration of 0.1 mM, folic acid with a final concentration of 0.02 mM, recombinant human interleukin-2 (recombinant IL-2) with a final concentration of 200 U / mL, horse serum (HS) with a final concentration of 12.5%, fetal bovine serum (FBS) with a final concentration of 12.5%, and penicillin / streptomycin (P / S) with a final concentration of 1.0%.

[0031] Example 1

[0032] The method for cryopreserving highly efficient and low-toxic natural killer cells in Example 1 includes the following steps:

[0033] S1: Take the NK-92 cell suspension, centrifuge to remove the supernatant, resuspend it with the cell cryopreservation solution, and save the NK-92 cells at a cooling rate of -1 °C / min to -80 °C in the manner of a cell concentration of 1×10 6 cells / mL and 1 mL per tube. The NK-92 cells are stored in liquid nitrogen for 3 to 7 days.

[0034] S2: Thaw and recover the cryopreserved NK cells in a 37 °C water bath until all ice crystals disappear. Then add 9 mL of fresh medium, centrifuge, and remove the supernatant. Resuspend the cell pellet with the fresh medium specifically for NK-92 cells, and add the fresh medium containing 200 μM of poloxamer (P188) within 0 h after the NK cells are recovered, and continue to culture the NK-92 cells for 7 days. Take the NK cell suspension at 0 to 72 h after recovery, use trypan blue staining to detect the survival rate of NK-92 cells, and take a picture of the growth state of NK cells under bright field.

[0035] The cell cryopreservation solution includes the medium applicable to NK cell culture, ethylene glycol, glucose, and polyvinylpyrrolidone.

[0036] Among them, ethylene glycol accounts for 3 w / v% of the cell cryopreservation solution, polyvinylpyrrolidone (24000M W ) accounts for 2 w / v% of the cell cryopreservation solution, and glucose (180.16M W ) accounts for 2 w / v% of the cell cryopreservation solution. The cell cryopreservation solution in Example 1 is named E3P2G2.

[0037] Example 2

[0038] The method for cryopreserving highly efficient and low-toxic natural killer cells in Example 2 is substantially the same as that in Example 1. The difference between the method for cryopreserving highly efficient and low-toxic natural killer cells in Example 2 and that in Example 1 is that the concentration of P188 in Example 2 is 250 μM.

[0039] Example 3

[0040] The efficient and low-toxic natural killer cell cryopreservation method of Example 3 is substantially the same as that of Example 1. The difference between the efficient and low-toxic natural killer cell cryopreservation method of Example 3 and that of Example 1 is that the concentration of P188 in Example 3 is 150 μM.

[0041] Example 4

[0042] The efficient and low-toxic natural killer cell cryopreservation method of Example 4 is substantially the same as that of Example 1. The difference between the efficient and low-toxic natural killer cell cryopreservation method of Example 4 and that of Example 1 is that ethylene glycol accounts for 1 w / v% of the cell cryopreservation solution, polyvinylpyrrolidone accounts for 1 w / v% of the cell cryopreservation solution, and glucose accounts for 1 w / v% of the cell cryopreservation solution. The cell cryopreservation solution of Example 4 is named E1P1G1.

[0043] Example 5

[0044] The efficient and low-toxic natural killer cell cryopreservation method of Example 5 is substantially the same as that of Example 1. The difference between the efficient and low-toxic natural killer cell cryopreservation method of Example 5 and that of Example 1 is that ethylene glycol accounts for 1 w / v% of the cell cryopreservation solution, polyvinylpyrrolidone accounts for 2 w / v% of the cell cryopreservation solution, and glucose accounts for 3 w / v% of the cell cryopreservation solution. The cell cryopreservation solution of Example 5 is named E1P2G3.

[0045] Example 6

[0046] The efficient and low-toxic natural killer cell cryopreservation method of Example 6 is substantially the same as that of Example 1. The difference between the efficient and low-toxic natural killer cell cryopreservation method of Example 6 and that of Example 1 is that ethylene glycol accounts for 1 w / v% of the cell cryopreservation solution, polyvinylpyrrolidone accounts for 3 w / v% of the cell cryopreservation solution, and glucose accounts for 2 w / v% of the cell cryopreservation solution. The cell cryopreservation solution of Example 6 is named E1P3G2.

[0047] Example 7

[0048] The efficient and low-toxic natural killer cell cryopreservation method of Example 7 is substantially the same as that of Example 1. The difference between the efficient and low-toxic natural killer cell cryopreservation method of Example 7 and that of Example 1 is that ethylene glycol accounts for 3 w / v% of the cell cryopreservation solution, polyvinylpyrrolidone accounts for 1 w / v% of the cell cryopreservation solution, and glucose accounts for 3 w / v% of the cell cryopreservation solution. The cell cryopreservation solution of Example 7 is named E3P1G3.

[0049] Example 8

[0050] The highly efficient and low-toxic natural killer cell cryopreservation method of Example 8 is substantially the same as that of Example 1. The difference between the highly efficient and low-toxic natural killer cell cryopreservation method of Example 8 and Example 1 is that ethylene glycol accounts for 3 w / v% of the cell cryopreservation solution, polyvinylpyrrolidone accounts for 3 w / v% of the cell cryopreservation solution, and glucose accounts for 1 w / v% of the cell cryopreservation solution. The cell cryopreservation solution of Example 8 is named E3P3G1.

[0051] Example 9

[0052] The highly efficient and low-toxic natural killer cell cryopreservation method of Example 9 is substantially the same as that of Example 1. The difference between the highly efficient and low-toxic natural killer cell cryopreservation method of Example 9 and Example 1 is that in the cell cryopreservation solution, ethylene glycol (5 w / v%), polyvinylpyrrolidone (1 w / v%), and glucose (2 w / v%). The cell cryopreservation solution of Example 9 is named E5P1G2.

[0053] Example 10

[0054] The highly efficient and low-toxic natural killer cell cryopreservation method of Example 10 is substantially the same as that of Example 1. The difference between the highly efficient and low-toxic natural killer cell cryopreservation method of Example 10 and Example 1 is that ethylene glycol accounts for 5 w / v% of the cell cryopreservation solution, polyvinylpyrrolidone accounts for 2 w / v% of the cell cryopreservation solution, and glucose accounts for 1 w / v% of the cell cryopreservation solution. The cell cryopreservation solution of Example 10 is named E5P2G1.

[0055] Example 11

[0056] The highly efficient and low-toxic natural killer cell cryopreservation method of Example 11 is substantially the same as that of Example 1. The difference between the highly efficient and low-toxic natural killer cell cryopreservation method of Example 11 and Example 1 is that ethylene glycol accounts for 5 w / v% of the cell cryopreservation solution, polyvinylpyrrolidone accounts for 3 w / v% of the cell cryopreservation solution, and glucose accounts for 3 w / v% of the cell cryopreservation solution. The cell cryopreservation solution of Example 11 is named E5P3G3.

[0057] Comparative Example 1

[0058] The highly efficient and low-toxic natural killer cell cryopreservation method of Comparative Example 1 is substantially the same as that of Example 1. The difference between the highly efficient and low-toxic natural killer cell cryopreservation method of Comparative Example 1 and Example 1 is that no plasma membrane repair molecule was added in Comparative Example 1.

[0059] Comparative Example 2

[0060] 10 wt% DMSO: Centrifuge the NK-92 cell suspension to remove the supernatant, and resuspend the cell pellet with 10 wt% DMSO prepared with fresh medium at a cell concentration of 1×10 6NK-92 cells were cryopreserved by the slow freezing method at a cell concentration of 1×10

[0061] From Figure 1 It can be seen that the survival rate of NK cells after thawing in Example 1 was 83%, which was not significantly different from that of 10 wt% DMSO (82%) in Comparative Example 2. From Figure 2 It can be seen that 12 h after resuscitation, the NK cells in Comparative Example 1 showed delayed death, while Examples 1-3 could significantly alleviate the phenomenon of delayed death of NK cells.

[0062] NK cell suspension was taken at 0-48 h after resuscitation, and trypan blue staining was used to detect the survival rate of NK-92 cells, and the growth state map of NK-92 cells under bright field was photographed. From Figure 3 It can be seen that after resuscitation, the fresh medium group supplemented with plasma membrane repair molecules in Example 1 could significantly inhibit the phenomenon of delayed cell death, the cell survival rate was significantly increased, and its survival rate and cell growth state were comparable to those in Comparative Example 2.

[0063] Experimental Example 1

[0064] The supernatant of the NK-92 cell suspension was removed by centrifugation. The cell cryopreservation solution of Example 1 and the fresh medium containing plasma membrane repair molecules were used to cryopreserve NK-92 cells at a cell concentration of 1×10 6 cells / mL and 1 mL per tube by the slow freezing method, including first cooling to -80°C at -1°C / min and then storing in liquid nitrogen. After water bath resuscitation of each group of NK-92 cells, 9 mL of fresh medium was added, the supernatant was removed by centrifugation, and then fresh medium was added, and the cells were continuously cultured in a suspension cell culture flask for 24 h. The NK-92 cell suspensions of each group were collected, centrifuged and the supernatant was removed, and then fresh medium was added to adjust the cell density to 2×10 6 cells / mL, 1×10 6 cells / mL and 2×10 5 cells / mL. The K562 cell suspension was collected, the supernatant was removed by centrifugation, resuspended with 1 mL of CFDASE labeling solution (1×), and then 1 μL of CFDA SE (2000X) was added to 1 mL of labeling solution, gently mixed, placed in an incubator at 37°C, and incubated in the dark for 30 min. Subsequently, the supernatant was removed by centrifugation, 2 mL of PBS (calcium- and magnesium-free) solution was added, the supernatant was removed after centrifugation, and this operation was repeated once, and then resuspended with 1640 medium (10% FBS + 1% double antibody) to adjust the cell density to 2×105 cells / mL. 100 μL of NK-92 cells and K562 cells were respectively taken and inoculated into a U-shaped 96-well plate with effector cells (NK-92 cells): target cells (K562 cells) = 0:1, 1:1, 5:1 and 10:1, and co-incubated in an incubator at 37 °C for 4 h. After co-incubation for 4 h, the cell suspension was pipetted and transferred to a centrifuge tube. The supernatant was removed by centrifugation, 50 μL of Assays Buffer (1×) and 5 μL of 7-AAD staining solution were added to resuspend the cells. After pipetting and mixing evenly, the cells were incubated at room temperature in the dark for 15 min. Then 450 μL of Assays Buffer (1×) was added to suspend the cells, and the cells were detected using a flow cytometer. The results are as Figure 4 shown. When the effector-to-target ratio was 1:1, the killing ability of the NK cells after resuscitation of the present invention against K562 cells was not significantly different from that of Comparative Example 2. When the effector-to-target ratios were 5:1 and 10:1, the killing ability of the NK cells after resuscitation of the present invention against K562 cells was significantly better than that of Comparative Example 2.

[0065] Killing rate (%) = Percentage of dead K562 cells co-incubated with NK-92 - Percentage of natural death of K562 cells.

[0066] Experimental Example 3

[0067] Take the NK-92 cell suspension, centrifuge to remove the supernatant, and respectively add cryopreservation reagents (10 wt% DMSO, E3P2G2, P188) prepared with fresh medium (Control), and adjust the cell density to 2×10 5 cells / mL, spread on a 24-well plate (200 μL per well), and placed in a refrigerator at 4 °C or an incubator at 37 °C respectively. Samples were taken at different culture times (0, 10, 30, 60 min), and the cell viability was detected by trypan blue staining. The results are as Figure 5 shown. Under the condition of 37 °C, compared with the fresh medium group, the viability of NK cells in the DMSO group at different incubation time points was significantly reduced, indicating that the presence of DMSO would cause toxic damage to NK cells. The viability of NK cells in the E3P2G2 and P188 groups was not significantly different from that in the fresh medium group, indicating that E3P2G2 and P188 have no obvious toxicity to cells.

[0068] Figure 6As shown, at 4°C, the cell viability of the fresh medium group gradually decreased with the prolongation of the incubation time, indicating that the cells were damaged by low temperature. Compared with the fresh medium group, the cell viability of the DMSO group decreased significantly, indicating that the cells were still damaged by the toxicity of DMSO under low temperature conditions. There was no significant difference in the viability of NK cells in the E3P2G2 group and the fresh medium group, indicating that E3P2G2 had no obvious toxicity to the cells. Compared with the fresh medium group, the viability of NK cells in the P188 group increased significantly, indicating that P188 could reduce low temperature damage and had a beneficial effect on the cells.

Claims

1. An efficient and low-toxic cryopreservation method for natural killer cells, characterized in that, It includes the following steps: S1: Resuspend NK cells with a cell cryopreservation solution and cryopreserve the resuspended NK cells; S2: Thaw and recover the cryopreserved NK cells, centrifuge to remove the cell cryopreservation solution, and then add fresh medium containing a plasma membrane repair molecule to culture the recovered NK cells; The cell cryopreservation solution includes polymers, organic permeable small molecules, and sugars.

2. The cryopreservation method for natural killer cells with high efficiency and low toxicity according to claim 1, wherein The plasma membrane repair molecule is one or more of poloxamer, carnosine, polyene phosphatidylcholine, MG53 protein, recombinant human MG53 protein, 16α-methyl-9,11-dehydroprednisolone, SNARE protein, S-100A11 protein, synaptotagmin, and ESCRT complex, and the concentration of the plasma membrane repair molecule is 0 - 500 μM.

3. The method for cryopreserving natural killer cells with high efficiency and low toxicity according to claim 1, characterized in that, The addition method of the fresh medium containing a plasma membrane repair molecule is: add it within 0 - 72 h after thawing and recovering the cryopreserved NK cells.

4. The method for cryopreserving natural killer cells with high efficiency and low toxicity according to claim 1, characterized in that, The cryopreservation density of the NK cells is 1×10 6 ~100×10 6 cells / mL.

5. The cryopreservation method for natural killer cells with high efficiency and low toxicity according to claim 1, characterized in that, The polymer is one or more of polyvinylpyrrolidone, dextran, polyvinyl alcohol, polyethylene glycol, polyproline, polyacrylamide, hyaluronic acid, hydroxyethyl starch, and sodium carboxymethylcellulose, and the polymer accounts for 0.5 - 10 w / v% of the cell cryopreservation solution; the organic permeable small molecule is one or more of glycerol, ethylene glycol, propylene glycol, glycerol, and urea, and the organic permeable small molecule accounts for 0.5 - 10 w / v% of the cell cryopreservation solution; the sugars include one or more of glucose, sorbose, trehalose, sucrose, fructose, xylitol, mannose, and lactose, and the sugars account for 0.5 - 10 w / v% of the cell cryopreservation solution.

6. The method for cryopreserving natural killer cells with high efficiency and low toxicity according to claim 1 or 5, characterized in that The cell cryopreservation solution further includes a medium suitable for NK cells.

7. The method for cryopreserving natural killer cells with high efficiency and low toxicity according to claim 6, characterized in that, The components of the medium suitable for NK cells include α-MEM, inositol with a final concentration of 0.18 - 0.22 mM, β-mercaptoethanol with a final concentration of 0.09 - 0.11 mM, folic acid with a final concentration of 0.018 - 0.02 mM, recombinant human interleukin-2 with a final concentration of 100 - 200 U / mL, horse serum with a final concentration of 10 - 13%, fetal bovine serum with a final concentration of 12 - 13%, and double antibody with a final concentration of 1 - 2%.

8. The highly efficient and low-toxic natural killer cell cryopreservation method according to claim 1, wherein The cryopreservation is to first cool down to -75 - 80 °C, store at -75 - 80 °C for 0 - 24 h, and then store in liquid nitrogen or a liquid nitrogen atmosphere; The cooling rate is -1 - 10 °C / min.

9. The high-efficiency and low-toxic natural killer cell cryopreservation method according to claim 1, characterized in that, The recovery is to thaw the cryopreserved NK cells under the water bath condition of 37 - 45 °C.

10. The method for cryopreserving natural killer cells with high efficiency and low toxicity according to claim 1, wherein Before adding the fresh medium containing a plasma membrane repair molecule in S2, it is necessary to resuspend the centrifuged NK cells.

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