NK cell cryopreservation liquid, NK cell cryopreservation method, medicine, combined medicine and application of NK cell cryopreservation liquid and NK cell cryopreservation method

Abstract

The invention provides an NK cell cryopreservation solution, an NK cell cryopreservation method, a drug, a combined drug and application of the NK cell cryopreservation solution and the NK cell cryopreservation method. The NK cell cryopreservation solution comprises a basic solution and human serum albumin, and the basic solution comprises methyl cellulose and dimethyl sulfoxide. According to the NK cell cryopreservation liquid, the activity recovery rate of the cryopreserved NK cells can be increased, cell damage is reduced, and the recovered and cultured NK cells still have high killing capacity and multiplication capacity. Moreover, the NK cell cryopreservation liquid is simple and reliable in component, can be directly infused into a human body, is easy to prepare and preserve, is suitable for large-scale cell cryopreservation and clinical transformation, and is high in application value.

Description

Technical Field

[0001] The present application relates to the field of biomedicine. Specifically, the present application relates to NK cell cryopreservation solutions, NK cell cryopreservation methods, drugs, combined drugs and their applications. Background Art

[0002] Natural killer (NK) cells are important immune cells that can recognize and kill tumor cells and virus-infected cells. In cell therapy and immunotherapy, NK cells are increasingly widely used. The killing activity of NK cells is not restricted by MHC and does not depend on antibodies, so it is called natural killing activity. NK cells are rich in cytoplasm and contain large azurophilic granules, and the content of the granules is positively correlated with the killing activity of NK cells. NK cells account for 5-10% of the lymphocyte population in the circulation and can secrete perforin and tumor necrosis factor to destroy target cells, which has important clinical significance and application prospects.

[0003] Cell cryopreservation is a technique for storing cells in a low-temperature environment to reduce cell metabolism and achieve long-term storage. During cryopreservation, appropriate operations and suitable cryopreservation conditions can reduce changes or losses in cell characteristics and play a role in cell preservation. Direct freezing of cells without any cryoprotectant will cause the occurrence of cryoinjury effects. Water in both the intracellular and extracellular environments will form ice crystals, which can cause mechanical damage, increased electrolytes, osmotic pressure changes, dehydration, pH changes, protein denaturation, etc. inside the cells, leading to cell death. Therefore, when cryopreserving, a cryoprotectant should be added to the culture medium to lower the freezing point. Under slow freezing conditions, intracellular water can permeate out of the cells before freezing, and storing in liquid nitrogen can reduce the formation of ice crystals.

[0004] In order to avoid problems such as the increased cost due to long-term culture, the decline in the growth state and activity of cells after the culture time exceeds the optimal logarithmic growth phase, which affects the treatment quality of cells, and the increased pain caused by multiple blood samplings from patients, etc., preserving cultured NK cells has also become one of the necessary methods. Therefore, it is particularly important to preserve the activity and function of NK cells by cryopreservation. Traditional cryopreservation methods often lead to a decline in cell activity, affecting their clinical application effects. Therefore, it is of great significance to develop an effective NK cell cryopreservation solution and cryopreservation method. Summary of the Invention

[0005] This application aims to solve at least to some extent the technical problems existing in the prior art. To this end, this application provides an NK cell cryopreservation solution, a combined preparation, a kit and its application, an NK cell cryopreservation method, a method for improving the proliferation ability and / or killing ability of NK cells after cryopreservation and resuscitation, an NK cell preparation, a drug, a combined drug and its application. The NK cell cryopreservation solution of this application can significantly improve the activity recovery rate of NK cells after cryopreservation, reduce cell damage, so that the NK cells still have high killing ability and proliferation ability after resuscitation culture. Moreover, the components of the NK cell cryopreservation solution are simple and reliable, can be directly infused into the human body, and are easy to configure and store, suitable for large-scale cell cryopreservation and clinical transformation, with high application value.

[0006] It should be noted that this application is completed based on the following discoveries of the inventors:

[0007] At present, the prior art mainly uses fetal bovine serum (FBS), dimethyl sulfoxide (DMSO) and RPMI-1640 medium to prepare cryopreservation solutions in different proportions for the preservation after NK cell culture, or uses autologous serum instead of fetal bovine serum for cryopreservation. Although the former method can preserve cells for a long time, the cryopreservation solution prepared with fetal bovine serum introduces exogenous proteins and increases the risk of pathogen contamination. Using autologous serum instead of fetal bovine serum can completely eliminate the risk of exogenous pollutant infection, but the activity of NK cells after cryopreservation and resuscitation culture is low, and it is extremely difficult to continue amplification culture. There are also some patents with overly complex disclosed components, which pose great challenges to the controllability of materials and clinical applications. Therefore, the current NK cell cryopreservation technology mainly has the following industrialization bottlenecks:

[0008] 1. The components of the cryopreservation solution are complex, including several or even more than a dozen components, which is not conducive to large-scale production and application; it contains exogenous animal protein components, increasing the risk of pathogen contamination; adding medium components cannot be directly applied to the human body;

[0009] 2. The cell viability and function after resuscitation are not good. Most studies only focus on the cell state, viability and function at the time after resuscitation, but the cells in the actual in vivo and in vitro application scenarios of cells need a recovery process. The cryopreservation solutions commonly used in the art result in a large number of cell deaths after several hours of culture, which is not the true effect of cell cryopreservation;

[0010] 3. The in vivo function maintenance of NK cells after resuscitation is often overlooked, while in vivo pharmacodynamic and pharmacokinetic experimental studies can directly reflect the true cryopreservation effect of cells.

[0011] In view of this, through in-depth research, analysis and screening, the inventors of the present application have obtained a cryopreservation solution for NK cells, which contains human serum albumin, methylcellulose and dimethyl sulfoxide. Human serum albumin can provide a stable protein environment and reduce cell damage during cryopreservation; methylcellulose has good biocompatibility and stability, and can form a protective gel layer to further protect the cell structure; dimethyl sulfoxide, as an antifreeze agent, can lower the freezing point of the solution and reduce ice crystal formation, thereby effectively protecting cells from freezing damage and increasing the cell activity recovery rate. The synergistic effect of these three components enables the cryopreservation solution to significantly increase the activity recovery rate of NK cells after cryopreservation, reduce cell damage, and the cells still have high cell killing ability and proliferation ability after resuscitation culture. The activity of cryopreserved and resuscitated NK cells is close to that of fresh NK cells before cryopreservation, and can exert good pharmacological effects in vivo. Moreover, these three components are all of GMP or pharmaceutical grade and can be directly infused into the human body, which greatly promotes the clinical application of NK cell products. It can not only improve the safety and stability of the clinical application of NK cell preparations, but also avoid the contamination risk brought by operations such as centrifugation and resuspension after cell resuscitation, avoid cell damage and the resulting quality differences before infusion, and can also lower the threshold of the clinical application of cell products. In addition, the cryopreservation solution has simple composition, low cost, does not introduce animal-derived components, and has high safety. Therefore, it is suitable for large-scale cell cryopreservation and clinical transformation, and has high application value.

[0012] Therefore, in one aspect of the present application, the present application proposes a cryopreservation solution for NK cells. According to an embodiment of the present application, the cryopreservation solution for NK cells includes: a basal solution and human serum albumin, and the basal solution includes methylcellulose and dimethyl sulfoxide. The synergistic effect of these three components enables the cryopreservation solution to significantly increase the activity recovery rate of NK cells after cryopreservation, reduce cell damage, and the cells still have high cell killing ability and proliferation ability after resuscitation culture. The activity of cryopreserved and resuscitated NK cells is close to that of fresh NK cells before cryopreservation, and can exert good pharmacological effects in vivo. Moreover, these three components are all of GMP or pharmaceutical grade and can be directly infused into the human body, which greatly promotes the clinical application of NK cell products. It is suitable for large-scale cell cryopreservation and clinical transformation, and has high application value.

[0013] According to an embodiment of the present application, the above-mentioned cryopreservation solution for NK cells may further have the following additional technical features:

[0014] According to an embodiment of the present application, based on the total volume of the cryopreservation solution for NK cells, the concentration of human serum albumin is 20 mg / mL to 30 mg / mL.

[0015] According to an embodiment of the present application, based on the total volume of the basal solution, the content of methylcellulose is 0.05% to 0.2%, and the content of dimethyl sulfoxide is 9% to 11%.

[0016] According to an embodiment of the present application, the base liquid further includes inorganic acid, sodium hydroxide, hydroxypropyl-β-cyclodextrin and water.

[0017] According to an embodiment of the present application, the NK cell cryopreservation solution further includes vitamin C; based on the total volume of the NK cell cryopreservation solution, the concentration of vitamin C is 200 μg / mL to 300 μg / mL.

[0018] In another aspect of the present application, the present application provides a combined preparation. According to an embodiment of the present application, the combined preparation includes: a first preparation, the first preparation includes the aforementioned NK cell cryopreservation solution; a second preparation, the second preparation includes cytokines.

[0019] According to an embodiment of the present application, the cytokines include at least one of IL-2, IL-12, IL-15, IL-18 and IL-7.

[0020] According to an embodiment of the present application, the NK cell cryopreservation solution further contains vitamin C; or the combined preparation further includes: a third preparation, the third preparation includes vitamin C.

[0021] In yet another aspect of the present application, the present application provides a kit. According to an embodiment of the present application, the kit includes: the aforementioned combined preparation.

[0022] In yet another aspect of the present application, the present application provides the use of the aforementioned NK cell cryopreservation solution, the combined preparation or the kit in at least one of the following: NK cell cryopreservation; improving the proliferation ability of NK cells after cryopreservation and resuscitation; improving the killing ability of NK cells after cryopreservation and resuscitation.

[0023] In yet another aspect of the present application, the present application provides a method for cryopreserving NK cells. According to an embodiment of the present application, the method includes: mixing NK cells with the aforementioned NK cell cryopreservation solution to obtain a mixed solution; subjecting the mixed solution to a freezing treatment.

[0024] According to an embodiment of the present application, the method is implemented using the aforementioned combined preparation or the aforementioned kit, and the method includes: performing a first mixing and culturing of NK cells with the second preparation, collecting the cultured cells; performing a second mixing and freezing treatment of the cultured cells with the first preparation.

[0025] According to an embodiment of the present application, in the mixed solution obtained by the first culture, the final concentration of each cytokine is 10 ng / mL to 300 ng / mL.

[0026] According to an embodiment of the present application, the culturing time is 12 h to 24 h, and the temperature is 35°C to 40°C.

[0027] According to an embodiment of the present application, the cryopreservation treatment includes: programmed cooling to -85°C to -95°C for cryopreservation.

[0028] According to an embodiment of the present application, the NK cell cryopreservation solution contains vitamin C; or the cells, the first preparation, and vitamin C are subjected to the second mixing.

[0029] In another aspect of the present application, the present application proposes a method for improving the proliferation ability and / or killing ability of NK cells after cryopreservation and resuscitation. According to an embodiment of the present application, the method includes: resuscitating the NK cells cryopreserved by the NK cell cryopreservation method described above to obtain a cell solution, and collecting the resuscitated NK cells in the cell solution; co-culturing the resuscitated NK cells with vitamin C.

[0030] According to an embodiment of the present application, the method further includes: co-culturing the resuscitated NK cells, vitamin C, and cytokines.

[0031] According to an embodiment of the present application, the cytokines include at least one of IL-2, IL-12, IL-21, and IL-15.

[0032] In another aspect of the present application, the present application proposes an NK cell preparation. According to an embodiment of the present application, the NK cell preparation includes: NK cells and the NK cell cryopreservation solution described above, and the NK cells are in the NK cell cryopreservation solution.

[0033] According to an embodiment of the present application, the NK cell cryopreservation solution includes: vitamin C.

[0034] In another aspect of the present application, the present application proposes a drug. According to an embodiment of the present application, the drug includes: the NK cell preparation described above.

[0035] In another aspect of the present application, the present application proposes a combined drug. According to an embodiment of the present application, the combined drug includes: the drug described above and vitamin C.

[0036] According to an embodiment of the present application, the combined drug further includes cytokines, and the cytokines include at least one of IL-2, IL-12, IL-21, and IL-15.

[0037] In another aspect of the present application, the present application proposes the use of the drug or the combined drug described above in the preparation of a pharmaceutical preparation. According to an embodiment of the present application, the pharmaceutical preparation is used for preventing or treating cancer.

[0038] According to an embodiment of the present application, the cancers include at least one of lung cancer, thyroid cancer, glioma, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, cholangiocarcinoma, acute lymphoblastic leukemia, lymphoma, multiple myeloma, neuroendocrine cancer, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, esophageal cancer, oral squamous cell carcinoma, and gastric cancer.

[0039] According to an embodiment of the present application, the pharmaceutical preparation is used to inhibit the expression of at least one of the following inhibitory receptor molecules by NK cells: CD94, TIGIT, CD158d, and TIM-3.

[0040] In another aspect of the present application, the present application provides a method for inhibiting the expression of inhibitory receptor molecules by NK cells, and the cell molecules include at least one of the following: CD94, TIGIT, CD158d, and TIM-3. According to an embodiment of the present application, the method includes: contacting NK cells, the aforementioned NK cell cryopreservation solution, and vitamin C.

[0041] Additional aspects and advantages of the present application will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present application. Description of the Drawings

[0042] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0043] Figure 1 Shows a flowchart of NK cell cryopreservation and resuscitation according to an embodiment of the present application;

[0044] Figure 2 Shows an analysis graph of NK cell survival rate and recovery rate according to Example 1 of the present application;

[0045] Figure 3 Shows an analysis graph of cell expansion ability according to Example 1 of the present application;

[0046] Figure 4 Shows a flow cytometry graph (percentage of NK cells) according to Example 2 of the present application;

[0047] Figure 5 Shows an analysis graph of killing efficiency, killing-related molecules, and percentage of NK cells according to Example 3 of the present application;

[0048] Figure 6 Shows an analysis graph of NK cell killing efficiency and cell number according to Example 4 of the present application;

[0049] Figure 7 Shows the analysis chart of different molecular expression levels of NK cells according to Embodiment 5 of the present application;

[0050] Figure 8 Shows the analysis chart of the tumor inhibition rate of mice according to Embodiment 7 of the present application;

[0051] Figure 9 Shows the statistics of the distribution and content change of NK cells in animals after infusion according to Embodiment 8 of the present application. Detailed implementation manners

[0052] The embodiments of the present application will be described in detail below. The embodiments described below are exemplary and are only used to explain the present application and should not be construed as a limitation to the present application.

[0053] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present application, unless otherwise stated, the meaning of "a plurality" is two or more.

[0054] In the ranges disclosed herein, the endpoints and any value are not limited to the exact range or value. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0055] In this document, the term "comprising" or "including" is an open expression, that is, it includes the content specified in the present application, but does not exclude other aspects of the content.

[0056] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where the events or conditions occur and the cases where the events or conditions do not occur.

[0057] The present application provides NK cell cryopreservation solutions, combined preparations, kits and their applications, NK cell cryopreservation methods, methods for improving the proliferation ability and / or killing ability of NK cells after cryopreservation and resuscitation, NK cell preparations, drugs, combined drugs and their applications, and will be described in detail below respectively.

[0058] NK cell cryopreservation solutions, combined preparations and kits

[0059] In one aspect of the present application, the present application provides a cryopreservation solution for NK cells. According to an embodiment of the present application, the cryopreservation solution for NK cells comprises: a basal solution and human serum albumin, and the basal solution comprises methylcellulose and dimethyl sulfoxide.

[0060] Human serum albumin can provide a stable protein environment and reduce cell damage during cryopreservation; methylcellulose has good biocompatibility and stability and can form a protective gel layer to further protect the cell structure; dimethyl sulfoxide, as an antifreeze agent, can lower the freezing point of the solution and reduce ice crystal formation, thereby effectively protecting cells from freezing damage and increasing the cell activity recovery rate. The synergistic effect of these three components enables the cryopreservation solution to significantly increase the activity recovery rate of NK cells after cryopreservation, reduce cell damage, such that the NK cells still have high killing ability and proliferation ability after resuscitation culture, and the activity of the cryopreserved and resuscitated NK cells is close to that of the fresh NK cells before cryopreservation. Moreover, these three components are all of GMP or pharmaceutical grade and can be directly administered to the human body, which greatly promotes the clinical application of NK cell products. It can not only improve the safety and stability of the clinical application of NK cell preparations, but also avoid the contamination risk brought by operations such as centrifugation and resuspension after cell resuscitation, avoid cell damage and the resulting quality differences before infusion, and can also lower the threshold of the clinical application of cell products. In addition, the cryopreservation solution has simple composition, low cost, does not introduce animal-derived components, and has high safety. Therefore, it is suitable for large-scale cell cryopreservation and clinical transformation and has high application value.

[0061] According to an embodiment of the present application, based on the total volume of the cryopreservation solution for NK cells, the concentration of human serum albumin is 20 mg / mL to 30 mg / mL, such as 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL. Thus, it can effectively stabilize the cell membrane structure and reduce the influence of mechanical damage and osmotic pressure changes on cells during cryopreservation, thereby increasing cell activity and survival rate. In addition, it can avoid increasing the viscosity of the solution due to too high a concentration, resulting in greater physical resistance to cells during cryopreservation and resuscitation, affecting cell diffusion and activity recovery, and can also reduce costs and operation complexity.

[0062] According to an embodiment of the present application, based on the total volume of the base liquid, the methylcellulose content is 0.05% to 0.2%, such as 0.05%, 0.08%, 0.1%, 0.12%, 0.15%, 0.18%, 0.2%, and the dimethyl sulfoxide content is 9% to 11%, such as 9%, 9.2%, 9.5%, 9.8%, 10%, 10.2%, 10.5%, 10.8%, 11%. Thus, when the methylcellulose content meets the above conditions, it helps methylcellulose form a protective gel layer around the cells, reducing the mechanical damage and the influence of osmotic pressure changes on the cells during cryopreservation. At the same time, it can also stabilize the cell membrane structure and maintain the cell morphology and function. When the dimethyl sulfoxide meets the above conditions, it helps to lower the freezing point of the solution and reduce the formation of ice crystals, thereby effectively preventing cell damage caused by the freezing of intracellular water and improving the cell activity recovery rate.

[0063] According to an embodiment of the present application, the base liquid further includes inorganic acid, sodium hydroxide, hydroxypropyl-β-cyclodextrin and water. Thus, it helps to further stabilize the cryopreservation solution and extend the shelf life. Exemplarily, the inorganic salt includes hydrochloric acid.

[0064] In some embodiments, the base liquid can be D10 (05-714-1A, sartorius, abbreviated as D10 or D10 cryopreservation solution in this article), which is a serum-free, chemically defined and animal-component-free cryopreservation solution. Its main components are 0.1% methylcellulose and 10% DMSO. Other materials used as solubilizers include hydrochloric acid, sodium hydroxide and hydroxypropyl-β-cyclodextrin. It is generally considered that when the DMSO concentration is less than 10%, the toxic effect of dimethyl sulfoxide is very small. However, during the treatment of diseases, short-term large-dose infusion of dimethyl sulfoxide will produce many toxic side effects. Therefore, on the premise of ensuring the cryopreservation effect, the addition of a lower concentration of DMSO is more beneficial for clinical use, which can avoid or reduce the adverse reactions of patients caused by the infusion of a large amount of DMSO. In the present application, by jointly preparing the D10 with human serum albumin into an NK cell cryopreservation solution, since human serum albumin is introduced, the DMSO concentration can be reduced, thereby improving the safety of the cryopreservation solution.

[0065] According to an embodiment of the present application, the NK cell cryopreservation solution further includes vitamin C. By adding vitamin C, the proliferation ability of NK cells in vivo and in vitro after cryopreservation can be improved.

[0066] According to an embodiment of the present application, based on the total volume of the NK cell cryopreservation solution, the concentration of vitamin C is 200 μg / mL to 300 μg / mL, such as 200 μg / mL, 220 μg / mL, 240 μg / mL, 250 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL. Thus, the proliferation ability of NK cells in vivo and in vitro after cryopreservation can be further improved.

[0067] In another aspect of the present application, the present application provides a combined preparation. According to an embodiment of the present application, the combined preparation includes: a first preparation, the first preparation includes the aforementioned NK cell cryopreservation solution; a second preparation, the second preparation includes cytokines. By using cytokines and NK cell cryopreservation solution in combination, before cryopreservation, NK cells are pre-stimulated with cytokines, which helps to activate NK cells, promote their proliferation and enhance NK cell activity, enabling them to maintain activity in the cryopreservation environment. Placing the cytokine-stimulated NK cells in the NK cell cryopreservation solution for cryopreservation helps to better improve the activity recovery rate of NK cells after cryopreservation, reduce cell damage, and the cells still have high cell killing ability and proliferation ability after resuscitation culture, and can exert better pharmacological effects in vivo.

[0068] According to an embodiment of the present application, the cytokine includes at least one of IL-2, IL-12, IL-15, IL-18, and IL-7. IL-2 is an important cytokine that induces the proliferation of NK cells. It can activate NK cells, promote the proliferation of NK cells and the production of cytokines. The functions of IL-15 and IL-7 are similar to those of IL-2. At the same time, they can also promote the directed differentiation of hematopoietic stem cells into NK cells by binding to the composite receptor γ chain expressed on the surface of NK cells, and play an important role in aspects such as the development, differentiation, and long-term in vitro survival of NK cells. The synergistic effect of IL-15 and IL-2 also enables the two to act jointly on the in vitro expansion of NK cells, which is the most traditional cytokine combination for the in vitro expansion of NK cells at present. It is reported that IL-18 can not only induce activated THi cells to secrete a large amount of IFN-γ, but also enhance the cytotoxicity of NK cells by promoting the opening of the Fas-FasL pathway, and shows a dose-dependent relationship.

[0069] According to an embodiment of the present application, the NK cell cryopreservation solution further contains vitamin C; or the combined preparation further includes: a third preparation, the third preparation includes vitamin C. During cryopreservation, by adding vitamin C, the proliferation ability of NK cells in vivo and in vitro after cryopreservation can be improved. The vitamin C can be added either to the cryopreservation solution or provided as an independent third preparation. When cryopreservation is needed, it is mixed with the cryopreservation solution for use.

[0070] In another aspect of the present application, the present application provides a kit. According to an embodiment of the present application, the kit includes the aforementioned combined preparation. Thus, by using the kit of the present application, the activity recovery rate of cryopreserved NK cells can be increased, cell damage can be reduced, and the cells still have high cell killing ability and proliferation ability after resuscitation culture, and can exert good pharmacodynamic effects in vivo.

[0071] It should be noted that the features and advantages described above for the NK cell cryopreservation solution also apply to the combined preparation and the kit, and will not be repeated here.

[0072] Applications and methods

[0073] In another aspect of the present application, the present application provides the use of the aforementioned NK cell cryopreservation solution, the combined preparation or the kit in at least one of the following: cryopreservation of NK cells; improving the proliferation ability of NK cells after cryopreservation and resuscitation; improving the killing ability of NK cells after cryopreservation and resuscitation. As described above, by using the NK cell cryopreservation solution, the combined preparation and the kit of the present application, the activity recovery rate of cryopreserved NK cells can be significantly increased, cell damage can be reduced, and the NK cells still have high killing ability and proliferation ability after resuscitation culture. Moreover, the composition of the NK cell cryopreservation solution is simple and reliable, can be directly infused into the human body, is easy to prepare and store, is suitable for large-scale cell cryopreservation and clinical transformation, and has high application value.

[0074] In another aspect of the present application, the present application provides a method for cryopreserving NK cells. According to an embodiment of the present application, the method includes: mixing NK cells with the aforementioned NK cell cryopreservation solution to obtain a mixed solution; and subjecting the mixed solution to a freezing treatment. As described above, by using the NK cell cryopreservation solution, the combined preparation and the kit of the present application, the activity recovery rate of cryopreserved NK cells can be significantly increased, cell damage can be reduced, and the NK cells still have high killing ability and proliferation ability after resuscitation culture.

[0075] According to an embodiment of the present application, the method is implemented using the aforementioned combined preparation or the aforementioned kit. The method includes: performing a first mixing and culturing of NK cells with the second preparation, and collecting the cultured cells; and performing a second mixing and freezing treatment of the cultured cells with the first preparation. By using the cytokine and the NK cell cryopreservation solution in combination, and pre-stimulating NK cells with the cytokine before cryopreservation, it helps to activate NK cells, promote their proliferation and enhance NK cell activity, so that they can maintain their activity in the cryopreservation environment. Cryopreserving the NK cells stimulated by the cytokine in the NK cell cryopreservation solution helps to better increase the activity recovery rate of cryopreserved NK cells, reduce cell damage, and the cells still have high cell killing ability and proliferation ability after resuscitation culture, and can exert good pharmacodynamic effects in vivo.

[0076] It should be noted that the cryopreserved NK cells need to be passaged in advance to make them have a certain cell activity. Exemplarily, the passage is performed before stimulating the NK cells with cytokines.

[0077] According to an embodiment of the present application, in the mixed solution obtained from the first culture, the final concentration of each cytokine is 10 ng / mL to 300 ng / mL, such as 10 ng / mL, 25 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 125 ng / mL, 150 ng / mL, 175 ng / mL, 200 ng / mL, 225 ng / mL, 250 ng / mL, 275 ng / mL, 300 ng / mL. Thus, it helps to activate NK cells, promote their proliferation and cytokine production, enabling them to maintain their activity in the cryopreservation environment.

[0078] According to an embodiment of the present application, the culture time is 12 h to 24 h, such as 12 h, 14 h, 15 h, 16 h, 18 h, 20 h, 22 h, 24 h, and the temperature is 35°C to 40°C, such as 35°C, 36°C, 37°C, 38°C, 39°C, 40°C. Thus, it helps to activate NK cells, promote their proliferation and cytokine production, enabling them to maintain their activity in the cryopreservation environment.

[0079] According to an embodiment of the present application, the cryopreservation treatment includes: programmed cooling to -85°C to -95°C for cryopreservation. Thus, by programmed cooling, the water in the cells has enough time to gradually drain out, reducing the formation of ice crystals, thereby effectively reducing the damage to the cells during freezing.

[0080] According to an embodiment of the present application, the NK cell cryopreservation solution contains vitamin C; or the cells, the first preparation and vitamin C are subjected to the second mixing. During cryopreservation, by adding vitamin C, the proliferation ability of NK cells in vivo and in vitro after cryopreservation can be improved. The addition method of vitamin C can be either added to the cryopreservation solution or provided independently, and when cryopreservation is needed, it is mixed with the cryopreservation solution for use.

[0081] In another aspect of the present application, the present application proposes a method for improving the proliferation ability and / or killing ability of NK cells after cryopreservation and resuscitation. According to an embodiment of the present application, the method includes: resuscitating the NK cells cryopreserved by the aforementioned NK cell cryopreservation method to obtain a cell solution, and collecting the resuscitated NK cells in the cell solution; co-culturing the resuscitated NK cells with vitamin C. Thus, by using the method of the present application, the activity recovery rate of NK cells after cryopreservation can be effectively improved, cell damage can be reduced, and the resuscitated and cultured NK cells still have high killing ability and proliferation ability.

[0082] According to an embodiment of the present application, the recovery includes placing the cryopreserved cells at 35°C to 40°C until the ice crystals in the cell suspension disappear.

[0083] According to an embodiment of the present application, the method further includes: co-culturing the recovered NK cells, vitamin C, and cytokines. By adding vitamin C, the proliferation ability and killing ability of NK cells after cryopreservation and recovery can be provided. Cytokines are used to maintain the normal growth of NK cells.

[0084] According to an embodiment of the present application, the cytokines include at least one of IL-2, IL-12, IL-21, and IL-15.

[0085] It should be noted that the features and advantages described above for the NK cell cryopreservation solution, combination preparation, and kit also apply to this application and method, and will not be elaborated here.

[0086] NK Cell Preparation, Drug, Combination Drug, and Application

[0087] In another aspect of the present application, the present application provides an NK cell preparation. According to an embodiment of the present application, the NK cell preparation includes: NK cells and the aforementioned NK cell cryopreservation solution, and the NK cells are located in the NK cell cryopreservation solution. As described above, the NK cell cryopreservation solution of the present application improves the activity recovery rate of NK cells after cryopreservation, reduces cell damage, and enables the NK cells after recovery culture to still have high killing ability and proliferation ability. Therefore, it can be applied to cell therapy, immunotherapy, and cell bank construction, etc., and has high application value.

[0088] It should be noted that the NK cell preparation of the present application can be an NK cell preparation containing NK cells and NK cell cryopreservation solution before cryopreservation, after cryopreservation and before recovery, or after cryopreservation and recovery. The NK cell preparation is in a liquid state before cryopreservation, in a solid state after cryopreservation and before recovery, and in a liquid state after cryopreservation and recovery.

[0089] According to an embodiment of the present application, the NK cell cryopreservation solution includes: vitamin C. By adding vitamin C, the proliferation ability and killing ability of NK cells after cryopreservation and recovery can be provided.

[0090] In another aspect of the present application, the present application provides a drug. According to an embodiment of the present application, the drug includes: the aforementioned NK cell cryopreservation solution and NK cells. Therefore, the NK cells in the drug of the present application have strong cell killing ability and proliferation ability, and can be applied to cell therapy, immunotherapy, and cell bank construction, etc., and have high application value. Exemplarily, in the drug of the present application, the NK cell cryopreservation solution and NK cells are in the form of a mixed solution and are obtained after cryopreservation and recovery.

[0091] In yet another aspect of the present application, the present application provides a combined drug. According to an embodiment of the present application, the combined drug comprises: the aforementioned drug and vitamin C. Under the action of vitamin C, the proliferation ability of NK cells cryopreserved with the aforementioned NK cell cryopreservation solution in vivo and in vitro can be improved, which helps the NK cells to exert better drug efficacy in vivo.

[0092] It should be noted that in the combined drug of the present application, the drug and vitamin C can be administered simultaneously or sequentially; both can be provided in the form of a pharmaceutical composition or separately as two medicaments.

[0093] According to an embodiment of the present invention, the combined drug further comprises a cytokine, and the cytokine comprises at least one of IL-2, IL-12, IL-21, and IL-15. Thus, it helps to maintain the normal growth of NK cells.

[0094] According to an embodiment of the present invention, the drug and / or the combined drug further comprises: a pharmaceutically acceptable excipient.

[0095] As used herein, the term "pharmaceutically acceptable" indicates that the pharmaceutical composition can be administered to a subject without producing adverse physiological reactions that impede the administration of the pharmaceutical composition. For example, "pharmaceutically acceptable excipients" refer to excipients useful in the preparation of generally safe, non-toxic, and desirable pharmaceutical compositions. Preferably, examples of these excipients or diluents include, but are not limited to: water, saline, Ringer's solution, glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, 5% human serum albumin, and liposomes and non-aqueous media such as fixed oils can also be used.

[0096] In yet another aspect of the present application, the present application provides the use of the aforementioned drug or the combined drug in the preparation of a pharmaceutical preparation. According to an embodiment of the present application, the pharmaceutical preparation is used for preventing or treating cancer. As described above, the NK cells of the present application have better cell killing ability and proliferation ability, and can exert better drug efficacy in vivo.

[0097] According to an embodiment of the present application, the cancer includes at least one of lung cancer, thyroid cancer, glioma, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, cholangiocarcinoma, acute lymphoblastic leukemia, lymphoma, multiple myeloma, neuroendocrine cancer, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, esophageal cancer, oral squamous cell carcinoma, and gastric cancer.

[0098] According to an embodiment of the present application, the pharmaceutical preparation is used to inhibit the expression of at least one of the following inhibitory receptor molecules by NK cells: CD94, TIGIT, CD158d, and TIM-3.

[0099] In another aspect of the present application, a method for inhibiting the expression of inhibitory receptor molecules by NK cells is proposed. The inhibitory receptor molecules include at least one of the following: CD94, TIGIT, CD158d, and TIM-3. According to an embodiment of the present application, the method includes: contacting NK cells, the aforementioned NK cell cryopreservation solution, and vitamin C.

[0100] Vitamin C exerts an immunomodulatory effect on immune cells mainly through two main mechanisms of action, namely antioxidant activity and epigenetic regulation (by providing ferrous ions to TET enzymes to keep them in a fully catalytic form, thus ensuring active DNA demethylation). It mainly affects the proliferation function of NK cells. However, for cryopreserved NK cells, vitamin C promotes NK cell activity by affecting the expression of inhibitory receptors (marker molecules such as CD94, TIGIT, or TIM3).

[0101] It should be noted that the features and advantages described above for the NK cell cryopreservation solution also apply to this NK cell preparation, drug, combination drug, and application, and will not be elaborated here.

[0102] The solution of the present application will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. For those where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this field or according to the product instructions are followed. For reagents or instruments where the manufacturer is not indicated, they are all conventional products that can be obtained through commercial purchase.

[0103] Example 1 Amplification, Cryopreservation, and Resuscitation of NK Cells

[0104] For the specific cryopreservation operation process, see the appendix Figure 1 .

[0105] I. Preparation and Cryopreservation of NK Cells

[0106] 1. Obtaining mononuclear cells (PBMC)

[0107] Centrifuge the donor's peripheral blood to obtain plasma and whole blood cells. The plasma is combined and inactivated in a 56°C water bath, then centrifuged to obtain clear plasma, which is stored at 2 - 8°C for later use; the whole blood cells are diluted 1:1 with physiological saline and slowly added to lymphocyte separation medium at a ratio of 1:1.5 to separate and extract mononuclear cells PBMC for later use;

[0108] 2. NK Cell Culture and Cryopreservation

[0109] 2.1 Cell culture on day D0

[0110] PBMCs were isolated from apheresis blood of different healthy donors and cultured according to the method described in paragraph

[0076] of Patent CN118325833A.

[0111] 2.2 Cell culture from day D3 to D17

[0112] On the 3rd day (D3), IL-2 was added to a final concentration of 5 ng / ml, and the cells were further cultured in an incubator at 37 °C, saturated humidity, and 5.0% CO2.

[0113] From the 5th day (D5), samples were taken and counted every other day until D21, and NK cell medium (KBM581 medium) was supplemented.

[0114] 2.3 Cell culture on day D21

[0115] The cells were divided into 4 portions and harvested and cryopreserved according to the protocols of each group:

[0116] Group 1 was cultured until D21, harvested, and the cells were resuspended in physiological saline containing 1.25 mg / ml human albumin.

[0117] Group 2 was cultured until D21, harvested, and cryopreserved using D10 basic cryopreservation solution.

[0118] Group 3 was cultured until D21, harvested, and cryopreserved using D10 basic cryopreservation solution containing 25 mg / mL human albumin at the final concentration.

[0119] For Group 4, 20 h before D21, IL-15 at a final concentration of 50 ng / mL and IL-18 at a final concentration of 250 ng / mL were added to the medium, and the cells were stimulated in an incubator at 37 °C and 5% CO2 for 20 h, then harvested and cryopreserved using D10 basic cryopreservation solution containing 25 mg / mL human albumin at the final concentration.

[0120] Cryopreservation was performed by placing the cell suspension in a sterile cryotube and cooling it at a programmed rate, and then the cells were transferred to liquid nitrogen for cryopreservation for 90 days.

[0121] The programmed cooling was as follows:

[0122] a. Wait at 20.0 °C until the sample is placed.

[0123] b. Cool the sample at a rate of 1.0 °C per minute to -12.0 °C.

[0124] c. Cool the chamber at a rate of 22.0 °C per minute to -50.0 °C.

[0125] d. The chamber temperature is decreased to -18.0 °C at a rate of 4 °C per minute;

[0126] e. The chamber temperature is decreased to -45.0 °C at a rate of 1 °C per minute;

[0127] f. The chamber temperature is decreased to -90.0 °C at a rate of 10.0 °C per minute;

[0128] g. The temperature reduction ends.

[0129] II. Amplification culture process of NK cells after resuscitation

[0130] 1. NK cell resuscitation

[0131] Take out the cells from the liquid nitrogen tank, quickly immerse the cryopreservation tube in a 37 °C constant temperature water bath, and take it out when the ice crystals in the cell suspension are about to completely disappear as observed with the naked eye. Centrifuge the mixed cell suspension, remove the supernatant after centrifugation, and resuspend the cells with KBM581 medium.

[0132] 2. NK cell culture and medium supplementation

[0133] According to the counting density and volume on the same day, calculate the KBM581 medium to be supplemented according to the formula (original volume on the same day × density / passage density = final passage volume).

[0134] 3. Experimental results

[0135] It can be seen from the experiment that regardless of the usage method, after using the cryopreservation solution of the present application, the viability and recovery rate of NK cell resuscitation are relatively high (cell viability = number of live cells / total number of cells; cell recovery rate = number of live cells after resuscitation / number of live cells before resuscitation), and the results are shown in Figure 2 . It can be seen that the cell recovery rates of groups 2, 3, and 4 are all above 95%, the cell viability at 0 h is above 85%, and the cell viability recovers to above 95% after 3 days of culture.

[0136] As Figure 3 shown, when the cells are cultured in vitro for 5 days, since group 1 is not cryopreserved, the NK cells still maintain high amplification ability. The amplification ability of NK cells decreases when cryopreserved in the basic cryopreservation solution (group 2) or the basic cryopreservation solution + HSA group (group 3) preservation solution, and among them, the cryopreservation effect of the D10 cryopreservation solution supplemented with human serum albumin (group 3) is relatively high. After the NK cells pre-stimulated with cytokines are cryopreserved in the basic cryopreservation solution + HSA preservation solution (group 4), their amplification ability is close to that of the fresh non-cryopreserved state.

[0137] Example 2. Detection of NK cell purity and killing-related molecules

[0138] 1) Experimental materials

[0139] Flow antibodies: Fluorescently labeled surface molecule marker antibodies: CD3-Percpcy5.5 (Catalog No.: 300430), CD56-BV785 (Catalog No.: 362550), FITC-CD3 (Catalog No.: 300406), CD56-Alexa Flour 647 (Catalog No.: 362514), NKG2D-APC (Catalog No.: 320808), Perforin-FITC (Catalog No.: 353310), Granzyme B-Alexa Flour 647 (Catalog No.: 515406), 4-1BB-PE / Cy7 (Catalog No.: 309818), CD107a-AF647 (Catalog No.: 328612), IFNγ-PE / Cy7 (Catalog No.: 506518), all purchased from Biolegend.

[0140] Blocking antibody: Mouse serum, purchased from Future, Catalog No. F001008.

[0141] Flow cytometry tubes: 5 ml transparent polystyrene or polypropylene round-bottom tubes with lids.

[0142] Monensin: Purchased from sigma, Catalog No. 22373-78-0.

[0143] Fixation / permeabilization Diluent: Purchased from ebioscience, Catalog No. 4298341.

[0144] Fixation / permeabilization concentrate: Purchased from ebioscience, Catalog No. 4311034.

[0145] Permeabilization buffer: Purchased from ebioscience, Catalog No. 4314840.

[0146] DAPI (4',6-Diamidino-2-Phenylindole, Dilactate): Purchased from Biolegend, Catalog No. 422801.

[0147] Flow cytometry washing solution: PBS.

[0148] Relevant reagents from companies of the same grade and flow antibodies with the same clone number can also be used.

[0149] 2) Main instruments and equipment

[0150] Centrifuge, laminar flow hood, flow cytometer (such as FACS Calibur, Beckman CytoFlex, etc.), RTCA detection system.

[0151] 3) Detection methods for cell activity-related molecules

[0152] ① Induction of intracellular molecularly labeled cells with monensin: Take 4×10 6 cells (2 ml) and place them in a 6-well plate. Add 10 μl of monensin (0.5 μg / μl) and incubate at 37°C in a 5% CO2 incubator for 4 hours.

[0153] ② Preparation of single-cell suspension: For surface molecularly labeled cells, take 9.0×10 6 cells and place them in a 15-ml centrifuge tube. Centrifuge at 400 g for 8 minutes to collect the cells, wash them twice with 10 ml of 1×PBS, and finally resuspend them in 0.81 ml of 1×PBS to form a single-cell suspension. After the induction of intracellular molecularly labeled cells with monensin is completed, add 5 ml of 1×PBS and wash twice, and finally resuspend them in 180 μl of 1×PBS to form a single-cell suspension.

[0154] ③ Blocking: Add 90 μl of mouse serum to the surface molecularly labeled cells and mix well. Add 20 μl of mouse serum to the intracellular molecularly labeled cells and mix well. Let stand at room temperature for 15 - 30 minutes.

[0155] ④ Labeling with antibodies: After blocking, aliquot the surface molecularly labeled cells into 9 flow cytometry tubes at 0.09 ml per tube. If multiple batches of cells are detected simultaneously, the cells in tubes 1 - 8 can be mixed with equal amounts of several batches of cells for labeling. The cell amount per tube is: 0.8×10 6 ~1×10 6 cells. Sample tube 1 is for surface molecular labeling, and the cell amount is: 0.8×10 6 ~1×10 6 cells. Sample tube 2 is for intracellular molecular labeling, and the cell amount is: 3×10 6 ~4×10 6 cells, and add the corresponding fluorescently labeled antibodies. Mix well and let stand at 4°C in the dark for 30 minutes (mix well every 15 minutes in the middle).

[0156] Table 1. Sample tubes and experimental operation table for the detection of NK cell surface molecules

[0157]

[0158]

[0159] ⑤ Washing: Add 1 ml of 1×PBS to each tube of surface molecule-labeled cells, centrifuge at 400 g for 8 minutes at 4°C to collect the cells. Repeat the washing 2 times with 1 ml of 1×PBS. Finally, add 200 μl of PBS to each tube to resuspend the labeled cells. Add 5 μl of DAPI (50 μg / ml, 40×) to the experimental group, transfer the tubes to the machine for detection.

[0160] Among them, sample tube 2 is the internal standard molecule. Therefore, after the labeling of the external standard molecules CD3, CD56, and CD45 is completed, first add 5 μl of DAPI, label for 3 minutes, add 1 ml of PBS to each tube and wash 2 times, discard the supernatant, and try to discard it as clean as possible;

[0161] ⑥ Add 300 μl of Fixation Buffer mix (Fixation / permeabilization concentrate: Fixation / permeabilization Diluent = 1:3), fix at 4°C in the refrigerator for 1 h; (mix once every 30 min). After completion, add 1 ml of Perm Wash Buffer (stock solution is 10×, diluted to 1× with double-distilled water), centrifuge at 500 g for 5 minutes at 4°C, and discard the supernatant;

[0162] ⑦ Resuspend the cells with 180 μl of Perm Wash Buffer, add 20 μl of mouse serum, and incubate at 4°C in the refrigerator for 20 min; after the blocking is completed, divide the cells into two equal parts, and label one part with the internal standard antibody.

[0163] ⑧ Label at 4°C in the dark for 1 hour (mix once in the middle). After the antibody labeling is completed, add 1 ml of Perm Wash Buffer, centrifuge at 500 g for 5 minutes at 4°C; and repeat the washing once again;

[0164] ⑨ Resuspend the cells with 200 μl of Perm Wash Buffer, transfer to the machine for detection;

[0165] ⑩ Detection: Calibrate and adjust the flow cytometer according to the instrument instruction manual. The blank control tube sample is used to adjust the forward scatter and side scatter voltages; the isotype control sample is used to adjust the voltages of each fluorescence channel so that the fluorescence is within the negative range. The single-label control sample is used to adjust the fluorescence compensation of each channel. After the cell gate is defined during detection, collect 1×10 4 cells in each sample gate.

[0166] 4) Judgment of the validity of experimental results

[0167] The positive rate of fluorescence staining of the blank control tube sample was <1%, the absolute cell count was less than 100, and the positive rates of the isotype controls of the five different fluorescently labeled antibodies were all <1%, with the absolute cell count being less than 100 cells; the experimental results meeting the above requirements were considered qualified; otherwise, the results were unqualified.

[0168] Percentage of positive cells (positive cells %) = (number of positive cells ÷ total number of counted cells 1 × 10 4 ) %

[0169] The results were as Figure 4 shown. When detecting the proportions of NK cells and T cells by flow cytometry, there were no significant differences between groups 2, 3, 4 and group 1, that is, cryopreservation did not affect the proportions of NK cells and T cells.

[0170] Detection method for cell killing activity in Example 3

[0171] Detection of NK cell killing by flow cytometry

[0172] Preparation of target cell suspension (K562 cells)

[0173] ① Cell counting: The cells were resuspended in 1640 medium containing 0.5% FBS (the medium used for target cells), counted, and the cell density was adjusted to 1 × 10 6 / mL;

[0174] ② CFSE staining: CFSE (working concentration 5 μM) was added to the cell suspension, immediately pipetted with a 1 mL pipette and vortexed thoroughly, incubated in a 37 °C incubator in the dark for 15 min, and vortexed every 5 min;

[0175] ③ Termination of staining: Five volumes of 4 °C pre-cooled complete medium (the medium used for target cells) were added to terminate the staining, incubated on ice for 5 minutes, centrifuged at 140 g, 4 °C for 5 min;

[0176] ④ Washing of cells: Resuspended with 4 °C pre-cooled complete medium (the medium used for target cells), centrifuged the cells at 140 g, 4 °C for 5 min; repeated the washing twice;

[0177] ⑤ Counting: The cells were resuspended, counted, and the cell density was adjusted to 2 × 10 5 / mL;

[0178] ⑥ Plating: Add 100 μl of the target cell suspension to each well of a 96-well round bottom plate to make the final cell number per well 20,000 cells / well;

[0179] Preparation and addition of NK cells

[0180] ⑦ Resuscitated NK cells from groups 1, 2, 3 and 4 were respectively prepared into NK cell suspensions;

[0181] ⑧ Take out the E-Plate 16 and place it in the laminar flow hood.

[0182] ⑨ Add 100 μl of NK cell suspension to the culture plate at different effector-to-target ratios (5:1, 2.5:1, 1.25:1). In addition, three groups of controls should be set up: target cells stained only with CFSE (to detect the natural death rate of target cells); effector cells only (to prove that effector cells do not contain non-specific CFSE staining); target cells + Tween-20 (as a positive control for target cell apoptosis).

[0183] ⑩ Incubate co-cultures. Add NK cells to each well in the pre-designed order. Centrifuge at 120 g at room temperature for 2 min to allow sufficient contact between effector and target cells; return to the 37 °C incubator and incubate for 4 hours.

[0184] After incubation, add 1 μl of 7-AAD, mix well, incubate in the dark for 5 min, and then perform detection on the machine.

[0185] Result analysis: Killing percentage = [((experimental group target cell death rate (%) - target cell death rate (%)) / (100% - target cell natural death rate (%)))] × 100%

[0186] The results are as Figure 5 shown. After cell cryopreservation and recovery, the proportion of NK cells did not change compared with that of the non-cryopreserved group (Group 1). The cell killing efficiency of Groups 3 and 4 after 72 h of recovery culture had almost no difference compared with that of the non-cryopreserved group (Group 1), and the killing efficiency of Groups 3 and 4 was higher than that of Group 2. Further, detection of cell killing-related molecules found that the expressions of 4-1BB, IFN-γ, CD107a, and perforin in Group 4 were all higher than those in Groups 2 and 3.

[0187] Example 4 Effect of VC (LAA) addition on NK cells (in vitro culture)

[0188] 1. Perform NK cell culture, cryopreservation, and recovery according to the method of Example 1. Among them, cryopreserve Group 4, and the culture conditions of the recovered NK cells are as follows:

[0189] Group 1: Resuspended the recovered NK cells in KBM581 medium, and supplemented KBM581 medium during the culture period.

[0190] Group 2: Resuspended the recovered NK cells in KBM581 medium containing 5 ng / mL IL-2, and supplemented KBM581 medium containing 5 ng / mL IL-2 during the culture period.

[0191] Group 3: The NK cells after resuscitation were resuspended in KBM581 medium containing 25 mg / mL human albumin, and KBM581 medium containing 25 mg / mL human albumin was supplemented during the culture.

[0192] Group 4: The NK cells after resuscitation were resuspended in KBM581 medium containing 5 ng / mL IL-2 and 25 mg / mL human albumin, cultured, and KBM581 medium containing 5 ng / mL IL-2 and 25 mg / mL human albumin was supplemented during the culture.

[0193] Group 5: The NK cells after resuscitation were resuspended in KBM581 medium containing 5 ng / mL IL-2, 25 mg / mL human albumin and 300 μg / ml VC, and KBM581 medium containing 5 ng / mL IL-2, 25 mg / mL human albumin and 300 μg / ml VC was supplemented during the culture.

[0194] 2. The cytotoxic activity of the NK cells after resuscitation and culture in the above 5 groups was detected by the method of Example 3, and the number of cells after resuscitation and culture was determined.

[0195] The results are as Figure 6 shown. It can be seen from the killing effect of NK cells that adding IL-2 during resuscitation and culture can improve the killing ability of NK cells, and adding VC can improve the proliferation ability of NK cells (indicated by the arrow).

[0196] Example 5 Effect of VC (LAA) addition on NK cells (in vitro detection)

[0197] 1. NK cells were cultured, cryopreserved and resuscitated according to the method of Example 1, except that the cryopreservation groups were as follows:

[0198] ST group: Cultured until D21, harvested, and cryopreserved using D10 basic cryopreservation solution.

[0199] ST + HSA group: Cultured until D21, harvested, and cryopreserved using D10 basic cryopreservation solution with a final concentration of 25 mg / mL human albumin.

[0200] ST + HSA + VC group: Cultured until D21, harvested, and cryopreserved using D10 basic cryopreservation solution with a final concentration of 25 mg / mL human albumin and 300 μg / ml VC.

[0201] 2. Immediately after resuscitation without culture, NK cells were collected for detection, and the surface receptors and related molecules of NK cells were detected. The detection methods are as follows:

[0202] (1) Experimental materials

[0203] Flow antibodies: Fluorescently labeled isotype control mouse monoclonal antibodies: mouse IgG1-FITC (Cat. No.: 400108), mouse IgG1-Percp cy5.5 (Cat. No.: 400150), mouse IgG1-APC (Cat. No.: 400120), mouse IgG1-BV785 (Cat. No.: 400170), mouse IgG1-PE (Cat. No.: 400114), all purchased from Biolegend; surface molecule marker antibodies: CD3-Percpcy5.5 (Cat. No.: 300430), CD56-BV785 (Cat. No.: 362550), Tim3-APC (Cat. No.: 345012), CD158d-PE (Cat. No.: 347006), CD94-FITC (Cat. No.: 305504), and TIGIT-APC (Cat. No.: 372706), all purchased from Biolegend; DAPI (Cat. No.: 422801) was purchased from Biolegend.

[0204] Blocking antibody: Mouse serum, purchased from Future, Cat. No. F001008.

[0205] Flow cytometry tubes: 5 ml transparent polystyrene or polypropylene round-bottom tubes with lids.

[0206] DAPI (4',6-Diamidino-2-Phenylindole, Dilactate): Purchased from Biolegend, Cat. No. 422801.

[0207] Flow cytometry washing solution: PBS.

[0208] Relevant reagents from companies of the same grade and flow antibodies with the same clone number can also be used.

[0209] (2) Main instrument and equipment

[0210] Centrifuge, laminar flow hood, flow cytometer (such as FACS Calibur, Beckman CytoFlex, etc.), RTCA detection system.

[0211] (3) Detection methods for cell activity-related molecules

[0212] [1] Preparation of single-cell suspension of surface molecule-labeled cells: Take 9.0×10 6 cultured NK cells and place them in a 15 ml centrifuge tube. Centrifuge at 400 g for 8 minutes to collect the cells, wash twice with 10 ml of 1×PBS, and finally resuspend them in 0.81 ml of 1×PBS to form a single-cell suspension.

[0213] [2]Blocking: Add 90 μl of mouse serum to the surface molecule-labeled cells obtained in [1], mix well, and let stand at room temperature for 15 - 30 minutes.

[0214] [3]Labeling antibody: Aliquot the blocked surface molecule-labeled cells obtained in step [2] into 10 flow cytometry tubes at 0.09 ml per tube. If multiple batches of cells are to be detected simultaneously, the cells in tubes 1 - 8 can be labeled with an equal mixture of several batches of cells. The cell dosage per tube is: 0.8 - 1×10 6 cells. The sample tube contains surface molecule-labeled cells, and the cell dosage is: 0.8 - 1×10 6 cells. Then add the corresponding fluorescently labeled antibody, mix well, and let stand at 4°C in the dark for 30 minutes (mix well every 15 minutes in the middle). The specific labeling method is shown in Table 1.

[0215] Table 1. Sample tubes and experimental operation table for detecting surface molecules of NK cells

[0216]

[0217]

[0218] [4]Washing: Add 1 ml of 1×PBS to each sample tube, centrifuge at 400 g for 8 minutes at 4°C to collect the cells. Repeat the washing 2 times with 1 ml of 1×PBS. Finally, add 200 μl of 1×PBS to each tube to resuspend the labeled cells. Add 5 μl of DAPI (50 μg / ml, 40×) to the experimental group and transfer the tubes to the instrument for detection.

[0219] [5]Detection: Calibrate and adjust the flow cytometer according to the instrument instruction manual. The blank control tube sample is used to adjust the voltages of forward scatter and side scatter. The isotype control sample is used to adjust the voltages of each fluorescence channel so that the fluorescence is within the negative range. The single-label control sample is used to adjust the fluorescence compensation of each channel. After defining the cell gate during detection, collect 1×10 4 cells in each sample gate.

[0220] The results are as Figure 7 shown. In terms of the expression of NK cell inhibitory receptor-related molecules such as CD94, TIM3, TIGIT, and CD158d molecules, the ST group is the highest, followed by the ST + HSA group, and the ST + HSA + VC group is the lowest, indicating that the addition of HSA and VC in the cryopreservation formula can improve the activity of NK cells after cryopreservation and recovery.

[0221] Example 6 Construction of animal model

[0222] 1 Construction of tumor model

[0223] 1) Grouping of experimental animals:

[0224] PBS group: Injected with PBS as a control;

[0225] NK-A group: Administered with the cryopreserved and revived cell preparation from Group 2 of Example 1;

[0226] NK-B group: Administered with the cryopreserved and revived cell preparation from Group 3 of Example 1;

[0227] NK-B + VC group: Administered with the cryopreserved and revived cell preparation from Group 3 of Example 1 + VC as an adjuvant;

[0228] NK-B + factor group: Administered with the cryopreserved (pretreated with cytokines: 50 ng / mL IL-15 and 250 ng / mL IL-18) and revived cell preparation from Group 4 of Example 1;

[0229] NK-B + VC + factor group: Administered with the cryopreserved (pretreated with cytokines: 50 ng / mL IL-15 and 250 ng / mL IL-18) and revived cell preparation from Group 4 of Example 1 + VC as an adjuvant.

[0230] 2) Collect SKOV3 cells in the logarithmic growth phase and wash them twice with PBS

[0231] After that, digest them with trypsin into a single-cell suspension.

[0232] 3) Wash the cell suspension with PBS and prepare a cell suspension of 8×10 7 cells / mL. Then mix it with Matrigel at a volume ratio of 9:1, and after mixing, aliquot it into 2 mL sterile cryogenic vials. All the above operations need to be carried out on ice chips.

[0233] 4) After preparation, place it on ice and send it to the animal house. After mixing the cells, subcutaneously inoculate them into healthy and qualified NOG mice.

[0234] 5) Before using the laminar flow hood, irradiate it with ultraviolet light for at least 20 min. Turn on the laminar flow hood and wait for it to run stably before operating.

[0235] 6) Use a 1 mL pipette to fully mix the cells, and then use a 1 mL insulin syringe to aspirate 100 μL (8×10 6 cells / mouse) of the cell suspension;

[0236] 7) Wipe the right back of the mouse with a 75% alcohol cotton ball, and hold the syringe in the right hand to subcutaneously inject the tumor cells to establish a tumor-bearing model.

[0237] 2 NK cell administration method

[0238] 1) Before using the laminar flow hood, irradiate it with ultraviolet light for at least 20 min. Turn on the laminar flow hood and wait for it to run stably before operating.

[0239] 2) After thoroughly mixing the cells with a 1-ml pipette, aspirate 250 μl of the cell suspension with a 1-ml insulin syringe.

[0240] 3) Wipe the mouse's tail with a 75% alcohol cotton ball. Hold the syringe in the right hand and infuse the preparation through the tail vein injection.

[0241] 4) Adjuvant administration: IL-2, 50,000 IU / mouse, administered intraperitoneally once every other day. VC is administered daily from D1 to D7, and intravenously once each on D9 and D11, 60 mg / kg.

[0242] i. Administration schedule for the pharmacodynamic model: Administer once every other day for 2 courses, i.e., on D1, D3, D5, D13, D15, D17, for a total of 6 administrations. The administration day is D1.

[0243] ii. Administration schedule for the pharmacokinetic model: Single administration.

[0244] Evaluation of the pharmacodynamic tumor model in Example 7

[0245] Based on the animal model and NK cell administration method constructed in Example 6, evaluate the pharmacodynamic tumor model.

[0246] 1) Pharmacodynamic observation: Observe 2 times a week (Day1, 5, 10, 13, 17, 20, 24, 27, for a total of 8 times) for tumor measurement.

[0247] 2) Tumor inhibition rate: Statistically analyze the tumor inhibition rate of different administration groups after cell reinfusion.

[0248] i. Measurement and calculation of tumor volume: Measure and record the long and short diameters of the tumor using a vernier caliper, and calculate the tumor volume according to the following formula: V = 1 / 2 × long diameter × short diameter²

[0249] ii. Tumor inhibition rate = (Average tumor volume in the Control group - Average tumor volume of each mouse in the test group) / Average tumor volume in the Control group %

[0250] The results are as Figure 8 shown. All different administration groups effectively inhibited tumor growth, and the pharmacodynamic ranking was NK-B + VC + factor group > NK-B + VC group ≈ NK-B + factor group > NK-B group > NK-A group. This indicates that NK cells cryopreserved with the D10 basic cryopreservation solution supplemented with HSA have good tumor killing ability, and adjuvant VC administration or cytokine pretreatment can further enhance this ability. Moreover, when cytokine pretreatment and adjuvant VC administration coexist, the pharmacodynamic effect can be optimized.

[0251] Evaluation of the pharmacokinetic model in Example 8

[0252] Based on the animal model and NK cell administration method constructed in Example 6, the pharmacokinetic model was evaluated.

[0253] 1. Experimental mouse dissection

[0254] 1) Place the mouse on the cage rack. Hold the mouse's tail with the right hand, and use the thumb and index finger of the left hand to press the skin around the mouse's eyes towards the back of the neck as much as possible to make the eyes congested and protrude.

[0255] 2) Use a curved forceps to clamp the root of the mouse's eyeball, slowly pull out the mouse's eyeball, and invert the mouse with its head down so that the blood flows vertically. At the same time, continuously press the mouse's heart to accelerate the heart pumping speed, and collect the mouse's peripheral blood into a 1.5 ml EP tube (the peripheral blood volume of 1 mouse is 200 - 1500 μl / mouse).

[0256] 3) After the collection, quickly invert and mix repeatedly.

[0257] 4) Euthanize the mouse by cervical dislocation.

[0258] 5) Use forceps to pick up the outer epidermis in the middle of the mouse's abdomen, cut open the outer epidermis with scissors, hold the outer epidermis at the cut with your hand, and tear the outer epidermis parallelly in two directions: towards the head and the tail of the mouse.

[0259] 6) Use forceps to pick up the inner membrane of the mouse's abdominal cavity and cut it open with scissors to expose the mouse's abdominal cavity.

[0260] 7) Use forceps to gently pick up the xiphoid process of the mouse, cut off the mouse's liver with scissors, put it into pre-cooled 1×PBS, and place it in a 4℃ refrigerator or on an ice box for standby.

[0261] 8) The red tissue adjacent to the stomach on the left side of the mouse is the mouse's spleen. Use forceps to gently pick up the spleen and cut off the mouse's spleen with scissors, put it into pre-cooled 1×PBS, and place it in a 4℃ refrigerator or on an ice box for standby.

[0262] 9) Use forceps to gently pick up the xiphoid process of the mouse, cut open the diaphragm with scissors, cut the mouse's ribs along the direction of the xiphoid process to expose the mouse's thoracic cavity; use forceps to hold the mouse's lung, cut the connection between the mouse's lung and the heart, etc. with scissors, remove the mouse's lung, put it into pre-cooled 1×PBS, and place it in a 4℃ refrigerator or on an ice box for standby.

[0263] 10) Use forceps to pick up the skin at the mouse's tumor site, carefully dissect the tumor tissue with an ophthalmic scissors, put it into pre-cooled 1×PBS, and place it in a 4℃ refrigerator or on an ice box for standby.

[0264] 11) Bring the samples back to the laboratory for pretreatment.

[0265] 2. Experimental mouse dissection

[0266] 2.2.1 Solid tumor and lung

[0267] 1) Remove the mouse solid tumor and lung tissues from 1×PBS, place them in a 6-well plate, cut the mouse lungs into tissue blocks of 1 mm3 size with scissors, add 3 mL of 1×PBS, add 30 μL of CollagenaseⅣ and 30 μL of DNASE I (1:100), mix well, and then place them in an incubator at 37°C and 5% CO2 for static digestion for 1 h (mix once every 30 min);

[0268] 2) Transfer the digested tumor and lung tissue cells to a tissue processing tube with a Pasteur pipette and perform semi-automatic dissociation using the Miltenyi tissue dissociation instrument;

[0269] 3) Discard the floating large jelly-like tissues;

[0270] 4) Centrifuge at 700×g, 4°C for 10 min;

[0271] 5) Pour off the supernatant and invert the centrifuge tube on a paper towel to suck away the excess liquid;

[0272] 6) Resuspend the precipitate with 4 mL of 42% Percoll solution, mix the cells well with a Pasteur pipette and gently add them to a 15 ml centrifuge tube containing 2 mL of 70% Percoll (add slowly to avoid causing fluctuations in the liquid level);

[0273] 7) Centrifuge at 1200×g for 30 min at 25°C with 6 rises and 2 drops;

[0274] 8) After centrifugation, aspirate the leukocytes in the middle layer into a 15 mL centrifuge tube and add 10 ml of 1×PBS;

[0275] 9) Centrifuge at 700×g, 4°C for 10 min;

[0276] 10) Discard the supernatant, filter through a 200-mesh filter, resuspend with 1 mL of 1×PBS and transfer to a 1.5 mL EP tube;

[0277] 11) Centrifuge at 700×g, 4°C for 10 min;

[0278] 12) Discard the supernatant and resuspend the cells with 200 μL of 1×PBS;

[0279] 13) Take 20 μl of the cell suspension for dilution and counting;

[0280] 2.2.2 Liver

[0281] 1) Transfer the liver tissue to a tissue processing tube, aspirate 3 mL of 1×PBS with a pipette, add 30 μL of CollagenaseⅣ and 30 μL of DNASE I (1:100), and perform semi-automatic dissociation using the Miltenyi tissue dissociation instrument;

[0282] 2) Centrifuge at 700×g, 4°C for 10 min;

[0283] 3) Pour off the supernatant and invert the centrifuge tube on a paper towel to aspirate the excess liquid;

[0284] 4) Resuspend the pellet in 4 mL of 42% Percoll solution, mix the cells thoroughly with a Pasteur pipette and gently add to a 15 ml centrifuge tube containing 2 mL of 70% Percoll (add slowly to avoid disturbing the liquid surface);

[0285] 5) Centrifuge at 1200×g, 25°C for 30 min with a setting of 6 up and 2 down;

[0286] 6) After centrifugation, aspirate the leukocytes in the middle layer into a 15 mL centrifuge tube and add 10 ml of 1×PBS;

[0287] 7) Centrifuge at 700×g, 4°C for 10 min;

[0288] 8) Discard the supernatant, filter through a 200-mesh sieve, resuspend in 1 mL of 1×PBS and transfer to a 1.5 mL EP tube;

[0289] 9) Centrifuge at 700×g, 4°C for 10 min;

[0290] 10) Discard the supernatant and resuspend the cells in 200 μL of 1×PBS;

[0291] 11) Take 20 μl of the cell suspension for dilution and counting;

[0292] 2.2.3 Spleen

[0293] 1) Transfer the spleen tissue to a tissue processing tube, pipette 3 mL of 1×PBS, add 30 μL of CollagenaseⅣ and 30 μL of DNASE I (1:100), and perform semi-automatic dissociation using a Miltenyi tissue dissociator;

[0294] 2) Centrifuge at 700×g, 4°C for 10 min;

[0295] 3) Pour off the supernatant and invert the centrifuge tube on a paper towel to aspirate the excess liquid;

[0296] 4) Add 1 mL of red blood cell lysate and incubate in the dark at 4°C for 5 min

[0297] 5) Add 10 mL of 1×PBS to terminate the lysis;

[0298] 6) Centrifuge at 700×g, 4°C for 10 min;

[0299] 7) After centrifugation, discard the supernatant, aspirate the excess supernatant on a paper towel, resuspend with 1 mL of 1×PBS, filter through a 200-mesh filter, and transfer the cells to a 1.5 mL EP tube;

[0300] 8) Centrifuge at 700×g, 4 °C for 10 min;

[0301] 9) After centrifugation, discard the supernatant completely with a 1 ml pipette, and resuspend the cell pellet with 200 μL of 1×PBS;

[0302] 10) Take 20 μl of the cell suspension for dilution and counting;

[0303] 2.2.4 Peripheral blood

[0304] 1) Centrifuge at 900×g, 4 °C for 5 min, let stand for 5 min, and discard the upper serum;

[0305] 2) Mix the cells thoroughly with a 1 ml pipette, take 100 μl of peripheral blood and put it into an EP tube containing 1 ml of red blood cell lysate, mix by inverting up and down, and lyse at room temperature for 5 min;

[0306] 3) Centrifuge at 800×g, 4 °C for 10 min;

[0307] 4) Discard the supernatant completely with a 1 m pipette, and add 1 mL of 1×PBS to resuspend;

[0308] 5) Centrifuge at 800×g, 4 °C for 10 min;

[0309] 6) Discard the supernatant completely with a 1 m pipette, and add 1 mL of 1×PBS to resuspend;

[0310] 7) Centrifuge at 800×g, 4 °C for 10 min;

[0311] 8) After centrifugation, discard the supernatant completely with a 1 ml pipette, and resuspend the cell pellet with 200 μL of 1×PBS;

[0312] 9) Take 20 μl of the cell suspension for dilution and counting;

[0313] 2.3 Detection of in vivo biodistribution of NK cells by flow cytometry

[0314] 2.3.1 Blocking

[0315] Adjust the cell density to less than 1×107 cells / ml, take 200 μL of the cell suspension, add mouse serum (1:10), and incubate at 4 °C for 15 min;

[0316] 2.3.2 Antibody labeling

[0317] 1) Add 20 μL of the premixed antibody to each tube, incubate at 4 °C for 30 min, and mix once in the middle

[0318] Antibody premixing:

[0319] Ac7 CD45, p-p CD45m, BV785 CD56, FITC CD3

[0320] 2) Add 1 mL of 1×PBS, centrifuge at 800×g for 10 min at 4°C, wash twice, and discard the supernatant.

[0321] 3) Resuspend the cells with 200 μL of 1×PBS.

[0322] Alternative step: Discard the supernatant, disperse the cells by flicking, and resuspend the cells with 200 μL of cell fixation solution.

[0323] 4) Add 2.5 μL of DAPI to each well and detect by flow cytometry.

[0324] 2.3.3 In vivo biodistribution evaluation indicators

[0325] 1) Number of NK cells = NK cell chimerism rate × tissue cell counting result

[0326] 2) NK cell chimerism rate = % of human NK+ cells × % of human CD45+ cells × 100 / % of murine CD45+ cells

[0327] The results are as Figure 9 shown. The peak time points and distribution patterns of NK cells in different groups in different visceral tissues in vivo are basically the same. The peak time of cells in tumor tissue is D22 days, and the peak time points of other visceral tissues are D3 or D5, indicating that tumor tissue is conducive to stimulating the proliferation and survival of NK cells. The in vivo distribution effect of the NK-B+VC+ factor group is the best (consistent with the pharmacodynamic results), and there are significant differences in the total content in the liver, peripheral blood, spleen, and tumor tissue near the peak compared with the NK-A group. The NK cell content (peak) of the NK-B+VC+ factor group and the NK-B+VC group in tumor tissue at D15 and D22 after drug administration is significantly higher than that of the NK-A group. Among them, the NK cell content of the NK-B+VC+ factor group is the highest, which is 11.72 times that of the NK-A group. Combining the in vivo pharmacodynamic data, it is indicated that pretreatment with cytokines and VC treatment can significantly promote the in vivo proliferation of NK cells, thereby exerting better pharmacodynamic effects.

[0328] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present application.

Claims

1. A cryopreservation solution for NK cells, characterized in that, Comprising: A basal solution and human serum albumin, wherein the basal solution comprises methylcellulose and dimethyl sulfoxide.

2. The NK cell cryopreservation solution according to claim 1, wherein Based on the total volume of the NK cell cryopreservation solution, the concentration of the human serum albumin is 20 mg / mL to 30 mg / mL; Optionally, based on the total volume of the basal solution, the methylcellulose content is 0.05% to 0.2%, and the dimethyl sulfoxide content is 9% to 11%; Optionally, the basal solution further comprises inorganic acid, sodium hydroxide, hydroxypropyl-β-cyclodextrin and water.

3. The NK cell cryopreservation solution according to claim 1, wherein Further comprising vitamin C; Based on the total volume of the NK cell cryopreservation solution, the concentration of the vitamin C is 200 μg / mL to 300 μg / mL.

4. A combined preparation, characterized in that, Comprising: A first preparation, the first preparation comprising the NK cell cryopreservation solution according to claim 1 or 2; A second preparation, the second preparation comprising cytokines.

5. The combined preparation according to claim 4, characterized in that, The cytokines include at least one of IL-2, IL-12, IL-15, IL-18 and IL-7.

6. The combined preparation according to claim 4 or 5, characterized in that, The NK cell cryopreservation solution further contains vitamin C; or The combined preparation further comprises: a third preparation, the third preparation comprising vitamin C.

7. A kit, characterized in that, Comprising: The combined preparation according to any one of claims 4 to 6.

8. Use of the NK cell cryopreservation solution according to any one of claims 1 to 3, the combined preparation according to any one of claims 4 to 6 or the kit according to claim 7 in at least one of the following: NK cell cryopreservation; Improving the proliferation ability of NK cells after cryopreservation and resuscitation; Improving the killing ability of NK cells after cryopreservation and resuscitation.

9. A method for cryopreserving NK cells, characterized in that, Comprising: Mixing NK cells with the NK cell cryopreservation solution according to claim 1 or 2 to obtain a mixed solution; Performing a freezing treatment on the mixed solution.

10. The method according to claim 9, characterized in that, The method is implemented by using the combined preparation according to claim 4 or 5 or the kit according to claim 7, and the method comprises: Performing a first mixing and culturing on NK cells with the second preparation, and collecting the cells after culturing; Performing a second mixing and freezing treatment on the cells after culturing with the first preparation; Optionally, in the mixed solution obtained by the first culturing, the final concentration of each of the cytokines is 10 ng / mL to 300 ng / mL; Optionally, the culturing time is 12 h to 24 h, and the temperature is 35°C to 40°C; Optionally, the freezing treatment comprises: programmed cooling to -85°C to -95°C for cryopreservation.

11. The method according to claim 9, wherein The NK cell cryopreservation solution contains vitamin C; or the second mixing is performed on the cells, the first preparation and vitamin C.

12. A method for improving the proliferation ability and / or killing ability of NK cells after cryopreservation and resuscitation, characterized in that, Comprising: Resuscitating the NK cells cryopreserved by using the NK cell cryopreservation method according to claim 8 or 9 to obtain a cell solution, and collecting the NK cells after resuscitation in the cell solution; Co-culturing the NK cells after resuscitation with vitamin C; Optionally, the method further comprises: co-culturing the NK cells after resuscitation, vitamin C and cytokines; Preferably, the cytokines include at least one of IL-2, IL-12, IL-21 and IL-15.

13. A NK cell preparation, characterized in that, Comprising: NK cells and the NK cell cryopreservation solution according to claim 1 or 2, wherein the NK cells are located in the NK cell cryopreservation solution.

14. The NK cell preparation according to claim 13, wherein The NK cell cryopreservation solution includes: vitamin C.

15. A drug, characterized in that, Includes: The NK cell preparation according to claim 13.

16. A combined drug, characterized in that, Includes: The drug according to claim 15 and vitamin C; Optionally, the combined drug further includes cytokines, and the cytokines include at least one of IL-2, IL-12, IL-21, and IL-15.

17. Use of the drug according to claim 15 or the combined drug according to claim 16 in the preparation of a pharmaceutical preparation, characterized in that, The pharmaceutical preparation is used for preventing or treating cancer; Optionally, the cancer includes at least one of lung cancer, thyroid cancer, glioma, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, cholangiocarcinoma, acute lymphoblastic leukemia, lymphoma, multiple myeloma, neuroendocrine cancer, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, esophageal cancer, oral squamous cell carcinoma, and gastric cancer; Optionally, the pharmaceutical preparation is used for inhibiting the expression of at least one of the following inhibitory receptor molecules by NK cells: CD94, TIGIT, CD158d, and TIM-3.

18. A method for inhibiting the expression of inhibitory receptor molecules on NK cells, wherein the inhibitory receptor molecules include at least one of the following: CD94, TIGIT, CD158d, and TIM-3, characterized in that, Includes: Contacting NK cells, the NK cell cryopreservation solution according to claim 1 or 2, and vitamin C.

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