NK cell cryopreservation solution, NK cell cryopreservation method, drug, and combination drug and use thereof

Abstract

An NK cell cryopreservation solution, an NK cell cryopreservation method, a drug, and a combination drug and the use thereof. The NK cell cryopreservation solution comprises a base solution and human serum albumin, wherein the base solution comprises methyl cellulose and dimethyl sulfoxide.

Description

NK cell cryopreservation solutions, NK cell cryopreservation methods, drugs and combination drugs and their applications Technical Field

[0001] This application relates to the field of biomedicine. Specifically, this application relates to NK cell cryopreservation solutions, NK cell cryopreservation methods, drugs and combination drugs, and their applications. Background Technology

[0002] Natural killer (NK) cells are important immune cells capable of recognizing and killing tumor cells and virus-infected cells. NK cells are increasingly used in cell therapy and immunotherapy. Their cytotoxic activity is not MHC-restricted and does not depend on antibodies, hence the term "natural killer activity." NK cells have abundant cytoplasm containing large azurophilic granules, and the granule content is positively correlated with their cytotoxic activity. NK cells constitute 5-10% of the circulating lymphocyte population and can secrete perforin and tumor necrosis factor to destroy target cells, demonstrating significant clinical importance and promising applications.

[0003] Cell cryopreservation is a technique that stores cells in a low-temperature environment to reduce cellular metabolism and achieve long-term preservation. During cryopreservation, proper handling and suitable freezing conditions can minimize changes or loss of cell characteristics, thus preserving the cell line. Direct freezing of cells without any cryoprotectants can lead to cryodamage, causing water in both the intracellular and extracellular environments to form ice crystals. This can result in mechanical damage, increased electrolyte levels, changes in osmotic pressure, dehydration, pH changes, protein denaturation, and ultimately, cell death. Therefore, cryoprotectants should be added to the culture medium to lower the freezing point. Slow freezing conditions allow intracellular water to permeate out of the cells before freezing, and storing cells in liquid nitrogen can reduce ice crystal formation.

[0004] To avoid the increased costs associated with prolonged culture, the decline in cell growth and activity beyond the optimal logarithmic growth phase, which could affect the quality of cell therapy, and the increased pain and trauma caused to patients by repeated blood draws, preserving cultured NK cells has become a necessary method. Therefore, preserving the activity and function of NK cells through cryopreservation is particularly important. Traditional cryopreservation methods often lead to decreased cell activity, affecting their clinical application. Therefore, developing an effective NK cell cryopreservation solution and method is of great significance. Summary of the Invention

[0005] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application proposes NK cell cryopreservation solutions, combination formulations, kits and their applications, NK cell cryopreservation methods, methods for improving the proliferation and / or killing ability of NK cells after cryopreservation and thawing, NK cell preparations, drugs, combination drugs and their applications. The NK cell cryopreservation solution of this application can significantly improve the activity recovery rate of NK cells after cryopreservation, reduce cell damage, and ensure that NK cells still possess high killing and proliferative abilities after thawing and culture. Furthermore, the NK cell cryopreservation solution has simple and reliable components, can be directly infused into the human body, and is easy to prepare and store, making it suitable for large-scale cell cryopreservation and clinical translation, with high application value.

[0006] It should be noted that this application is based on the inventor's following discoveries:

[0007] Currently, existing technologies mainly involve preparing cryopreservation solutions using fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), and RPMI-1640 medium in different proportions for the preservation of NK cells after culture, or using autologous serum instead of FBS for cryopreservation. While the former method preserves cells for a longer period, the use of FBS-based cryopreservation solutions introduces exogenous proteins, increasing the risk of pathogen contamination. Using autologous serum completely eliminates the risk of exogenous contaminant infection, but the viability of NK cells after cryopreservation and thawing is low, making further expansion culture extremely difficult. Furthermore, some patents disclose overly complex compositions, posing significant challenges to material controllability and clinical application. Therefore, the current NK cell cryopreservation technology faces the following industrialization bottlenecks:

[0008] 1. The cryopreservation solution has a complex composition, containing several or even more than a dozen components, which is not conducive to large-scale production and application; it contains exogenous animal protein components, which increases the risk of pathogen contamination; and it contains culture medium components, so it cannot be directly applied to humans.

[0009] 2. Poor cell viability and function after thawing. Most studies only focus on the cell state, viability and function at the time of thawing. However, in real in vitro and in vivo applications, cells need a recovery process. The cryopreservation solution commonly used in this field results in a large number of cell deaths after a few hours of culture, which is not the true effect of cell cryopreservation.

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

[0011] In view of this, the inventors of this application, through in-depth research, analysis, and screening, have obtained an NK cell cryopreservation solution containing human serum albumin, methylcellulose, and dimethyl sulfoxide. Human serum albumin provides a stable protein environment, reducing cell damage during cryopreservation; methylcellulose has good biocompatibility and stability, forming a protective gel layer to further protect cell structure; dimethyl sulfoxide, as an antifreeze agent, lowers the freezing point of the solution and reduces ice crystal formation, thereby effectively protecting cells from freezing damage and improving cell viability recovery rate. The synergistic effect of these three components enables the cryopreservation solution to significantly improve the viability recovery rate of NK cells after cryopreservation, reduce cell damage, and ensure that cells retain high cytotoxic and proliferative capacity after thawing and culture. The activity of cryopreserved and thawed NK cells is close to that of fresh NK cells before cryopreservation, allowing them to exert good pharmacological effects in vivo. Furthermore, all three components are GMP or pharmaceutical grade, allowing for direct infusion into the human body. This significantly advances the clinical application of NK cell products, improving the safety and stability of NK cell preparations in clinical use. It also avoids the contamination risks associated with post-resuscitation procedures such as centrifugation and resuscitation, preventing cell damage and resulting quality variations before infusion. Additionally, it lowers the barrier to entry for clinical application of cell products. Moreover, the cryopreservation solution is simple in composition, low in cost, and does not introduce animal-derived components, ensuring high safety. Therefore, it is suitable for large-scale cell cryopreservation and clinical translation, demonstrating high application value.

[0012] Therefore, in one aspect of this application, an NK cell cryopreservation solution is proposed. According to an embodiment of this application, the NK cell cryopreservation solution comprises: a base solution and human serum albumin, wherein the base solution comprises methylcellulose and dimethyl sulfoxide. The synergistic effect of these three components enables the cryopreservation solution to significantly improve the activity recovery rate of NK cells after cryopreservation, reduce cell damage, and ensure that the cells retain high cell-killing and proliferative capabilities after thawing and culture. The activity of cryopreserved and thawed NK cells is close to that of fresh NK cells before cryopreservation, allowing them to exert good pharmacological effects in vivo. Furthermore, all three components are GMP or pharmaceutical grade, suitable for direct infusion into the human body, which greatly promotes the clinical application of NK cell products, is suitable for large-scale cell cryopreservation and clinical translation, and has high application value.

[0013] According to embodiments of this application, the above-mentioned NK cell cryopreservation solution may also have the following additional technical features:

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

[0015] According to an embodiment of this application, based on the total volume of the base liquid, the methylcellulose content is 0.05%~0.2%, and the dimethyl sulfoxide content is 9%~11%.

[0016] According to embodiments of this application, the base solution further comprises an inorganic acid, sodium hydroxide, hydroxypropyl-β-cyclodextrin, and water.

[0017] According to an embodiment of this 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 this application, a combination formulation is proposed. According to an embodiment of this application, the combination formulation comprises: a first formulation comprising the aforementioned NK cell cryopreservation solution; and a second formulation comprising cytokines.

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

[0020] According to embodiments of this application, the NK cell cryopreservation solution further contains vitamin C; or the combined formulation further includes a third formulation, the third formulation comprising vitamin C.

[0021] In another aspect of this application, a kit is provided. According to an embodiment of this application, the kit comprises the aforementioned combination formulation.

[0022] In another aspect of this application, this application proposes the use of the aforementioned NK cell cryopreservation solution, the combined formulation, or the kit in at least one of the following: NK cell cryopreservation; enhancing the proliferation capacity of NK cells after cryopreservation and thawing; enhancing the killing capacity of NK cells after cryopreservation and thawing.

[0023] In another aspect of this application, a method for cryopreserving NK cells is proposed. According to an embodiment of this application, the method includes: mixing NK cells with the aforementioned NK cell cryopreservation solution to obtain a mixture; and freezing the mixture.

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

[0025] According to an embodiment of this application, the final concentration of each cytokine in the culture mixture is 10 ng / mL to 300 ng / mL.

[0026] According to an embodiment of this application, the culture time is 12 h to 24 h, and the temperature is 35℃ to 40℃.

[0027] According to an embodiment of this application, the freezing process includes: programmed cooling to... Store frozen at 85℃ to -95℃.

[0028] According to an embodiment of this application, the NK cell cryopreservation solution contains vitamin C; or the cells, the first preparation, and vitamin C are mixed in the second manner.

[0029] In another aspect of this application, a method is proposed to improve the proliferative capacity and / or cytotoxic capacity of NK cells after cryopreservation and thawing. According to an embodiment of this application, the method includes: thawing NK cells cryopreserved using the aforementioned NK cell cryopreservation method to obtain cell fluid, collecting the thawed NK cells from the cell fluid; and co-culturing the thawed NK cells with vitamin C.

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

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

[0032] In another aspect of this application, an NK cell preparation is provided. According to an embodiment of this application, the NK cell preparation comprises: NK cells and the aforementioned NK cell cryopreservation solution, wherein the NK cells are located in the NK cell cryopreservation solution.

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

[0034] In another aspect of this application, a medicament is proposed. According to an embodiment of this application, the medicament comprises: the aforementioned NK cell preparation.

[0035] In another aspect of this application, a combination drug is proposed. According to an embodiment of this application, the combination drug comprises: the aforementioned drug and vitamin C.

[0036] According to embodiments of this application, the combined drug further comprises cytokines, the cytokines including at least one of IL-2, IL-12, IL-21, and IL-15.

[0037] In another aspect of this application, the use of the foregoing medicament or the combination of medicaments in the preparation of a pharmaceutical formulation is proposed. According to embodiments of this application, the pharmaceutical formulation is used for the prevention or treatment of cancer.

[0038] In another aspect of this application, a method for preventing or treating cancer is proposed, the method comprising administering the aforementioned drug or the aforementioned combination of drugs to a subject.

[0039] According to embodiments of this application, the cancer includes at least one of the following: lung cancer, thyroid cancer, glioma, colon cancer, rectal cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer, acute lymphoblastic leukemia, lymphoma, multiple myeloma, neuroendocrine carcinoma, 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.

[0040] According to embodiments of this application, the pharmaceutical preparation is used to inhibit the expression of at least one of the following inhibitory receptor molecules on NK cells: CD94, TIGIT, CD158d, and TIM-3.

[0041] In another aspect of this application, a method for inhibiting the expression of inhibitory receptor molecules by NK cells is provided, said cellular molecules including at least one of the following: CD94, TIGIT, CD158d, and TIM-3. According to an embodiment of this application, the method includes contacting NK cells, the aforementioned NK cell cryopreservation solution, and vitamin C.

[0042] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0043] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0044] Figure 1 shows a flowchart of NK cell cryopreservation and thawing according to an embodiment of this application;

[0045] Figure 2 shows the NK cell survival rate and recovery rate analysis according to Example 1 of this application;

[0046] Figure 3 shows a cell expansion capacity analysis diagram according to Example 1 of this application;

[0047] Figure 4 shows a cell flow cytometry plot (percentage of NK cells) according to Example 2 of this application.

[0048] Figure 5 shows the analysis of killing efficiency, killing-related molecules, and NK cell percentage according to Example 3 of this application;

[0049] Figure 6 shows the NK cell killing efficiency and cell number analysis according to Example 4 of this application;

[0050] Figure 7 shows the analysis of different molecular expression levels in NK cells according to Example 5 of this application;

[0051] Figure 8 shows the analysis of tumor inhibition rate in mice according to Example 7 of this application;

[0052] Figure 9 shows the distribution and content changes of NK cells in an animal after infusion according to Example 8 of this application. Embodiments of the present invention

[0053] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0054] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0055] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0056] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0057] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0058] This application discloses NK cell cryopreservation solutions, combination preparations, kits and their applications, NK cell cryopreservation methods, methods for improving the proliferation and / or killing ability of NK cells after cryopreservation and thawing, NK cell preparations, drugs, combination drugs and their applications, which will be described in detail below.

[0059] NK cell cryopreservation solutions, combination formulations, and kits

[0060] In one aspect of this application, an NK cell cryopreservation solution is provided. According to an embodiment of this application, the NK cell cryopreservation solution comprises: a base solution and human serum albumin, wherein the base solution comprises methylcellulose and dimethyl sulfoxide.

[0061] Human serum albumin provides a stable protein environment, reducing cell damage during cryopreservation; methylcellulose exhibits good biocompatibility and stability, forming a protective gel layer to further protect cell structure; dimethyl sulfoxide, as an antifreeze agent, lowers the freezing point of the solution and reduces ice crystal formation, effectively protecting cells from freezing damage and improving cell viability recovery. The synergistic effect of these three components allows the cryopreservation solution to significantly improve the viability recovery rate of NK cells after cryopreservation, reduce cell damage, and ensure that NK cells retain high killing and proliferative capabilities after thawing and culture. The activity of cryopreserved and thawed NK cells is close to that of fresh NK cells before cryopreservation. Furthermore, all three components are GMP or pharmaceutical grade, directly applicable to human use. This greatly advances the clinical application of NK cell products, not only improving the safety and stability of NK cell preparations in clinical applications but also avoiding the contamination risks associated with centrifugation and resuspension after cell thawing, preventing cell damage and resulting quality differences before infusion, and lowering the barrier to entry for clinical application of cell products. Moreover, this cryopreservation solution has a simple composition, low cost, does not introduce animal-derived components, and is highly safe. Therefore, it is suitable for large-scale cell cryopreservation and clinical translation, and has high application value.

[0062] According to embodiments of this application, based on the total volume of the NK cell cryopreservation solution, the concentration of human serum albumin is 20 mg / mL to 30 mg / mL, for example, 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, and 30 mg / mL. This effectively stabilizes the cell membrane structure, reduces the mechanical damage and osmotic pressure changes experienced by cells during cryopreservation, thereby improving cell activity and survival rate. Furthermore, it avoids increasing solution viscosity due to excessively high concentrations, which could lead to greater physical resistance to cell diffusion and activity recovery during cryopreservation and thawing, thus reducing costs and operational complexity.

[0063] According to embodiments of this application, based on the total volume of the base solution, the methylcellulose content is 0.05%~0.2%, for example 0.05%, 0.08%, 0.1%, 0.12%, 0.15%, 0.18%, 0.2%, and the dimethyl sulfoxide content is 9%~11%, for example 9%, 9.2%, 9.5%, 9.8%, 10%, 10.2%, 10.5%, 10.8%, 11%. Thus, the methylcellulose content meeting the above conditions helps to form a protective gel layer around the cells, reducing the mechanical damage and osmotic pressure changes experienced by the cells during cryopreservation, while also stabilizing the cell membrane structure and maintaining cell morphology and function. The dimethyl sulfoxide meeting the above conditions helps to lower the freezing point of the solution, reduce ice crystal formation, thereby effectively preventing cell damage caused by intracellular water freezing and improving the cell viability recovery rate.

[0064] According to embodiments of this application, the base solution further comprises an inorganic acid, sodium hydroxide, hydroxypropyl-β-cyclodextrin, and water. This helps to further stabilize the cryopreservation solution and extend its shelf life. Exemplarily, the inorganic salt includes hydrochloric acid.

[0065] In some embodiments, the base solution can be NutriFreez® D10 (05-714-1A, Sartorius, hereinafter referred to as D10 or D10 cryopreservation solution), which is a serum-free, chemically defined, and animal-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 believed that dimethyl sulfoxide (DMSO) has minimal toxicity when the DMSO concentration is less than 10%. However, during disease treatment, short-term, high-volume infusions of DMSO can produce numerous toxic side effects. Therefore, while ensuring cryopreservation effectiveness, adding a lower concentration of DMSO is more beneficial for clinical use, avoiding or reducing adverse reactions caused by large-volume DMSO infusions. This application prepares an NK cell cryopreservation solution by co-preparing D10 with human serum albumin. The introduction of human serum albumin reduces the DMSO concentration, thereby improving the safety of the cryopreservation solution.

[0066] According to embodiments of this application, the NK cell cryopreservation solution further includes vitamin C. Adding vitamin C can enhance the proliferation capacity of NK cells in vivo and in vitro after cryopreservation.

[0067] According to embodiments of this 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, for example, 200 μg / mL, 220 μg / mL, 240 μg / mL, 250 μg / mL, 260 μg / mL, 280 μg / mL, or 300 μg / mL. This further enhances the proliferation capacity of cryopreserved NK cells both in vivo and in vitro.

[0068] In another aspect of this application, a combination formulation is proposed. According to an embodiment of this application, the combination formulation comprises: a first formulation comprising the aforementioned NK cell cryopreservation solution; and a second formulation comprising cytokines. Using cytokines and the NK cell cryopreservation solution in combination, pre-stimulating NK cells with cytokines before cryopreservation helps activate NK cells, promote their proliferation, and enhance NK cell activity, enabling them to maintain activity under cryopreservation conditions. Cryopreserving NK cells stimulated by cytokines in the NK cell cryopreservation solution helps to better improve the activity recovery rate of NK cells after cryopreservation, reduce cell damage, and ensure that the cells retain high cell-killing and proliferative capabilities after thawing and culture, thus exerting a better pharmacological effect in vivo.

[0069] According to embodiments of this application, the cytokines include at least one of IL-2, IL-12, IL-15, IL-18, and IL-7. IL-2 is an important cytokine that induces NK cell proliferation; it can activate NK cells, promote NK cell proliferation, and increase cytokine production. IL-15 and IL-7 have similar effects to IL-2, and they can also promote the directed differentiation of hematopoietic stem cells into NK cells by binding to the complex receptor γ chain expressed on the surface of NK cells, and play an important role in the development, differentiation, and long-term in vitro survival of NK cells. The synergistic effect of IL-15 and IL-2 also enables their combined use for the in vitro expansion of NK cells, making it the most traditional cytokine combination for in vitro expansion of NK cells. It has been reported that IL-18 not only induces activated Th1 cells to secrete large amounts of IFN-γ, but also enhances the cytotoxicity of NK cells by promoting the opening of the Fas-FasL pathway in a dose-dependent manner.

[0070] According to embodiments of this application, the NK cell cryopreservation solution further contains vitamin C; or the combined formulation further includes a third formulation comprising vitamin C. During cryopreservation, the addition of vitamin C can enhance the proliferation capacity of NK cells in vivo and in vitro after cryopreservation. Vitamin C can be added either to the cryopreservation solution or provided as a separate third formulation, which is then mixed with the cryopreservation solution when cryopreservation is required.

[0071] In another aspect of this application, a reagent kit is provided. According to an embodiment of this application, the reagent kit comprises the aforementioned combination formulation. Therefore, using the reagent kit of this application can improve the activity recovery rate of NK cells after cryopreservation, reduce cell damage, and ensure that the cells retain high cell-killing and proliferative capabilities after thawing and culture, thus exerting a better pharmacological effect in vivo.

[0072] It should be noted that the characteristics and advantages described above for NK cell cryopreservation solutions also apply to combination formulations and kits, and will not be repeated here.

[0073] Applications and methods

[0074] In another aspect of this application, the NK cell cryopreservation solution, the combined formulation, or the kit described above are proposed for use in at least one of the following: NK cell cryopreservation; improving the proliferation capacity of NK cells after cryopreservation and thawing; and improving the cytotoxic capacity of NK cells after cryopreservation and thawing. As mentioned above, the NK cell cryopreservation solution, combined formulation, and kit of this application can significantly improve the activity recovery rate of NK cells after cryopreservation, reduce cell damage, and ensure that NK cells still possess high cytotoxic and proliferative capacity after thawing and culture. Furthermore, the NK cell cryopreservation solution has a simple and reliable composition, can be directly infused into the human body, and is easy to prepare and store, making it suitable for large-scale cell cryopreservation and clinical translation, and thus has high application value.

[0075] In another aspect, this application proposes a method for cryopreserving NK cells. According to an embodiment of this application, the method includes: mixing NK cells with the aforementioned NK cell cryopreservation solution to obtain a mixture; and freezing the mixture. As mentioned above, using the NK cell cryopreservation solution, combination preparation, and kit of this application can significantly improve the viability recovery rate of NK cells after cryopreservation, reduce cell damage, and ensure that NK cells retain high killing and proliferative capabilities after thawing and culture.

[0076] According to embodiments of this application, the method is implemented using the aforementioned combination formulation or the aforementioned kit. The method includes: first mixing and culturing NK cells with the second formulation, and collecting the cultured cells; second mixing and freezing the cultured cells with the first formulation. Using cytokines and NK cell cryopreservation solution in combination, pre-stimulating NK cells with cytokines before cryopreservation helps activate NK cells, promote their proliferation, and enhance NK cell activity, enabling them to maintain activity under cryopreservation conditions. Cryopreserving NK cells stimulated by cytokines in NK cell cryopreservation solution helps to better improve the activity recovery rate of NK cells after cryopreservation, reduce cell damage, and ensure that the cells still have high cell-killing and proliferative capabilities after thawing and culture, thus exerting a better pharmacological effect in vivo.

[0077] It should be noted that the NK cells that have been cryopreserved need to be passaged in advance to give them a certain degree of cell viability. For example, passaged culture is carried out before cytokine stimulation of NK cells.

[0078] According to embodiments of this application, the final concentration of each cytokine in the first culture mixture is 10 ng / mL to 300 ng / mL, for example, 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, and 300 ng / mL. This helps to activate NK cells, promote their proliferation and cytokine production, and enable them to maintain their activity under cryopreservation conditions.

[0079] According to embodiments of this application, the culture time is 12 h to 24 h, for example, 12 h, 14 h, 15 h, 16 h, 18 h, 20 h, 22 h, or 24 h, and the temperature is 35℃ to 40℃, for example, 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃. This helps to activate NK cells, promote their proliferation and cytokine production, and enable them to maintain their activity under cryopreservation conditions.

[0080] According to an embodiment of this application, the freezing process includes: programmed cooling to... Freeze at 85℃ to -95℃. This gradual cooling allows sufficient time for water to drain from the cells, reducing ice crystal formation and effectively minimizing cell damage during freezing.

[0081] According to embodiments of this application, the NK cell cryopreservation solution contains vitamin C; or the cells, the first preparation, and vitamin C are mixed in a second manner. During cryopreservation, the addition of vitamin C can enhance the proliferation capacity of NK cells in vivo and in vitro after cryopreservation. Vitamin C can be added either to the cryopreservation solution or provided independently and mixed with the cryopreservation solution when needed.

[0082] In another aspect, this application proposes a method for improving the proliferation and / or cytotoxicity of NK cells after cryopreservation and thawing. According to an embodiment of this application, the method includes: thawing NK cells cryopreserved using the aforementioned NK cell cryopreservation method to obtain cell fluid, collecting the thawed NK cells from the cell fluid; and co-culturing the thawed NK cells with vitamin C. Therefore, the method of this application can effectively improve the activity recovery rate of NK cells after cryopreservation, reduce cell damage, and ensure that the thawed NK cells still possess high cytotoxicity and proliferation capacity.

[0083] According to an embodiment of this application, the revival includes placing the frozen cells at 35°C to 40°C until the ice crystals in the cell suspension disappear.

[0084] According to an embodiment of this application, the method further includes: co-culturing the revived NK cells, vitamin C, and cytokines. The addition of vitamin C enhances the proliferative and cytotoxic abilities of the cryopreserved NK cells. Cytokines are used to maintain the normal growth of NK cells.

[0085] According to embodiments of this application, the cytokines include at least one of IL-2, IL-12, IL-21, and IL-15.

[0086] It should be noted that the features and advantages described above for NK cell cryopreservation solutions, combination preparations, and kits also apply to this application and method, and will not be repeated here.

[0087] NK cell preparations, drugs, combination therapies and applications

[0088] In another aspect, this application proposes an NK cell preparation. According to an embodiment of this application, the NK cell preparation comprises: NK cells and the aforementioned NK cell cryopreservation solution, wherein the NK cells are located in the NK cell cryopreservation solution. As previously mentioned, the NK cell cryopreservation solution of this application improves the activity recovery rate of NK cells after cryopreservation, reduces cell damage, and ensures that NK cells retain high killing and proliferative capabilities after thawing and culture. Therefore, it can be applied to cell therapy, immunotherapy, and cell bank construction, etc., and has high application value.

[0089] It should be noted that the NK cell preparation of this application can be an NK cell preparation containing NK cells and NK cell cryopreservation solution before cryopreservation, after cryopreservation and without thawing, or after cryopreservation and thawing. The NK cell preparation is in a liquid state before cryopreservation, in a solid state after cryopreservation and without thawing, and in a liquid state after cryopreservation and thawing.

[0090] According to an embodiment of this application, the NK cell cryopreservation solution includes vitamin C. Adding vitamin C can enhance the proliferative and cytotoxic abilities of NK cells after cryopreservation and thawing.

[0091] In another aspect, this application proposes a drug. According to an embodiment of this application, the drug comprises: the aforementioned NK cell cryopreservation solution and NK cells. Therefore, the NK cells in the drug of this application possess strong cell-killing and proliferative capabilities, and can be applied to cell therapy, immunotherapy, and cell bank construction, etc., exhibiting high application value. Exemplarily, in the drug of this application, the NK cell cryopreservation solution and NK cells are obtained in the form of a mixture after cryopreservation and thawing.

[0092] In another aspect of this application, a combination drug is proposed. According to an embodiment of this application, the combination drug comprises: the aforementioned drug and vitamin C. Under the action of vitamin C, the proliferation capacity of NK cells cryopreserved in the aforementioned NK cell cryopreservation solution can be enhanced both in vivo and in vitro, helping NK cells to better exert their therapeutic effects in vivo.

[0093] It should be noted that in the combined drug of this application, the drug and vitamin C can be administered simultaneously or sequentially; both can be provided in the form of a drug composition or as two separate agents.

[0094] According to embodiments of the present invention, the combined drug further comprises cytokines, said cytokines including at least one of IL-2, IL-12, IL-21, and IL-15. This helps maintain normal NK cell growth.

[0095] According to embodiments of the present invention, the drug and / or combination drug further comprises: pharmaceutically acceptable excipients.

[0096] In this document, the term "pharmaceutically acceptable" indicates that a pharmaceutical composition can be administered to a subject without producing adverse physiological reactions that would impede the administration of the pharmaceutical composition. For example, "pharmaceuticalally acceptable excipients" refers to excipients useful in the preparation of generally safe, non-toxic, and desirable pharmaceutical compositions. Preferably, examples of such excipients or diluents include, but are not limited to: water, saline, Ringer's solution, glucose, mannitol, dextran, 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 mediators, such as non-volatile oils, may also be used.

[0097] In another aspect of this application, the use of the aforementioned drug or the combination of drugs in the preparation of pharmaceutical formulations is proposed. According to embodiments of this application, the pharmaceutical formulation is used for the prevention or treatment of cancer. As previously stated, the NK cells of this application possess superior cell-killing and proliferative capabilities, and can exert good pharmacological effects in vivo.

[0098] According to embodiments of this application, the cancer includes at least one of the following: lung cancer, thyroid cancer, glioma, colon cancer, rectal cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer, acute lymphoblastic leukemia, lymphoma, multiple myeloma, neuroendocrine carcinoma, 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.

[0099] According to embodiments of this application, the pharmaceutical preparation is used to inhibit the expression of at least one of the following inhibitory receptor molecules on NK cells: CD94, TIGIT, CD158d, and TIM-3.

[0100] In addition, this application also proposes a method for preventing or treating cancer, the method comprising: administering the aforementioned drug or the combined drug to a subject.

[0101] In another aspect of this application, a method for inhibiting the expression of inhibitory receptor molecules by NK cells is provided, said inhibitory receptor molecules including at least one of the following: CD94, TIGIT, CD158d, and TIM-3. According to an embodiment of this application, the method includes: contacting NK cells, the aforementioned NK cell cryopreservation solution, and vitamin C.

[0102] Vitamin C exerts its immunomodulatory effects on immune cells primarily through two main mechanisms: antioxidant activity and epigenetic regulation (by providing ferrous iron to the TET enzyme, ensuring its complete catalytic form and thus guaranteeing active DNA demethylation). For NK cells, it mainly affects their proliferative function. However, for cryopreserved NK cells, vitamin C can promote NK cell activity by influencing the expression of inhibitory receptors (markers such as CD94, TIGIT, or TIM-3).

[0103] It should be noted that the characteristics 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 repeated here.

[0104] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0105] Example 1: Expansion, cryopreservation and thawing of NK cells

[0106] The specific cryopreservation procedure is shown in Figure 1.

[0107] I. Preparation and cryopreservation of NK cells

[0108] 1. Obtaining Personal Boreal Cells (PBMCs)

[0109] The donor's peripheral blood was centrifuged to obtain plasma and whole blood cells. The plasma was combined and inactivated in a 56°C water bath, then centrifuged to obtain clear plasma, which was stored at 2–8°C for later use. The whole blood cells were diluted 1:1 with physiological saline and then slowly added to the lymphocyte separation medium at a ratio of 1:1.5 to separate and extract PBMCs for later use.

[0110] 2. NK cell culture and cryopreservation

[0111] 2.1 Cell culture day D0

[0112] PBMCs were isolated from apheresis blood from different healthy donors, in accordance with paragraph

[0076] of the specification of patent CN118325833A;

[0113] 2.2 Cell culture days D3-D17

[0114] Incubate until day 3 (D3), supplement with IL. 2. The final concentration was increased to 5 ng / ml, and the cells were placed in an incubator at 37°C, saturated humidity, and 5.0% CO2 for further culture.

[0115] Starting from day 5 (D5), samples were taken and counted every two days until day 21, and NK cell culture medium (KBM581 medium) was added.

[0116] 2.3 Cell culture day 21

[0117] The cells were divided into four groups, and each group was harvested and cryopreserved according to its respective protocol:

[0118] Group 1 cells were cultured to day 21, harvested, and resuspended in physiological saline containing 1.25 mg / ml human serum albumin.

[0119] Group 2 was cultured to day 21, harvested, and cryopreserved using the D10 basal cryopreservation solution;

[0120] Group 3 was cultured to day 21, harvested, and cryopreserved using D10 basal cryopreservation solution with a final concentration of 25 mg / mL human serum albumin;

[0121] In Group 4, during the first 20 hours of culture up to D21, IL-15 and IL-18 at a final concentration of 50 ng / mL and 250 ng / mL respectively were added to the culture medium and the culture was stimulated for 20 hours in a 37℃, 5% CO2 incubator. The culture was then harvested and cryopreserved using D10 basal cryopreservation solution with a final concentration of 25 mg / mL human serum albumin.

[0122] Cryopreservation involves placing a cell suspension in a sterile cryovial and then cooling it under a controlled temperature, before transferring the cells to liquid nitrogen for 90 days of cryopreservation.

[0123] The cooling process is as follows:

[0124] a. Wait at 20.0℃ until the sample is placed;

[0125] b. The sample temperature decreases at a rate of 1.0℃ per minute. 12.0℃;

[0126] c. The cavity temperature drops at a rate of 22.0℃ per minute. 50.0℃;

[0127] d. The cavity temperature drops at a rate of 4°C per minute. 18.0℃;

[0128] e. The cavity temperature drops at a rate of 1°C per minute. 45.0℃;

[0129] f. The cavity temperature drops at a rate of 10.0°C per minute. 90.0℃;

[0130] g. Cooling has ended.

[0131] II. NK Cell Resuscitation and Expansion Culture Process

[0132] 1. NK cell resuscitation

[0133] Remove the cells from the liquid nitrogen tank and quickly immerse the cryovial in a 37°C constant temperature water bath. Remove the cryovial when the ice crystals in the cell suspension are almost completely gone, as observed by the naked eye. Centrifuge the mixed cell suspension, remove the supernatant, and resuspend the cells in KBM581 medium.

[0134] 2. NK cell culture and fluid replacement

[0135] Based on the count density and volume of the day, calculate the amount of KBM581 culture medium that needs to be added according to the formula (original volume of the day × density / subculture density = final subculture volume).

[0136] 3. Experimental Results

[0137] The experiment showed that, regardless of the method of use, the NK cell viability and recovery rate were high after using the cryopreservation solution of this application (cell viability = number of viable cells / total number of cells; cell recovery rate = number of viable cells after thawing / number of viable cells before thawing), as shown in Figure 2. It can be seen that the cell recovery rates of groups 2, 3, and 4 were all above 95%, the cell viability at 0 h was above 85%, and the cell viability recovered to above 95% after 3 days of culture.

[0138] As shown in Figure 3, after 5 days of in vitro cell culture, NK cells in group 1 maintained high proliferative capacity because they were not cryopreserved. The proliferative capacity of NK cells cryopreserved in either the basal cryopreservation solution (group 2) or the basal cryopreservation solution + HSA solution (group 3) decreased, with the D10 cryopreservation solution containing human serum albumin (group 3) showing a relatively higher cryopreservation effect. NK cells pre-stimulated with cytokines, cryopreserved in the basal cryopreservation solution + HSA solution (group 4), exhibited proliferative capacity close to that of their fresh, un-cryopreserved state.

[0139] Example 2: Detection of NK cell purity and killing-related molecules

[0140] 1) Experimental materials

[0141] Flow cytometry antibodies: Fluorescently labeled surface molecular 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.

[0142] Blocking antibody: mouse serum, purchased from Future, catalog number F001008.

[0143] Flow cytometry tubes: 5ml transparent polystyrene or polypropylene round-bottomed capped test tubes.

[0144] Monensin: Purchased from Sigma, catalog number 22373-78-0.

[0145] Fixation / permeabilization Diluent: Purchased from ebioscience, catalog number 4298341.

[0146] Fixation / permeabilization concentrate: purchased from eBioscience, catalog number 4311034.

[0147] Permeabilization buffer: purchased from ebioscience, catalog number 4314840.

[0148] DAPI (4',6-Diamidino-2-Phenylindole, Dilactate): Purchased from Biolegend, part number 422801.

[0149] Flow cytometry wash buffer: PBS.

[0150] Alternatively, reagents from the same company and flow cytometry antibodies with the same clone number can be used.

[0151] 2) Main instruments and equipment

[0152] Centrifuges, clean benches, flow cytometers (such as FACS Calibur, Beckman CytoFlex, etc.), and RTCA detection systems.

[0153] 3) Cell viability-related molecular detection methods

[0154] ① Intracellular molecular marker monensin induction: Take 4×10 cells from cultured cells 6 Each sample (2 ml) was placed in a 6-well plate, and 10 μl of monensin (0.5 μg / μl) was added. The plate was then incubated at 37°C in a 5% CO2 incubator for 4 hours.

[0155] ② Preparation of single-cell suspension: 9.0 × 10⁹ cells from cultured cells were used for surface molecular marker cell preparation. 6 Each cell was placed in a 15 ml centrifuge tube, centrifuged at 400 g for 8 minutes to collect the cells, washed twice with 10 ml of 1×PBS, and finally resuspended in 0.81 ml of 1×PBS to form a single-cell suspension. After the intracellular molecular marker monensin induction was completed, the cells were washed twice with 5 ml of 1×PBS and finally resuspended in 180 μl of 1×PBS to form a single-cell suspension.

[0156] ③ Blocking: Add 90 μl of mouse serum to surface molecular marker cells, mix well, add 20 μl of mouse serum to intracellular molecular marker cells, mix well, and let stand at room temperature for 15-30 minutes.

[0157] ④ Labeling with antibodies: After blocking, aliquot the surface-labeled cells into 9 flow cytometry tubes at a rate of 0.09 ml per tube. If multiple batches of cells are to be tested simultaneously, the cells from tubes 1-8 can be mixed in equal volumes from several batches and labeled. The cell volume per tube is 0.8 × 10⁸. 6 ~1×10 6 Sample tube 1 is labeled with surface molecular markers, and the cell dosage is 0.8 × 10⁻⁶. 6 ~1×10 6 Sample tube 2 contains intracellular molecular markers; the cell dosage is 3 × 10⁻⁶ cells. 6 ~4×10 6 Add the corresponding fluorescently labeled antibody, mix well, and incubate at 4°C in the dark for 30 minutes (mixing once every 15 minutes).

[0158] Table 1. Sample tubes and experimental procedures for NK cell surface molecular detection

[0159]

[0160] ⑤ Washing: Add 1 ml of 1×PBS to each tube of surface-labeled cells, centrifuge at 400g for 8 minutes at 4℃ to collect cells. Wash twice with 1 ml of 1×PBS. Finally, add 200 μl of PBS to each tube to resuspend the labeled cells. For the experimental group, add 5 μl of DAPI (50 µg / ml, 40×). Transfer to the instrument for detection.

[0161] Sample tube 2 is an internal standard molecule. Therefore, after labeling the external standard molecules CD3, CD56, and CD45, add 5 μl of DAPI and label for 3 mins. Add 1 ml of PBS to each tube and wash twice, discarding the supernatant as thoroughly as possible.

[0162] ⑥ Add 300 μl of Fixation Buffer mix (Fixation / permeabilization concentrate: Fixation / permeabilization diluent = 1:3), fix at 4℃ for 1 h (mix every 30 min), after which add 1 ml of Perm Wash Buffer (stock solution is 10×, dilute with double-distilled water to 1× for use) 500 g, 4℃, 5 min, discard the supernatant;

[0163] ⑦ Resuspend the cells in 180 µl Perm Wash Buffer, add 20 μl of mouse serum and block at 4°C for 20 min; after blocking, divide the cells into two portions, one of which is labeled with internal standard antibody.

[0164] ⑧ Label at 4℃ in the dark for 1 hour (mix once in between). After antibody labeling is complete, add 1 ml of Perm Wash Buffer, 500g, 4℃, 5 min; and repeat the washing once more.

[0165] 9. Resuspend cells in 200 µl of Perm Wash Buffer and perform analysis.

[0166] ⑩ Detection: The flow cytometer was calibrated and adjusted according to the instrument's instruction manual. Blank control samples were used to adjust the forward and side-scattering voltages; isotype control samples were used to adjust the voltages of each fluorescence channel to ensure fluorescence was within the negative range. Single-label control samples were used to adjust fluorescence compensation for each channel. During detection, after delineating the cell gates, 1×10⁻⁶ samples were collected from each sample gate. 4 Each cell.

[0167] 4) Determination of the validity of experimental results

[0168] The blank control tube sample has a positive rate of <1% in fluorescence staining and an absolute cell count of less than 100. The positive rate of each of the five isotype controls with different fluorescently labeled antibodies is <1% and the absolute cell count is less than 100. The experimental results are considered qualified if they meet the above requirements; otherwise, the results are considered unqualified.

[0169] Percentage of positive cells (positive cells %) = (Number of positive cells ÷ Total number of cells counted) × 10⁻⁶ 4 )%

[0170] As shown in Figure 4, the proportions of NK cells and T cells detected by flow cytometry showed no significant differences between groups 2, 3, and 4 and group 1, indicating that cryopreservation does not affect the proportions of NK cells and T cells.

[0171] Example 3: Method for Detecting Cell Killing Activity

[0172] Flow cytometry detection of NK cell killing

[0173] Preparation of target cell suspension (K562 cells)

[0174] ① Cell counting: Resuspend cells in 1640 medium (the medium used for target cells) containing 0.5% FBS, count the cells, and adjust the cell density to 1×10⁻⁶. 6 / mL;

[0175] ②CFSE staining: Add CFSE (working concentration of 5 μM) to the cell suspension, immediately mix with a 1 mL pipette, vortex thoroughly, and incubate in a 37℃ incubator in the dark for 15 min. Take it out and vortex once every 5 min.

[0176] ③Terminate staining: Add 5 times the volume of 4°C pre-cooled complete culture medium (the culture medium used for target cells) to terminate staining, incubate on ice for 5 minutes, and centrifuge at 140 g, 4°C, for 5 minutes.

[0177] ④ Washing cells: Resuspend cells in pre-cooled complete culture medium (the medium used for target cells) at 4℃, centrifuge at 140 g, 4℃, for 5 min; repeat washing twice.

[0178] ⑤ Counting: Resuspend cells, count them, and adjust the cell density to 2 × 10⁻⁶. 5 / mL;

[0179] ⑥ Plating: Add 100 μl of target cell suspension to each well of a 96-well round-bottom plate to make the final cell count 20,000 cells / well;

[0180] Preparation and addition of NK cells

[0181] ⑦ Prepare NK cell suspensions from the resuscitated NK cells of groups 1, 2, 3 and 4 respectively;

[0182] ⑧ Remove E-Plate 16 and place it in the clean bench;

[0183] 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 control groups 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); and target cells + Tween-20 (as a positive control for target cell apoptosis).

[0184] ⑩ Co-incubation: Add NK cells to each well in the pre-designed order. Centrifuge at 120 g at room temperature for 2 min to ensure sufficient contact between the effector and target cells; then incubate at 37°C for 4 hours.

[0185] After incubation, add 1 μl of 7-AAD, mix well, incubate in the dark for 5 min, and then perform instrumental analysis.

[0186] Results analysis: Kill percentage = [(Target cell mortality rate in experimental group (%) - Target cell mortality rate (%)) / (100% - Natural target cell mortality rate (%))] × 100%

[0187] The results are shown in Figure 5. The proportion of NK cells after cryopreservation and thawing remained unchanged compared to the unfrozen group (group 1). The cell-killing efficiency of groups 3 and 4 after 72 h of thawing and culture was almost identical to that of the unfrozen group (group 1), and the killing efficiency of groups 3 and 4 was higher than that of group 2. Further analysis of cell-killing related molecules revealed that the expression of 4-1BB, IFN-γ, CD107a, and perforin in group 4 was higher than that in groups 2 and 3.

[0188] Example 4: Effects of VC (LAA) on NK cells (in vitro culture)

[0189] 1. NK cells were cultured, cryopreserved, and thawed according to the method in Example 1. Cryopreservation was performed in group 4. The NK cell culture conditions after thawing were as follows:

[0190] Group 1: After resuscitation, NK cells were resuspended in KBM581 medium and supplemented with KBM581 medium during culture.

[0191] Group 2: After resuscitation, NK cells were resuspended in KBM581 medium containing 5 ng / mL IL-2, and KBM581 medium containing 5 ng / mL IL-2 was added during culture.

[0192] Group 3: After resuscitation, NK cells were resuspended in KBM581 medium containing 25 mg / mL human serum albumin. During culture, KBM581 medium containing 25 mg / mL human serum albumin was added.

[0193] Group 4: After resuscitation, NK cells were resuspended in KBM581 medium containing 5 ng / mL IL-2 and 25 mg / mL human serum albumin and cultured. During the culture period, KBM581 medium containing 5 ng / mL IL-2 and 25 mg / mL human serum albumin was added.

[0194] Group 5: After resuscitation, NK cells were resuspended in KBM581 medium containing 5 ng / mL IL-2, 25 mg / mL human serum albumin and 300 μg / mL VC. During culture, KBM581 medium containing 5 ng / mL IL-2, 25 mg / mL human serum albumin and 300 μg / mL VC was added.

[0195] 2. The cell-killing activity of the five groups of NK cells after resuscitation and culture were tested and the number of cells after resuscitation and culture was determined using the method in Example 3.

[0196] The results are shown in Figure 6. It can be seen from the NK cell killing effect that adding IL-2 during the resuscitation culture can improve the killing ability of NK cells, and adding VC can improve the proliferation ability of NK cells (indicated by arrows).

[0197] Example 5: Effects of VC (LAA) on NK cells (in vitro detection)

[0198] 1. NK cells were cultured, cryopreserved, and thawed according to the method in Example 1, except that the cryopreserved cells were grouped as follows:

[0199] ST group: cultured to day 21, harvested, and cryopreserved using D10 basic cryopreservation solution;

[0200] ST+HSA group: cultured to D21, harvested, and cryopreserved using D10 basal cryopreservation solution with a final concentration of 25 mg / mL human serum albumin;

[0201] ST+HSA+VC group: cultured to D21, harvested, and cryopreserved using D10 basal cryopreservation solution with a final concentration of 25 mg / mL human serum albumin and 300 μg / mL VC.

[0202] 2. NK cells were collected immediately after resuscitation without prior culture for testing. The detection methods for NK cell surface receptors and related molecules are as follows:

[0203] (1) Experimental materials

[0204] Flow cytometry antibody: Fluorescently labeled isotype control mouse monoclonal antibodies: mouse IgG1-FITC (Catalog No.: 400108), mouse IgG1-Percp cy5.5 (Catalog No.: 400150), mouse IgG1-APC (Catalog No.: 400120), mouse IgG1-BV785 (Catalog No.: 400170), mouse IgG1-PE (catalog number: 400114) was purchased from Biolegend. Surface molecular marker antibodies: CD3-Percpcy5.5 (catalog number: 300430), CD56-BV785 (catalog number: 362550), Tim3-APC (catalog number: 345012), CD158d-PE (catalog number: 347006), CD94-FITC (catalog number: 305504), and TIGIT-APC (catalog number: 372706) were all purchased from Biolegend. DAPI (catalog number: 422801) was purchased from Biolegend.

[0205] Blocking antibody: mouse serum, purchased from Future, catalog number F001008.

[0206] Flow cytometry tubes: 5ml transparent polystyrene or polypropylene round-bottomed capped test tubes.

[0207] DAPI (4',6-Diamidino-2-Phenylindole,Dilactate): Purchased from Biolegend, part number 422801.

[0208] Flow cytometry wash buffer: PBS.

[0209] Alternatively, reagents from the same company and flow cytometry antibodies with the same clone number can be used.

[0210] (2) Main instruments and equipment

[0211] Centrifuges, clean benches, flow cytometers (such as FACS Calibur, Beckman CytoFlex, etc.), and RTCA detection systems.

[0212] (3) Cell viability-related molecular detection methods

[0213] [1] Preparation of single-cell suspension of surface molecular marker cells: 9.0 × 10⁹ cultured NK cells were used. 6 Each cell was placed in a 15ml centrifuge tube, centrifuged at 400g for 8 minutes to collect the cells, washed twice with 10ml of 1×PBS, and finally resuspended in 0.81ml of 1×PBS to form a single-cell suspension.

[0214] [2] Blocking: Add 90 μl of mouse serum to the surface molecular marker cells obtained in [1] and mix well; let stand at room temperature for 15-30 minutes.

[0215] [3] Labeling antibody: The blocked surface molecularly labeled cells obtained in step [2] were dispensed into 10 flow cytometry tubes at a rate of 0.09 ml / tube. If multiple batches of cells are to be detected at the same time, the cells in tubes 1-8 can be mixed with several batches of cells in equal amounts and labeled. The cell volume per tube is: 0.8-1×10 6 Each sample tube contains surface-labeled cells, with a cell dosage of 0.8-1 × 10⁻⁶ cells. 6 Each sample was prepared by adding the corresponding fluorescently labeled antibody, mixing well, and then incubating at 4°C in the dark for 30 minutes (mixing every 15 minutes). Specific labeling methods are shown in Table 1.

[0216] Table 1. Sample tubes and experimental procedures for NK cell surface molecular detection

[0217]

[0218] [4] Washing: Add 1 ml of 1×PBS to each sample tube, centrifuge at 400g for 8 minutes at 4℃ to collect cells. Wash twice 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 to the instrument for detection.

[0219] [5] Detection: The flow cytometer was calibrated and adjusted according to the instrument manual. Blank control samples were used to adjust the forward and side scattering voltages. Isotype control samples were used to adjust the voltages of each fluorescence channel to ensure that the fluorescence was within the negative range. Single-label control samples were used to adjust the fluorescence compensation of each channel. After delineating the cell gates during detection, 1×10⁻⁶ samples were collected from each sample gate. 4 Each cell.

[0220] As shown in Figure 7, the ST group showed the highest expression of NK cell inhibitory receptor-related molecules such as CD94, TIM-3, TIGIT, and CD158d, followed by the ST+HSA group, while the ST+HSA+VC group showed the lowest expression. This indicates that the addition of HSA and VC to the cryopreservation formulation can improve the activity of NK cells after cryopreservation and thawing.

[0221] Example 6: Construction of an animal model

[0222] 1. Construction of tumor models

[0223] 1) Grouping of experimental animals:

[0224] PBS group: Control group injected with PBS;

[0225] NK-A group: The drug administered was the cell preparation that was frozen and thawed from group 2 of Example 1;

[0226] NK-B group: The drug administered was the cell preparation that was frozen and thawed from group 3 of Example 1;

[0227] NK-B+VC group: The administration consisted of the cell preparation that was frozen and thawed in Group 3 of Example 1, plus VC as an adjunct to the administration.

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

[0229] NK-B+VC+Factor Group: The administration consisted of cryopreserved cells (pretreated with cytokines: 50 ng / mL IL-15 and 250 ng / mL IL-18) and revived cell preparations plus VC as an adjunct to administration, as described in Example 1 Group 4.

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

[0231] Then, trypsin digests the cells into a single-cell suspension.

[0232] 3) Wash the cell suspension with PBS and prepare 8×10⁸ ppm. 7 Cell suspensions of cells / mL were prepared and then mixed with matrix gel at a volume ratio of 9:1. After thorough mixing, the mixture was dispensed into 2 mL sterile cryovials. All of the above operations must be performed on crushed ice.

[0233] 4) After preparation, place on ice and send to the animal room. After mixing the cells, subcutaneously inoculate them into healthy NOG mice that have passed the health check.

[0234] 5) Irradiate the laminar flow hood with ultraviolet light for at least 20 minutes before use. Turn on the laminar flow hood and wait for it to stabilize before proceeding with operations.

[0235] 6) After thoroughly mixing the cells with a 1ml pipette, use a 1ml insulin syringe to draw 100 μl (8×10⁸ cells / mL). 6 Cell suspension (cells / each);

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

[0237] 2. NK cell drug delivery method

[0238] 1) Irradiate the laminar flow hood with ultraviolet light for at least 20 minutes before use. Turn on the laminar flow hood and wait for it to stabilize before proceeding with operations.

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

[0240] 3) Wipe the mouse's tail with a cotton ball soaked in 75% alcohol, and administer the preparation via the tail vein using a syringe held in the right hand.

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

[0242] i. Dosing regimen for the pharmacodynamic model: Administer once every other day for 2 courses of treatment, namely D1, D3, D5, D13, D15, and D17, for a total of 6 administrations. The day of administration is D1.

[0243] ii. Dosing regimen for pharmacokinetic models: single dose.

[0244] Example 7 Evaluation of the drug-efficacy tumor model

[0245] The efficacy of the drug in the tumor model was evaluated based on the animal model and NK cell administration method constructed in Example 6.

[0246] 1) Observation of drug efficacy: Tumor measurement was performed twice a week (Day 1, 5, 10, 13, 17, 20, 24, 27, for a total of 8 times).

[0247] 2) Tumor inhibition rate: The tumor inhibition rate of different drug administration groups after cell reinfusion was statistically analyzed.

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

[0249] ii. Tumor inhibition rate = (mean tumor volume in the control group - mean tumor volume per mouse in the experimental group) / mean tumor volume in the control group %

[0250] The results are shown in Figure 8. All treatment groups effectively inhibited tumor growth, with the efficacy ranking as follows: 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 using D10-based cryopreservation solution supplemented with HSA have good tumor-killing capabilities, and adjuvant VC administration or cytokine pretreatment can further enhance this capability. Moreover, the efficacy is optimal when both cytokine pretreatment and adjuvant VC administration are present simultaneously.

[0251] Example 8 Evaluation of the pharmacokinetic model

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

[0253] 1. Dissection of laboratory mice

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

[0255] 2) Use curved forceps to grasp the base of the mouse's eyeball, slowly pull out the eyeball, and invert the mouse with its head down so that the blood flows out vertically. At the same time, continuously press the mouse's heart to accelerate the heart's pumping speed. Collect the mouse's peripheral blood in a 1.5 ml EP tube (200~1500 µl of peripheral blood per mouse).

[0256] 3) After collection, quickly and repeatedly invert and mix thoroughly;

[0257] 4) Euthanasia of mice with cervical dislocation;

[0258] 5) Use tweezers to pick up the outer skin in the middle of the mouse's abdomen, cut the outer skin with scissors, pinch the cut edge of the outer skin with your hand, and tear the outer skin parallel to the mouse's head and tail.

[0259] 6) Use tweezers to grasp the peritoneum of the mouse and use scissors to cut it open to expose the mouse's peritoneum;

[0260] 7) Gently grasp the mouse's xiphoid process with tweezers, cut off the mouse's liver with scissors, place it in pre-cooled 1×PBS, and store it in a 4°C refrigerator or ice box for later use;

[0261] 8) The red tissue adjacent to the stomach on the left side of the mouse is the mouse spleen. Gently pick up the spleen with tweezers, cut off the mouse spleen with scissors, put it in pre-cooled 1×PBS, and put it in a 4°C refrigerator or ice box for later use.

[0262] 9) Gently grasp the mouse's xiphoid process with tweezers, cut open the diaphragm with scissors, cut open the mouse's ribs along the direction of the xiphoid process, and expose the mouse's thoracic cavity; grasp the mouse's lungs with tweezers, cut open the connection between the mouse and the heart with scissors, remove the mouse's lungs, put them in pre-cooled 1×PBS, and put them in a 4℃ refrigerator or ice box for later use.

[0263] 10) Use tweezers to pick up the skin at the tumor site of the mouse, carefully dissect the tumor tissue with ophthalmic scissors, put it into pre-cooled 1×PBS, and place it in a 4°C refrigerator or ice box for later use;

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

[0265] 2. Dissection of laboratory mice

[0266] 2.2.1 Solid tumors and lung

[0267] 1) Remove mouse solid tumors and lung tissue from 1×PBS, place them in a 6-well plate, and cut the mouse lungs into 1 mm pieces with scissors. 3 Add 3 mL of 1×PBS to the tissue block of the size, add 30 μL of Collagenase IV and 30 μL of DNASE I (1:100), mix thoroughly, and then place in a 37℃, 5% CO2 incubator for static digestion for 1 h (mix once every 30 min).

[0268] 2) The digested tumor and lung tissue cells were transferred to a tissue processing tube using a Pasteur pipette, and semi-automatic dissociation was performed using a Miltenyi tissue dissociation instrument;

[0269] 3) Discard any large, floating, gelatinous tissue.

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

[0271] 5) Discard the supernatant and invert the centrifuge tube onto a paper towel to absorb any excess liquid;

[0272] 6) Resuspend the precipitate with 4 mL of 42% Percoll solution, mix the cells thoroughly with a Pasteur pipette, and gently add the mixture to a 15 mL centrifuge tube containing 2 mL of 70% Percoll (add slowly to avoid turbulence).

[0273] 7) Centrifuge at 1200×g for 30 min, with a lift of 6 and a drop of 2.

[0274] 8) After centrifugation, aspirate the white blood cells from the intermediate layer into a 15 mL centrifuge tube and add 10 mL of 1×PBS;

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

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

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

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

[0279] 13) Take 20 μl of cell suspension, dilute and count;

[0280] 2.2.2 Liver

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

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

[0283] 3) Discard the supernatant and invert the centrifuge tube onto a paper towel to absorb excess liquid;

[0284] 4) Resuspend the precipitate with 4 mL of 42% Percoll solution, mix the cells thoroughly with a Pasteur pipette, and gently add the mixture to a 15 mL centrifuge tube containing 2 mL of 70% Percoll (add slowly to avoid turbulence).

[0285] 5) Centrifuge at 1200×g for 30 min, with a lift of 6 and a drop of 2.

[0286] 6) After centrifugation, aspirate the white blood cells from the intermediate layer into a 15 mL centrifuge tube and add 10 mL of 1×PBS;

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

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

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

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

[0291] 11) Take 20 μl of cell suspension, dilute and count;

[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 Collagenase IV and 30 μL of DNASE I (1:100), and perform semi-automatic dissociation using the Miltenyi tissue dissociation instrument;

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

[0295] 3) Discard the supernatant and invert the centrifuge tube onto a paper towel to absorb excess liquid;

[0296] 4) Add 1 mL of erythrocyte lysis buffer and lyse at 4°C in the dark for 5 min.

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

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

[0299] 7) After centrifugation, discard the supernatant, aspirate excess supernatant onto a paper towel, resuspend in 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℃, for 10 min;

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

[0302] 10) Take 20 μl of cell suspension, dilute and count;

[0303] 2.2.4 Peripheral blood

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

[0305] 2) Mix the cells thoroughly with a 1ml pipette, take 100 µl of peripheral blood and put it into an EP tube containing 1ml of erythrocyte lysis buffer, invert the tube to mix, and lyse at room temperature for 5 min.

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

[0307] 4) Discard the supernatant with a 1ml pipette and resuspend in 1mL of 1×PBS;

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

[0309] 6) Discard the supernatant with a 1ml pipette and resuspend in 1mL of 1×PBS;

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

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

[0312] 9) Take 20 μl of cell suspension, dilute and count;

[0313] 2.3 Flow cytometry detection of NK cell biodistribution in vivo

[0314] 2.3.1 Closure

[0315] Regulating cell density less than 1×10 7 Cells / ml, take 200 μL of cell suspension and add mouse serum (1:10), incubate at 4℃ for 15 min;

[0316] 2.3.2 Antibody labeling

[0317] 1) Add 20 μL of premixed antibody to each tube, incubate at 4°C for 30 min, mixing once in between.

[0318] Antibody premix:

[0319] Ac7 CD45, pp CD45m, BV785 CD56, FITC CD3

[0320] 2) Add 1 mL of 1×PBS, 800×g, 4℃, 10 min, wash twice, discard the supernatant.

[0321] 3) Resuspend the cells in 200 μL of 1×PBS;

[0322] Alternative steps: Discard the supernatant, disperse the cells, and resuspend the cells in 200 μL of cell fixative.

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

[0324] 2.3.3 In vivo biodistribution assessment indicators

[0325] 1) NK cell count = NK cell chimerism rate × tissue cell count result

[0326] 2) NK cell chimerism rate = (Human NK+ cells % × Human CD45+ % × 100) / (Mouse CD45+ %)

[0327] As shown in Figure 9, the peak time and distribution patterns of NK cells in different organs and tissues were basically consistent across different groups. The peak time for cells in tumor tissue was day 22, while the peak time for other organs and tissues was day 3 or day 5, suggesting that tumor tissue is conducive to stimulating NK cell proliferation and survival. The NK-B+VC+ factor group showed the best in vivo distribution effect (consistent with the efficacy results), with the total content in the liver, peripheral blood, spleen, and tumor tissue showing significant differences compared to the NK-A group near the peak value. The NK-B+VC+ factor group and the NK-B+VC group showed significantly higher NK cell content (peak value) in tumor tissue at days 15 and 22 after drug treatment than the NK-A group, with the highest NK cell content in the NK-B+VC+ factor group being 11.72 times that of the NK-A group. Combined with the in vivo efficacy data, this suggests that cytokine pretreatment and VC treatment can significantly promote NK cell proliferation in vivo, thereby achieving better drug efficacy.

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

Claims

1. An NK cell cryopreservation solution, characterized in that, include: The base solution and human serum albumin, wherein the base solution comprises methylcellulose and dimethyl sulfoxide.

2. The NK cell cryopreservation solution according to claim 1, characterized in that, 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.

3. The NK cell cryopreservation solution according to claim 1, characterized in that, Based on the total volume of the base solution, the methylcellulose content is 0.05%~0.2%, and the dimethyl sulfoxide content is 9%~11%.

4. The NK cell cryopreservation solution according to claim 1, characterized in that, The base solution further comprises an inorganic acid, sodium hydroxide, hydroxypropyl-β-cyclodextrin, and water.

5. The NK cell cryopreservation solution according to claim 1, characterized in that, 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.

6. A combination formulation, characterized in that, include: A first formulation, comprising the NK cell cryopreservation solution according to any one of claims 1-5; The second formulation includes cytokines.

7. The combination formulation according to claim 6, characterized in that, The cytokines include at least one of IL-2, IL-12, IL-15, IL-18 and IL-7.

8. The combination formulation according to claim 6 or 7, characterized in that, The NK cell cryopreservation solution further contains vitamin C; or The combined formulation further includes a third formulation, wherein the third formulation includes vitamin C.

9. A reagent kit, characterized in that, include: The combination formulation according to any one of claims 6 to 8.

10. The use of the NK cell cryopreservation solution according to any one of claims 1 to 5, the combination formulation according to any one of claims 6 to 8, or the kit according to claim 9 in at least one of the following: NK cell cryopreservation; To enhance the proliferative capacity of NK cells after cryopreservation and thawing; To enhance the NK cell killing ability after cryopreservation and thawing.

11. A method for cryopreserving NK cells, characterized in that, include: NK cells are mixed with the NK cell cryopreservation solution according to any one of claims 1 to 5 to obtain a mixture; The mixture is then subjected to freezing treatment.

12. The method according to claim 11, characterized in that, The method is implemented using the combination formulation of any one of claims 6-8 or the kit of claim 9, and the method includes: NK cells are mixed with the second preparation and cultured, and the cultured cells are collected. The cultured cells are then mixed with the first formulation and subjected to a second freezing treatment.

13. The method according to claim 12, characterized in that, In the culture mixture, the final concentration of each cytokine is 10 ng / mL to 300 ng / mL.

14. The method according to claim 12, characterized in that, The culture time is 12 h to 24 h, and the temperature is 35℃ to 40℃.

15. The method according to claim 12, characterized in that, The freezing process includes: programmed cooling to... Store frozen at 85℃ to -95℃.

16. The method according to claim 12, characterized in that, The NK cell cryopreservation solution contains vitamin C; the cells, the first preparation, and vitamin C are then mixed in the second step.

17. A method for improving the proliferative capacity and / or cytotoxic capacity of NK cells after cryopreservation and thawing, characterized in that, include: NK cells cryopreserved using the NK cell cryopreservation method described in any one of claims 11-16 are revived to obtain cell fluid, and the revived NK cells in the cell fluid are collected. The revived NK cells were co-cultured with vitamin C.

18. The method according to claim 17, characterized in that, The method further includes: co-culturing the resuscitated NK cells, vitamin C, and cytokines.

19. The method according to claim 18, characterized in that, The cytokines include at least one of IL-2, IL-12, IL-21, and IL-15.

20. An NK cell preparation, characterized in that, include: NK cells and the NK cell cryopreservation solution according to any one of claims 1-5, wherein the NK cells are located in the NK cell cryopreservation solution.

21. The NK cell preparation according to claim 20, characterized in that, The NK cell cryopreservation solution includes: Vitamin C.

22. A drug, characterized in that, include: The NK cell preparation according to claim 20 or 21.

23. A combination drug, characterized in that, include: The drug and vitamin C as described in claim 22.

24. The combined medicament according to claim 23, characterized in that, The combined drug further includes cytokines, including at least one of IL-2, IL-12, IL-21, and IL-15.

25. The use of the drug of claim 22 or the combined drug of claim 23 in the preparation of a pharmaceutical formulation, characterized in that, The pharmaceutical preparation is used to prevent or treat cancer.

26. The application according to claim 25, characterized in that, The cancers mentioned include at least one of the following: 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, bile duct cancer, acute lymphoblastic leukemia, lymphoma, multiple myeloma, neuroendocrine carcinoma, 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.

27. The application according to claim 25, characterized in that, 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.

28. A method for inhibiting the expression of inhibitory receptor molecules on NK cells, wherein the inhibitory receptor molecules comprise at least one of the following: CD94, TIGIT, CD158d, and TIM-3, characterized in that, include: Contact NK cells, the NK cell cryopreservation solution according to any one of claims 1-5, and vitamin C.

29. A method for preventing or treating cancer, characterized in that, include: The subject is given the drug of claim 22 or the combination drug of claim 23.

30. The method according to claim 29, characterized in that, The cancers mentioned include at least one of the following: 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, bile duct cancer, acute lymphoblastic leukemia, lymphoma, multiple myeloma, neuroendocrine carcinoma, 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.

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