A cell cryopreservation solution and use thereof

Abstract

The application discloses a kind of cell cryopreservation solution and its application, belong to biotechnology field, the component of cell cryopreservation solution in the application is DMSO 5~10%, dextran 40 glucose injection 50%~75%, human blood albumin 15%~45%, compound electrolyte solution 2.5%~5%, hydroxyethyl starch electrolyte injection 5~20%, vitamin C 10~40ng / mL, vitamin B6 10~40ng / mL.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a cell cryopreservation solution and its applications. Background Technology

[0002] NK cells, also known as natural killer cells, are important components of the immune system. They can recognize and attack abnormal cells in the body, such as tumor cells and virus-infected cells, without causing graft-versus-host disease (GVHD). Due to these unique functions, NK cells have enormous application potential in areas such as cancer immunotherapy and antiviral therapy. With the passage of time, the deepening of medical research, and the development of medical technology, research on NK cell anti-cancer applications has grown exponentially, becoming a major area of ​​innovation in immunotherapy.

[0003] Because the timing of in vitro culture and clinical use cannot be guaranteed to be completely consistent, some patients cannot receive effective treatment in the first instance. Therefore, cryogenic storage is crucial for the clinical application of cell immunotherapy to achieve cross-regional cell transport.

[0004] Based on differences in cryopreservation techniques and methods, cell freezing methods are mainly divided into slow freezing and rapid freezing. Both rapid and slow freezing require cryoprotectants.

[0005] Cryoprotectants are classified into two types: permeable and non-permeable. Both can lower the freezing point and electrolyte concentration of a solution, reducing ice crystal formation. Permeable cryoprotectants are generally small molecules, mainly composed of dimethyl sulfoxide (DMSO), glycerol (GLY), methanol (METH), ethylene glycol (GE), propylene glycol (PG), dimethylformamide (DMF), and dimethylacetamide (DMA). The time it takes for them to penetrate into the cell varies depending on the freezing material and the type of cryoprotectant. Non-permeable cryoprotectants, also known as extracellular protectants, include fructose, sucrose (SUC), trehalose (TRE), honey, and some high molecular weight compounds such as polyvinylpyrrolidone (PVP), dextran, egg yolk, and albumin. These substances cannot cross the cell membrane; their function is to protect the cell by maintaining cell membrane stability. Non-permeable cryoprotectants increase extracellular osmotic pressure, causing water to rapidly drain from the cells during freezing. This reduces intracellular water content, thereby minimizing ice crystal formation and protecting cells from mechanical damage caused by ice crystals. Furthermore, during cell thawing, the hypertonic concentration of the non-permeable cryoprotectant in the extracellular fluid effectively prevents swelling caused by rapid water ingress, which could disrupt cryopreservation.

[0006] Cell cryopreservation is one of the most critical steps in the storage and transportation of cells during cell immunotherapy. The quality of cryopreservation determines the survival quality and functional state of cells after thawing, thus affecting the efficacy of cell therapy. The composition of different cryopreservation solutions and the resulting osmotic pressure are among the main factors affecting cell viability and function during cryopreservation and thawing. Most FDA-approved CAR-T products are typically cryopreserved using a solution containing 5-10% DMSO, serum, and human serum albumin, followed by thawing in a 37°C water bath. Compared to CAR-T products, NK cells are more sensitive to cryopreservation and thawing than T cells. NK cells are more difficult to cryopreserve in a highly active state (or the thawing effect after cryopreservation is worse), and using conventional cryopreservation solutions makes it difficult to guarantee the viability, yield, and function of NK cells after thawing. Using simple, efficient, and clinically compliant cryopreservation solutions to maintain high activity and cytotoxic effects after cryopreservation and thawing has been one of the main obstacles to the clinical application of NK cells. Summary of the Invention

[0007] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0008] This invention provides a cell cryopreservation solution, wherein the cryopreservation solution comprises 5-10% DMSO, 50%-75% dextran 40 glucose injection, 15%-45% human serum albumin, 2.5%-5% compound electrolyte solution, 5-20% hydroxyethyl starch electrolyte injection, 10-40 ng / mL vitamin C, and 10-40 ng / mL vitamin B6.

[0009] Furthermore, the DMSO is USP-grade high-purity dimethyl sulfoxide; the dextran 40 injection is dextran 40 glucose injection; and the human serum albumin is human serum albumin injection with an initial mass concentration of 20%.

[0010] Furthermore, the dextran 40 glucose injection consists of 30g dextran 40 and 25g glucose.

[0011] Furthermore, the concentration of DMSO is 5% or 7.5%.

[0012] Furthermore, the concentrations of dextran 40 glucose injection are 75% and 60%.

[0013] Furthermore, the vitamin C concentration is 40 ng / mL.

[0014] Furthermore, the concentration of vitamin B6 is 20 ng / mL.

[0015] Furthermore, the concentration of the compound electrolyte solution is 2.5% or 5%.

[0016] Furthermore, the cells are NK cells and PBMC cells.

[0017] The term "cell cryopreservation" as used in this application refers to one of the main methods of cell preservation. Cryopreservation technology involves placing cells in low or ultra-low temperatures to preserve their cellular characteristics while temporarily removing them from their growth state, allowing them to be thawed and used when needed. Furthermore, appropriately preserving a certain quantity of cells can prevent the loss of cultured cells due to contamination or other unforeseen events, thus serving the purpose of cell preservation.

[0018] In some implementation schemes, dextran 40 glucose injection is a commonly used plasma substitute, primarily used to treat hypovolemia caused by blood loss, trauma, burns, etc. Dextran 40 is a commonly used blood volume expander, and clinically, the two are often used in combination to treat conditions such as cerebral infarction, soft tissue injuries of bones and joints, refractory ascites due to cirrhosis, and vascular vertigo.

[0019] In some implementations, DMSO is dimethyl sulfoxide, a sulfur-containing organic compound with the molecular formula C2H6OS. It is a colorless, odorless, transparent liquid at room temperature and is a hygroscopic and flammable liquid.

[0020] In some embodiments, the compound electrolyte solution typically contains components such as sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride. In the embodiments of the present invention, the compound electrolyte solution used is commercially available.

[0021] The cell cryopreservation solution of this invention is suitable for cryopreserving human cells, including but not limited to peripheral blood mononuclear cells, lymphocytes (including activated lymphocytes), umbilical cord blood stem cells, peripheral blood hematopoietic stem cells, bone marrow hematopoietic hepatocytes, and human-derived cell lines (such as HepG11, A549, SK-BR-3, and other human tumor cell lines). The cells can be directly isolated from the human body or expanded in vitro. Cells thawed after cryopreservation, such as activated lymphocytes, can be further cultured and expanded or directly reinfused clinically for disease treatment, such as tumor therapy.

[0022] In some embodiments, the concentration of DMSO is 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%. In some embodiments, the concentration of dextran 40 glucose injection is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%. In some implementations, the concentration of human serum albumin is 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%. In some embodiments, the concentration of vitamin C is 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, or 40 ng / mL. In some embodiments, the concentration of vitamin B6 is 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, or 40 ng / mL.

[0023] This invention provides a method for cryopreserving cells, comprising the following steps: centrifuging and washing cells with 0.9% sodium chloride injection; pre-cooling the aforementioned cryopreservation solution with an appropriate amount; resuspending the cells in the pre-cooled cryopreservation solution; and adjusting the cell suspension density to 5 × 10⁻⁶. 6 / mL-1×108 / mL, the cell suspension is filled into cell cryopreservation bags, cooled by programmed temperature, and then stored in gaseous liquid nitrogen.

[0024] Furthermore, after resuspending the cells in the pre-cooled cryopreservation solution, the cell density needs to be counted.

[0025] Furthermore, the pre-cooling temperature is 2-8℃.

[0026] Furthermore, the cells are NK cells and PBMC cells.

[0027] The term "NK cell" as used in this application generally refers to bone marrow lymphoid stem cells, whose differentiation and development depend on the bone marrow and thymus microenvironment. They are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs, and lymph nodes, and can also be induced to differentiate from PBMCs (peripheral blood mononuclear cells). Unlike T cells and B cells, NK cells are a type of lymphocyte that can non-specifically kill tumor cells and virus-infected cells without prior sensitization.

[0028] As used in this application, the term "PBMC" refers to peripheral blood mononuclear cells, which are cells in peripheral blood that have a single nucleus, including lymphocytes and monocytes.

[0029] This invention provides a method for thawing and inoculating cryopreserved cells obtained based on the aforementioned cryopreservation method. The method comprises: thawing cells in a 37°C water bath, and then inoculating 1.0-3.0 × 10⁻⁶ cells with water. 6 Cells at a density of [number] cells / mL were seeded into activation medium. After 3 days of culture, amplification medium 1 was used for replenishment, with the replenishment volume being 1 times the initial inoculation volume. After 2 days of culture following this replenishment, amplification medium 2 was used for a second replenishment, with the replenishment volume being 1 times the initial inoculation volume. After the second replenishment, after 1 day of culture, amplification medium 2 was used for a third replenishment, with the replenishment volume being 1 times the initial inoculation volume.

[0030] After three rehydration cycles, the cells were cultured for one day and then replenished with amplification medium 2 for a fourth time. After the fourth rehydration, the cell density was adjusted to 1.0-2.0 × 10⁻⁶ cells / day. 6 cells / mL

[0031] After four rehydration cycles, the cells were cultured for two days and then replenished with amplification medium three times. After five rehydration cycles, the cell density was adjusted to 1.0-2.0 × 10⁻⁶ cells / year. 6 Cells / mL were added five times, and after 2 days of culture, amplification medium was used for six more additions. After six additions, the cell density was adjusted to 1.0-2.0 × 10⁶ cells / mL. 6Cells / mL were added six times, and after 2 days of culture, amplification medium was used for seven more additions. After seven additions, the cell density was adjusted to 1.0-2.0 × 10⁶ cells / mL. 6 Cells / mL were added seven times, followed by two days of culture with amplification medium. Then, eight more additions were made, and the cell density was adjusted to 1.0-2.0 × 10⁶ cells / mL after the initial eight additions. 6 Cells were cultured for 2 days after eight infusions at a density of 1 / mL; the cells were NK cells and PBMC cells.

[0032] Furthermore, the activation medium is an NK basal medium containing cytokines IL-2, IL-15, IL-21, humanized CD137 monoclonal antibody, humanized CD3 monoclonal antibody, and vitamins.

[0033] Furthermore, the NK basal culture medium is NK MACS, Miltenyi, or Tongli Haiyuan AS01-2 or X-VIVO15, Lonza or GT-T551H3, TAKARA or cellgrowSCGM, Cellgenix or AIM-V, or GIBCO.

[0034] Furthermore, the IL-2 is 1000-10000 IU / mL, IL-1 is 550-2000 IU / mL, IL-21 is 0.1-5 IU / mL, CD13 is 75-15 μg / mL, CD3 is 5-15 μg / mL, vitamin is 0-1000 ng / mL, and 3-10% serum substitute is added simultaneously.

[0035] Furthermore, the vitamin contains one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, and vitamin C / L-ascorbic acid.

[0036] Furthermore, the serum substitute is CTS. TM Immune cell serum substitutes.

[0037] Furthermore, the amplification medium 1 is an NK basal medium containing 3-10% serum substitute, IL-2 1000-10000 IU / mL, IL-1 550-2000 IU / mL, IL-2 1 0.1-5 IU / mL, and vitamin 0-1000 ng / mL.

[0038] Furthermore, the vitamin contains one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, and vitamin C / L-ascorbic acid.

[0039] Furthermore, the serum substitute is CTS. TM Immune cell serum substitutes.

[0040] Furthermore, the NK basal culture medium is NK MACS, Miltenyi, or Tongli Haiyuan AS01-2 or X-VIVO15, Lonza or GT-T551H3, TAKARA or cellgrowSCGM, Cellgenix or AIM-V, or GIBCO.

[0041] Furthermore, the amplification medium 2 is an NK basal medium containing 3-10% serum substitute, IL-2 1000-10000 IU / mL, IL-1 550-2000 IU / mL, and vitamin 0-1000 ng / mL.

[0042] Furthermore, the vitamin contains one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, and vitamin C / L-ascorbic acid.

[0043] Furthermore, the serum substitute is CTS. TM Immune cell serum substitutes.

[0044] Furthermore, the NK basal culture medium is NK MACS, Miltenyi, or Tongli Haiyuan AS01-2 or X-VIVO15, Lonza or GT-T551H3, TAKARA or cellgrowSCGM, Cellgenix or AIM-V, or GIBCO.

[0045] Furthermore, the amplification medium 3 is an NK basal medium containing 3-10% serum substitute, IL-2 1000-10000 IU / mL, IL-1 550-2000 IU / mL, and vitamin 0-1000 ng / mL.

[0046] Furthermore, the vitamin contains one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, and vitamin C / L-ascorbic acid.

[0047] Furthermore, the serum substitute is CTS. TM Immune cell serum substitutes.

[0048] Furthermore, the NK basal culture medium is NK MACS, Miltenyi, or Tongli Haiyuan AS01-2 or X-VIVO15, Lonza or GT-T551H3, TAKARA or cellgrowSCGM, Cellgenix or AIM-V, or GIBCO.

[0049] As used in this invention, the term "serum substitute" refers to a reagent used in cell culture as an alternative to serum (e.g., FBS) to maintain the undifferentiated state of cells and their culture. Examples of serum substitutes include KNOCKOUT™ SR (KnockOut™ serum substitute or KSR; Gibco), StemSure serum substitute (SSR; WakoPure Chemical Industries, Ltd.), N-2 additive (Wako Pure Chemical Industries, Ltd.), and CTS™ immune cell serum substitute. In a specific embodiment of this invention, the serum substitute is CTS™ immune cell serum substitute.

[0050] This invention provides the application of the cryopreservation method or the resuscitation and inoculation method described above in the preparation of cells that are preserved for a long time and whose functions are intact, wherein the cells are NK cells or PBMC cells.

[0051] Advantages and beneficial effects of the present invention:

[0052] This invention provides a ready-to-use cryopreservation solution free of animal-derived components for cell therapy. This cryopreservation solution uses human serum albumin, free of animal-derived proteins, as a nutrient preservative, solving the problem of animal-derived substances activating immune cells to generate an immune response. The use of dextran, a high-molecular-weight cryopreservative, reduces the concentration of low-molecular-weight solutes in the cryopreservation solution, mitigating salt damage. The addition of vitamin C effectively ensures cell survival and recovery rates after thawing, as well as their in vitro activation and expansion capabilities and in vitro cytotoxic activity. Attached Figure Description

[0053] Figure 1 This is a graph showing the cell recovery rate of PBMCs after thawing in different cryopreservation solutions.

[0054] Figure 2 This is a graph showing the cell viability results of PBMCs after thawing in different cryopreservation solutions.

[0055] Figure 3 This is a graph showing the purity of NK cells after thawing in PBMCs from different cryopreservation solutions.

[0056] Figure 4 This is a graph showing the NK cell expansion capacity of PBMCs after thawing in different cryopreservation solutions.

[0057] Figure 5 This is a graph showing the purity of NK cells expanded 9 days after PBMC thawing in different cryopreservation solutions.

[0058] Figure 6 This is a graph showing the purity of NK cells expanded 13 days after PBMC thawing in different cryopreservation solutions.

[0059] Figure 7 This is a graph showing the purity of NK cells expanded 17 days after PBMC thawing in different cryopreservation solutions.

[0060] Figure 8 This image shows the expression results of NKG2D on the surface of NK cells after 17 days of expansion and culture following thawing of PBMCs in different cryopreservation solutions.

[0061] Figure 9 This image shows the expression of NKp46 on the surface of NK cells after 17 days of expansion and culture following thawing of PBMCs in different cryopreservation solutions.

[0062] Figure 10 This is a graph showing the cytotoxic activity of NK cells against K562 cells after PBMCs in different cryopreservation solutions were thawed and expanded for 17 days.

[0063] Figure 11 This is a graph showing the cell recovery rate after NK cell resuscitation in different cryopreservation solutions.

[0064] Figure 12 This is a graph showing the cell viability results of NK cells after thawing in different cryopreservation solutions.

[0065] Figure 13 This is a graph showing the cell purity results after NK cell resuscitation in different cryopreservation solutions.

[0066] Figure 14 This is a graph showing the apoptosis results of NK cells after thawing in different cryopreservation solutions.

[0067] Figure 15 This is a graph showing the cytotoxic activity of NK cells against K562 cells after thawing in different cryopreservation solutions.

[0068] Figure 16 This is a graph showing the in vitro proliferation capacity of NK cells after thawing in different cryopreservation solutions.

[0069] Figure 17 These are images showing the apoptosis results of NK cells after 1 month and 6 months of cryopreservation.

[0070] Figure 18 This is a graph showing the in vitro expansion capacity of NK cells after thawing from cryopreservation for 1 month and 6 months. Detailed Implementation

[0071] The present invention will now be described in detail with reference to embodiments and accompanying drawings to enable those skilled in the art to understand and implement the invention, and to further recognize its advantages. Unless otherwise defined in this specification, all technical terms herein are used according to conventional definitions commonly used and understood by those skilled in the art. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0072] Example 1: Preparation of Cell Cryopreservation Solution

[0073] NK cell cryopreservation solution comprises the following components: DMSO 5-10%, dextran 40 glucose injection 50-75%, human serum albumin 15-45%, compound electrolyte solution 2-20%, hydroxyethyl starch electrolyte injection 5-20%, vitamin C 10-40 ng / mL, and vitamin B6 10-40 ng / mL. The DMSO is USP-grade high-purity dimethyl sulfoxide; the dextran 40 injection is dextran 40 glucose injection (30g dextran 40 and 25g glucose); and the human serum albumin is a 20% initial concentration human serum albumin injection.

[0074] Specifically, the experiments were conducted using the NK cell cryopreservation solutions shown in Table 1.

[0075] Table 1. Frozen solution formulation

[0076]

[0077] Example 2: Study on the suitability of cryopreservation solution for PBMC

[0078] 1. Isolation of peripheral blood mononuclear cells (PBMCs)

[0079] (1) 40 mL of the patient’s peripheral blood was centrifuged at room temperature using a low-speed differential centrifuge machine for 30 minutes to separate the plasma and blood cells.

[0080] (2) Mix the blood cell pellet with an equal volume of physiological saline and separate peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient centrifugation.

[0081] (3) Carefully add the above mixture to a 50 mL centrifuge tube containing the Ficoll layer and centrifuge at room temperature for 20 minutes. Aspirate the PBMC layer, trying to remove as much of the cell layer as possible from the interface between the two liquids, add physiological saline and mix by pipetting, then centrifuge at 1500 rpm for 10 minutes at room temperature.

[0082] (4) After discarding the supernatant, resuspend the cells with physiological saline, bring the volume to 40 mL, and take a 100 μL sample for counting.

[0083] (5) Take 1×10 6 NK cell purity was determined by flow cytometry.

[0084] (6) Centrifuge at 1500 rpm for 10 minutes at room temperature, discard the supernatant, and obtain peripheral blood PBMC precipitate.

[0085] 2. PBMC cryopreservation

[0086] (1) Resuspend the PBMC precipitate in the pre-cooled cell cryopreservation solution in Table 1.

[0087] (2) Take a 100 μL sample for counting. Based on the counting results, adjust the cell suspension density to 2 × 10⁻⁶. 7 / mL.

[0088] (3) Dispense PBMC into cryovials at a rate of 1.5 mL / vial.

[0089] (4) Place the cryovials in a programmed cooling box and leave them at -80°C overnight before transferring them to a liquid nitrogen tank for storage.

[0090] 3. PBMC resuscitation and vaccination

[0091] (1) After one month of freezing, the frozen PBMCs were removed from the liquid nitrogen tank.

[0092] (2) Thaw the cells rapidly in a 37°C water bath.

[0093] (3) After the cells have completely melted, transfer the cell suspension to a 50ml centrifuge tube containing NK MACS and Miltenyi medium, and centrifuge at 1500rpm for 10 minutes at room temperature.

[0094] (4) After centrifugation, discard the supernatant and take 5 mL of NK cell activation culture medium (containing 5% CTS). TM NK cells were resuspended in Miltenyi MACS (immune cell serum substitutes) with IL-2 1000 IU / mL, IL-1 5300 IU / mL, IL-2 10.2 IU / mL, CD1 375 μg / mL, CD3 5 μg / mL, and vitamin C 100 ng / mL.

[0095] (5) Take 100 μL of cell suspension for counting, and take 1×10 6 NK cell purity was determined by flow cytometry.

[0096] (6) According to 1.0×10 6 Seeds were seeded at a cell density of [number] cells / mL at T75cm. 2 Cell culture flasks, with a final volume of 15 mL, are placed in a 37℃, 5% CO2 cell culture incubator.

[0097] 4. First IV fluid resuscitation

[0098] On day 3 after cell seeding, fluid replacement was performed, supplementing with amplification medium 1 (containing 5% CTS). TMImmune cell serum substitutes, IL-2 1000 IU / mL, IL-15 300 IU / mL, IL-21 0.2 IU / mL, and vitamin C 100 ng / mL (NK MACS, Miltenyi). The rehydration volume was 15 mL.

[0099] 5. Second fluid resuscitation

[0100] On day 5 of culture, a second replenishment of medium was performed, adding amplification medium 2 (containing 5% CTS). TM Immune cell serum substitute, IL-2 1000 IU / mL, IL-15 300 IU / mL, vitamin C 100 ng / mL (NKMACS, Miltenyi). The replacement volume was 15 mL.

[0101] 6. Third fluid resuscitation

[0102] On day 6 of culture, a third replenishment of medium was performed, adding amplification medium 2 (containing 5% CTS). TM Immune cell serum substitute, IL-2 1000 IU / mL, IL-15 300 IU / mL, vitamin C 100 ng / mL (NKMACS, Miltenyi). The replacement fluid volume was 15 mL.

[0103] 7. Fourth fluid resuscitation

[0104] On day 7, cell density was measured at a rate of 1×10⁻⁶. 6 Cell density of cells / mL supplemented with amplification medium 2 (containing 5% CTS) TM Immune cell serum substitutes, IL-2 1000 IU / mL, IL-15 300 IU / mL, and vitamin C 100 ng / mL in NK cells (MACS, Miltenyi), and the cell culture flasks were placed in an incubator for further culture.

[0105] 8. Fifth fluid resuscitation

[0106] On day 9, cell density was measured, and 1×10⁻⁶ cells were collected. 6 1 cell, flow cytometry was used to detect the expression of NK and CD16.

[0107] Press 1×10 6 Cell density of cells / mL supplemented with amplification medium 3 (containing 5% CTS) TM The cell culture flasks containing immune cell serum substitutes, IL-2 1000 IU / mL, IL-15 300 IU / mL, and vitamin C 100 ng / mL (Tongli Haiyuan AS01-2) were placed in an incubator for further culture.

[0108] 9. Sixth fluid resuscitation

[0109] On day 11, cell density was measured at a rate of 1×10⁻⁶. 6 Cell density of cells / mL supplemented with amplification medium 3 (containing 5% CTS) TM The cell culture flasks containing immune cell serum substitutes, IL-2 1000 IU / mL, IL-15 300 IU / mL, and vitamin C 100 ng / mL (Tongli Haiyuan AS01-2) were placed in an incubator for further culture.

[0110] 10. Seventh fluid resuscitation

[0111] On day 13, cell density was measured, and 1×10⁶ cells were collected. 6 1 cell, flow cytometry was used to detect the expression of NK and CD16.

[0112] Press 1×10 6 Cell density of cells / mL supplemented with amplification medium 3 (containing 5% CTS) TM The cell culture flasks containing immune cell serum substitutes, IL-2 1000 IU / mL, IL-15 300 IU / mL, and vitamin C 100 ng / mL (Tongli Haiyuan AS01-2) were placed in an incubator for further culture.

[0113] 11. Eighth fluid resuscitation

[0114] On day 15, cell density was measured at a rate of 1×10⁻⁶. 6 Cell density of cells / mL supplemented with amplification medium 3 (containing 5% CTS) TM The cell culture flasks containing immune cell serum substitutes, IL-2 1000 IU / mL, IL-15 300 IU / mL, and vitamin C 100 ng / mL (Tongli Haiyuan AS01-2) were placed in an incubator for further culture.

[0115] 12. NK cell collection and functional testing

[0116] On day 17, cell density was measured, and 1×10⁻⁶ cells were collected. 6 Flow cytometry was used to detect the expression of NK cells, CD16, and the activation receptors NKp46 and NKG2D in individual cells.

[0117] Take 5×10 6 The in vitro cytotoxic activity of NK cells against K562 was detected by Calcein assay.

[0118] Experimental results:

[0119] Table 2 Results of PBMC cell recovery rate (%) in different cryopreservation solutions

[0120] JDNK001-1 77.50 81.75 79.63±3.01 JDNK001-2 71.00 84.17 77.59±9.31 CS5 53.50 63.33 58.42±6.95

[0121] After thawing, PBMCs in different cryopreservation media all showed some cell number loss. There was no difference in cell recovery rate and cell viability between the JDNK001-1 and JDNK001-2 groups, both of which were higher than the CS5 group. (See Tables 2 and 3.) Figure 1 and Figure 2 (As shown).

[0122] Table 3. Results of PBMC cell viability (%) in different cryopreservation solutions

[0123]

[0124]

[0125] After thawing, there was no difference in the purity of NK cells in PBMCs from different cryopreservation solutions (as shown in Table 4). Figure 3 (As shown).

[0126] Table 4. Results of NK cell purity (% white blood cells) in PBMCs from different cryopreservation solutions

[0127] JDNK001-1 17.38 31.56 24.47±10.03 JDNK001-2 17.65 31.90 24.78±10.08 CS5 16.15 32.03 24.09±11.23

[0128] After thawing, PBMCs in each cryopreservation group were activated and expanded to produce NK cells. The expansion capacity of PBMCs and the purity of NK cells were tested on days 9, 13 and 17, respectively.

[0129] Table 5. Results of NK cell amplification capacity detection in PBMCs with different cryopreservation solutions

[0130]

[0131] The results showed that there was no significant difference in the expansion capacity of PBMCs among the groups at 9 days; however, there were significant differences in expansion capacity at 13 days and 9 days, with JDNK001-1 group > JDNK001-2 group > CS5 group. But there was no significant difference in NK cell purity among the groups (as shown in Tables 5 and 6). Figure 4 , Figure 5 , Figure 6 , Figure 7 (As shown).

[0132] Table 6. NK cell purity detection results after PBMC amplification in different cryopreservation solutions (% viable cells)

[0133]

[0134]

[0135] Seventeen days after culture, cells were collected to evaluate NK cell activity. Firstly, the expression of the NK cell surface activating receptor NKG2D showed no significant change, remaining above 95% (as shown in Table 7). Figure 8 (As shown). Table 7 shows the expression of NKG2D on the surface of NK cells (%NK cells) after 17 days of PBMC amplification culture in different cryopreservation solutions.

[0136] JDNK001-1 99.90 99.98 99.94±0.06 JDNK001-2 99.90 99.98 99.94±0.06 CS5 99.88 99.94 99.91±0.04

[0137] The expression of NK cell surface activating receptor NKp46 was significantly higher in the JDNK001-1 and JDNK001-2 groups than in the CS5 group (as shown in Table 8). Figure 9 (As shown).

[0138] Table 8. Detection of NKp46 expression (%NK cells) on the surface of NK cells after 17 days of PBMC amplification culture in different cryopreservation solutions.

[0139] JDNK001-1 84.28 89.75 87.02±3.87 JDNK001-2 82.06 90.91 86.49±6.26 CS5 65.81 88.14 76.98±15.79

[0140] In vitro cytotoxic activity showed no significant differences among the groups (as shown in Table 9 and...). Figure 10 (As shown).

[0141] Table 9. Detection of NK cell cytotoxicity (%) against K562 cells after 17 days of PBMC amplification culture in different cryopreservation solutions.

[0142]

[0143]

[0144] The above results indicate that, considering the suitability of cryopreservation for PBMCs, the protective effect of CS5 is significantly weaker than that of the JDNK001-1 group and the JDNK001-2 group; the activation and expansion capacity of NK cells after thawing of PBMCs cryopreserved in the JDNK001-1 group is better than that in the JDNK001-2 group.

[0145] Example 3: Study on the suitability of cryopreservation solution for NK cells

[0146] 1. Cryopreservation of NK cells

[0147] (1) Cell collection:

[0148] ① Transfer the NK cell suspension to a 250mL centrifuge tube and centrifuge at 1800rpm for 10min at room temperature to obtain cell pellet.

[0149] ② Discard the supernatant, resuspend the cells in 100ml of 0.9% sodium chloride injection, and centrifuge at 1800rpm for 10min at room temperature.

[0150] ③ Discard the supernatant, resuspend the cells in 100ml of 0.9% sodium chloride injection, filter the mixed cell suspension through a 70μm cell sieve, and centrifuge at 1800rpm for 10min at room temperature to obtain the precipitate.

[0151] (2) Preparation of cell / cryopreservation suspension:

[0152] ① Discard the supernatant and remove as much of the supernatant as possible to reduce the dilution of the cryopreservation solution.

[0153] ② Resuspend the cells in an appropriate amount of pre-cooled (2-8℃) cryopreservation solution from Example 1, stain with AOPI and count the cells.

[0154] ③ Based on the counting results, add pre-cooled (2-8℃) cryopreservation solution and adjust the density to 3×10⁻⁶. 7 cells / mL.

[0155] ④ Fill the cell / cryopreservation solution mixture into cell cryopreservation bags at 20ml / bag ratio.

[0156] (3) Program cooling:

[0157] ① Place the cryopreservation bag into the fixing box and quickly place it into the programmed cooling device, fixing the temperature probe under the cryopreservation bag.

[0158] ②Start the programmed cooling device to perform programmed cooling.

[0159] ③ After the cooling process is completed, immediately remove the frozen cells from the programmed cooling instrument and quickly transfer them to a gas phase tank for storage.

[0160] 2. NK cell resuscitation

[0161] (1) Start the constant temperature water bath and keep its temperature constant at 37℃.

[0162] (2) Locate the cryopreservation location, remove each group of cells from the gas phase liquid nitrogen tank, quickly place the cryopreservation bag into a 37°C constant temperature water bath, and shake it from time to time to thaw it as soon as possible.

[0163] (3) After the cells have completely thawed, wipe the outside of the cryopreservation bag with lint-free paper soaked in alcohol.

[0164] (4) Mix the cell suspensions of each group in a biosafety cabinet and quickly transfer the mixed cell suspensions to 50mL centrifuge tubes.

[0165] 2. Functional evaluation of cells after resuscitation

[0166] (1) Take 100 μL of suspension, stain with AO / PI and count the cells, and record the cell density, volume and cell viability.

[0167] (2) Take 1×10 6Flow cytometry was used to detect the expression of NK and T cells, CD16 expression, and NK cell apoptosis.

[0168] Experimental results:

[0169] Table 10 Detection of NK cell recovery rate (%) in different cryopreservation solutions

[0170] JDNK001-1 94.00 94.00 94.00±0.00 JDNK001-2 92.33 93.67 93.00.±0.95 CS5 87.33 83.00 85.17±3.06

[0171] After NK cell resuscitation in different cryopreservation solutions, cell counts decreased in all groups. There was no significant difference in cell recovery rates between the JDNK001-1 and JDNK001-2 groups, but both were higher than the CS5 group. Cell viability in the JDNK001-1 group was higher than that in the JDNK001-2 and CS5 groups (see Tables 10, 11, and 12). Figure 11 , Figure 12 (As shown).

[0172] Table 11 Detection of NK cell viability (%) in different cryopreservation solutions

[0173] JDNK001-1 85.46 74.83 80.15±7.52 JDNK001-2 73.69 74.83 74.26±0.81 CS5 72.48 74.83 73.66±1.66

[0174] After NK cell resuscitation in different cryopreservation solutions, there was no significant difference in NK cell purity (as shown in Table 12 and...). Figure 13 (As shown).

[0175] Table 12 Detection of NK cell purity (%) in different cryopreservation solutions

[0176] JDNK001-1 94.91 97.42 96.17±1.77 JDNK001-2 94.60 97.34 95.97±1.94 CS5 94.55 97.35 95.95±1.98

[0177] Apoptosis data showed that the apoptosis rate in the JDNK001-1 group was significantly lower than that in the JDNK001-2 group and the CS5 group (as shown in Table 13 and 14). Figure 14 (As shown).

[0178] Table 13 Detection of NK cell apoptosis (%) in different cryopreservation solutions

[0179] JDNK001-1 5.85 3.67 4.76±1.54 JDNK001-2 14.21 10.41 12.31±2.69 CS5 9.38 13.4 11.39±2.84

[0180] (3) Killing efficiency against target cells

[0181] This embodiment takes the detection of the killing efficiency of NK cells against target cells K562 in different cryopreservation solution groups as an example.

[0182] ① Target cell preparation:

[0183] Take a sufficient amount of target cells (K562), centrifuge at 500g for 10 min, discard the supernatant, resuspend in 1640 basal medium, count and assess viability. Adjust the target cell density to 1×10⁻⁶ cells according to the counting density. 6Cells / mL, add 7.5 μL / 1 × 10⁻⁶ Calcein-AM solution at a concentration of 1 mM. 6 Stain with PBS (cells / mL) for 30 min. After staining, wash twice with PBS, resuspend in 1640 basal medium, and count to determine viability. Adjust the target cell density to 2 × 10⁶ cells / mL according to the counting density. 5 Cells / mL, for later use.

[0184] ②Effective cell preparation:

[0185] Take a sufficient amount of NK cells, centrifuge at 500g for 10 min, discard the supernatant, resuspend in 1640 basal medium, count and assess viability. Dilute effector cells to the appropriate concentration according to the effector-to-target ratio. Effector-to-target ratio: 5:1.

[0186] ③Planning:

[0187] Spontaneous wells (100 μL culture medium + 100 μL target cells)

[0188] Maximum well (100 μL target cells + 50 μL maximum release well + 50 μL basal culture medium)

[0189] Experimental wells (100 μL target cells + 100 μL effector cells)

[0190] Set up 3 replicates and incubate in a CO2 incubator in the dark for 4 hours.

[0191] Instrumentation: Add 50 μL of Triton-X-100 to the maximum release well to a final concentration of 0.1%, centrifuge at 400g for 5 min, transfer 150 μL of supernatant to an opaque microplate, and perform instrumentation (excitation wavelength: 488 nm, emission wavelength: 520 nm).

[0192] Experimental results:

[0193] Table 14. In vitro cytotoxic activity of NK cells before and after cryopreservation (%)

[0194]

[0195]

[0196] The cytotoxic ability of NK cells against target cells is an important indicator of NK cell activity. Post-resuscitation cytotoxicity assays showed that the cytotoxic activity of the JDNK001-1 group was higher than that of the JDNK001-2 group and the CS5 group (as shown in Table 14). Figure 15 (As shown).

[0197] (4) In vitro proliferation of cells after resuscitation

[0198] ① Add the remaining cells from each group to 40ml of NK cell basal culture medium (equilibrated to room temperature), and centrifuge at room temperature to wash and obtain cell pellet.

[0199] ② Discard the supernatant, resuspend the cells in an appropriate amount of NK cell expansion medium (NK MACS, Miltenyi containing 5% serum substitute, 1000 IU / mL IL-2, and 500 IU / mL IL-15), take samples, stain with AOPI, and count the cells.

[0200] ③ Based on the counting results, according to 2.0 × 10 6 Cells were seeded at a density of cells / mL in T75 cell culture flasks.

[0201] ④ Samples were taken on day 3 of cell culture, stained with AOPI and counted. The counts were then calculated at a ratio of 1.0 × 10⁻⁶. 6 Expand the culture by cell density of cells / mL.

[0202] ⑤ Subsequent sampling and counting on the following day, based on the counting results, at a rate of 1.0 × 10⁻⁶. 6 Replenish fluid at a cell density of cells / mL. Plot a proliferation curve.

[0203] Experimental results:

[0204] Table 15. Detection of in vitro expansion capacity of NK cells after thawing in different cryopreservation solutions.

[0205]

[0206]

[0207] The results of NK cell proliferation assays after resuscitation showed that the in vitro proliferation capacity of CS5 group > JDNK001-1 group > JDNK001-2 group (as shown in Table 15 and 16). Figure 16 (As shown). Considering factors such as NK cell viability, apoptosis, cytotoxic activity, and in vivo and in vitro expansion capacity after resuscitation, JDNK001-1 provides stronger protection for NK cells.

[0208] Based on the data from studies on the suitability of cryopreservation solutions for PBMCs and NK cells, JDNK001-1 is selected as the preferred cryopreservation solution formulation for NK cell culture.

[0209] Example 4: Stability study of cryopreserved NK cells using the preferred JDNK001-1 cell type.

[0210] NK cells frozen in JDNK001-1 were thawed after 1 month and 6 months of storage, and the viable cell density, cell viability, NK cell purity, NK cell apoptosis, cell killing activity, and in vitro expansion capacity after thaw were measured.

[0211] 1. NK cell resuscitation

[0212] (1) Start the constant temperature water bath and keep its temperature constant at 37℃.

[0213] (2) Locate the cryopreservation location, remove each group of cells from the gas phase liquid nitrogen tank, quickly place the cryopreservation bag into a 37°C constant temperature water bath, and shake it from time to time to thaw it as soon as possible.

[0214] (3) After the cells have completely thawed, wipe the outside of the cryopreservation bag with lint-free paper soaked in alcohol.

[0215] (4) Mix the cell suspensions of each group in a biosafety cabinet and quickly transfer the mixed cell suspensions to 50mL centrifuge tubes.

[0216] 2. Functional evaluation of cells after resuscitation

[0217] (1) Take 100 μL of suspension, stain with AO / PI and count the cells, and record the cell density, volume and cell viability.

[0218] (2) Take 1×10 6 Flow cytometry was used to detect the expression of NK and T cells, CD16 expression, and NK cell apoptosis.

[0219] (3) Killing efficiency against target cells

[0220] ① Target cell preparation:

[0221] Take a sufficient amount of target cells (K562), centrifuge at 500g for 10 min, discard the supernatant, resuspend in 1640 basal medium, count and assess viability. Adjust the target cell density to 1×10⁻⁶ cells according to the counting density. 6 Cells / mL, add 7.5 μL / 1 × 10⁻⁶ Calcein-AM solution at a concentration of 1 mM. 6 Stain with PBS (cells / mL) for 30 min. After staining, wash twice with PBS, resuspend in 1640 basal medium, and count to determine viability. Adjust the target cell density to 2 × 10⁶ cells / mL according to the counting density. 5 Cells / mL, for later use.

[0222] ②Effective cell preparation:

[0223] Take a sufficient amount of NK cells, centrifuge at 500g for 10 min, discard the supernatant, resuspend in 1640 basal medium, count and assess viability. Dilute effector cells to the appropriate concentration according to the effector-to-target ratio. Effector-to-target ratio: 5:1.

[0224] ③Planning:

[0225] Spontaneous wells (100 μL culture medium + 100 μL target cells)

[0226] Maximum well (100 μL target cells + 50 μL maximum release well + 50 μL basal culture medium)

[0227] Experimental wells (100 μL target cells + 100 μL effector cells)

[0228] Set up 3 replicates and incubate in a CO2 incubator in the dark for 4 hours.

[0229] ④ Detection: Add 50 μL of Triton-X-100 to the maximum release well to a final concentration of 0.1%, centrifuge at 400g for 5 min, transfer 150 μL of supernatant to an opaque microplate, and detect using the microplate (excitation wavelength: 488 nm, emission wavelength: 520 nm).

[0230] Experimental results: The stability data of WM086 are shown in Table 16.

[0231] Table 16 Stability Data for WM086

[0232]

[0233] The stability data of WM088 is shown in Table 17.

[0234] Table 17 Stability Data for WM088

[0235]

[0236] The 6-month stability results showed that the NK cells cryopreserved by JDNK001-1 did not show significant changes in cell viability after 6 months of storage in liquid nitrogen, and were all greater than 80%; the purity of NK cells and the in vitro killing activity of NK cells did not show significant changes (as shown in Tables 16 and 17).

[0237] (4) In vitro proliferation of cells after resuscitation

[0238] ① Add the remaining cells from each group to 40ml of NK cell basal culture medium (equilibrated to room temperature), and centrifuge at room temperature to wash and obtain cell pellet.

[0239] ② Discard the supernatant, resuspend the cells in an appropriate amount of NK cell expansion medium (NK MACS, Miltenyi containing 5% serum substitute, 1000 IU / mL IL-2, and 500 IU / mL IL-15), take samples, stain with AOPI, and count the cells.

[0240] ③ Based on the counting results, according to 2.0 × 10 6 Cells were seeded at a density of cells / mL in T75 cell culture flasks.

[0241] ④ Samples were taken on day 3 of cell culture, stained with AOPI and counted. The counts were then calculated at a ratio of 1.0 × 10⁻⁶. 6 Expand the culture by cell density of cells / mL.

[0242] ⑤ Subsequent sampling and counting on the following day, based on the counting results, at a rate of 1.0 × 10⁻⁶. 6 Replenish fluid at a cell density of cells / mL. Plot a proliferation curve.

[0243] Experimental results: The in vitro amplification capacity of WM086 after revival is shown in Table 18.

[0244] Table 18. Detection of in vitro amplification capacity of WM086 after recovery.

[0245]

[0246] The in vitro amplification capacity of WM088 after resuscitation is shown in Table 19.

[0247] Table 19. Detection of in vitro amplification capacity of WM088 after recovery.

[0248]

[0249]

[0250] As cryopreservation time increases, apoptosis shows an increasing trend (e.g., Figure 17 (As shown in Tables 18 and 19); however, the in vitro amplification capacity after thawing was not affected by the cryopreservation time, and it still maintained good in vitro amplification capacity after 6 months of cryopreservation (as shown in Tables 18 and 19). Figure 18 (As shown).

Claims

1. A cell cryopreservation solution for the preservation of NK cells and / or PBMC cells, wherein the cryopreservation solution comprises 7.5% DMSO, 75% dextran 40 glucose injection, 15% human serum albumin, 2.5% compound electrolyte solution, 40 ng / mL vitamin C, and 20 ng / mL vitamin B6.

2. The cryopreservation solution as described in claim 1, wherein DMSO is USP grade high-purity dimethyl sulfoxide; dextran 40 injection is dextran 40 glucose injection; and human serum albumin is human serum albumin injection with an initial mass concentration of 20%.

3. The cryopreservation solution as described in claim 2, wherein the dextran 40 glucose injection solution comprises 30g dextran 40 and 25g glucose.

4. A method for cryopreserving cells, the method comprising the following steps: centrifuging and washing cells with 0.9% sodium chloride injection solution; pre-cooling the cryopreservation solution according to any one of claims 1-3; resuspending the cells in the pre-cooled cryopreservation solution; and adjusting the cell suspension density to 5 × 10⁻⁶. 6 / mL-1×10 8 / mL, the cell suspension is filled into cell cryopreservation bags, cooled by programmed temperature, and then stored in gaseous liquid nitrogen.

5. In the cryopreservation method as described in claim 4, the cell density needs to be counted after the cells are resuspended in the pre-cooled cryopreservation solution.

6. The cryopreservation method as described in claim 4, wherein the pre-cooling temperature is 2-8°C.

7. The cryopreservation method according to claim 4, wherein the cells are NK cells or PBMC cells.

8. A method for thawing and inoculating cryopreserved cells obtained based on the cryopreservation method according to any one of claims 4-7, wherein the method comprises: thawing cells in a 37°C water bath, and inoculating 1.0-3.0 × 10⁻⁶ cells with water. 6 Cells at a density of [number] cells / mL were seeded into activation medium and cultured for 3 days. Then, amplification medium 1 was used to replenish the cells, with the replenishment volume being twice the initial seeding volume. Two days after inoculation, the culture medium was replenished a second time using amplification medium 2, with the replenishment volume being 1 times the initial inoculation volume. After the second replenishment of fluid, the culture was incubated for one day, and then replenished a third time with amplification medium 2, with the replenishment volume being 1 times the initial inoculation volume. After three rehydration cycles, the cells were cultured for one day and then replenished with amplification medium 2 for a fourth time. After the fourth rehydration, the cell density was adjusted to 1.0-2.0 × 10⁻⁶ cells / day. 6 cells / mL After four rehydration cycles, the cells were cultured for two days and then amplified with a third culture medium. A fifth rehydration cycle was performed, and the cell density was adjusted to 1.0-2.0 × 10⁻⁶ cells / day. 6 cells / mL After five rehydration cycles, the cells were cultured for two days and then replenished with amplification medium three times. After six rehydration cycles, the cell density was adjusted to 1.0-2.0 × 10⁻⁶ cells / year. 6 cells / mL After six rehydration cycles, the cells were cultured for two days and then replenished with amplification medium three times. After seven rehydration cycles, the cell density was adjusted to 1.0-2.0 × 10⁻⁶ cells / year. 6 cells / mL After seven rehydration cycles, the cells were cultured for two days and then subjected to an eighth rehydration cycle using expansion medium. After the eighth rehydration cycle, the cell density was adjusted to 1.0-2.0 × 10⁻⁶ cells / year. 6 Cells were cultured for 2 days after eight infusions at a density of 1 / mL; the cells were NK cells and PBMC cells.

9. The resuscitation inoculation method as described in claim 8, wherein the activation culture medium is an NK basal culture medium containing cytokines IL-2, IL-15, IL-21, humanized CD137 monoclonal antibody, humanized CD3 monoclonal antibody, and vitamins.

10. The resuscitation inoculation method as described in claim 9, wherein the NK basal culture medium is NK MACS, Miltenyi, or Tongli Haiyuan AS01-2 or X-VIVO15, Lonza or GT-T551H3, TAKARA or cellgrowSCGM, Cellgenix or AIM-V, or GIBCO.

11. The resuscitation inoculation method as described in claim 9, wherein the IL-2 is 1000-10000 IU / mL, IL-15 is 50-2000 IU / mL, IL-21 is 0.1-5 IU / mL, CD137 is 5-15 μg / mL, CD3 is 5-15 μg / mL, and vitamin is 0-1000 ng / mL, and 3-10% serum substitute is added simultaneously.

12. The resuscitation inoculation method as described in claim 11, wherein the vitamin contains one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, and vitamin C / L-ascorbic acid.

13. The resuscitation inoculation method as described in claim 11, wherein the serum substitute is CTS™ immune cell serum substitute.

14. The resuscitation inoculation method as described in claim 8, wherein the amplification culture medium 1 is an NK basal culture medium containing 3-10% serum substitute, IL-2 1000-10000 IU / mL, IL-15 50-2000 IU / mL, IL-21 0.1-5 IU / mL, and vitamin 0-1000 ng / mL.

15. The resuscitation inoculation method as described in claim 14, wherein the vitamin contains one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, and vitamin C / L-ascorbic acid.

16. The resuscitation inoculation method as described in claim 14, wherein the serum substitute is CTS™ immune cell serum substitute.

17. The resuscitation inoculation method as described in claim 14, wherein the NK basal culture medium is NK MACS, Miltenyi, or Tongli Haiyuan AS01-2 or X-VIVO15, Lonza or GT-T551H3, TAKARA or cellgrowSCGM, Cellgenix or AIM-V, or GIBCO.

18. The resuscitation inoculation method according to claim 8, wherein the amplification culture medium 2 is an NK basal culture medium containing 3-10% serum substitute, IL-2 1000-10000 IU / mL, IL-15 50-2000 IU / mL, and vitamin 0-1000 ng / mL.

19. The resuscitation inoculation method as described in claim 18, wherein the vitamin contains one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, and vitamin C / L-ascorbic acid.

20. The resuscitation inoculation method of claim 18, wherein the serum substitute is CTS™ immune cell serum substitute.

21. The resuscitation inoculation method as described in claim 18, wherein the NK basal culture medium is NK MACS, Miltenyi, or Tongli Haiyuan AS01-2 or X-VIVO15, Lonza or GT-T551H3, TAKARA or cellgrowSCGM, Cellgenix or AIM-V, or GIBCO.

22. The resuscitation inoculation method as described in claim 8, wherein the amplification culture medium 3 is an NK basal culture medium containing 3-10% serum substitute, IL-2 1000-10000 IU / mL, IL-15 50-2000 IU / mL, and vitamin 0-1000 ng / mL.

23. The resuscitation inoculation method as described in claim 22, wherein the vitamin contains one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, and vitamin C / L-ascorbic acid.

24. The resuscitation inoculation method as described in claim 22, wherein the serum substitute is CTS™ immune cell serum substitute.

25. The resuscitation inoculation method as described in claim 22, wherein the NK basal culture medium is NK MACS, Miltenyi, or Tongli Haiyuan AS01-2 or X-VIVO15, Lonza or GT-T551H3, TAKARA or cellgrowSCGM, Cellgenix or AIM-V, or GIBCO.

26. The application of the cryopreservation method according to any one of claims 4-7, or the resuscitation and inoculation method according to any one of claims 8-25, in the preparation of cells that are preserved for a long time and whose functions are intact, wherein the cells are NK cells or PBMC cells.

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