Cell freezing medium and application thereof

Abstract

The invention discloses a cell freezing medium and application thereof, and belongs to the technical field of biology, the cell freezing medium comprises the following components: 5-10% of DMSO, 50-75% of dextran 40 glucose injection, 15-45% of human serum albumin, 2.5-5% of compound electrolyte solution, 5-20% of hydroxyethyl starch electrolyte injection, 10-40ng / mL of vitamin C, and 10-40ng / mL of vitamin B6.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a cell cryopreservation solution and its application. Background Art

[0002] NK cells, also known as Natural Killer Cells, are an important component of the immune system. They can recognize and attack abnormal cells in the body, such as tumor cells and virus-infected cells, without triggering graft-versus-host disease (GVHD). Due to these unique functions of NK cells, they have great application potential in the fields of cancer immunotherapy and antiviral therapy. With the passage of time, the in-depth development of medical research, and the progress of medical technology, the anti-cancer research on NK cells has increased exponentially and has become a major area of innovation in immunotherapy.

[0003] Since it is impossible to ensure the complete unity of the in vitro culture and the clinical use time node, some patients cannot receive effective treatment in a timely manner. Therefore, cryopreservation is crucial for the clinical application of cell immunotherapy to achieve cross-regional cell transportation.

[0004] According to the technical differences in cryopreservation and the methods of cell cryopreservation, cell freezing methods are mainly divided into slow freezing and rapid freezing. Whether it is rapid freezing or slow freezing, cryoprotectants are required.

[0005] Cryoprotectants are divided into two types: permeable and non-permeable. Both can reduce the freezing point of the solution and the electrolyte concentration, and reduce the formation of ice crystals. Permeable cryoprotectants are generally small molecule substances, mainly including dimethyl sulfoxide (DMSO), glycerol (GLY), methanol (METH), ethylene glycol (GE), propylene glycol (PG), dimethylformamide (DMF), and dimethylacetamide (DMA), etc. The time for them to penetrate into the cells varies depending on the types of frozen materials and cryoprotectants. Non-permeable cryoprotectants are also called extracellular protectants, such as fructose, sucrose (SUC), trehalose (TRE), honey, and some high molecular compounds, such as polyvinylpyrrolidone (PVP), dextran, yolk, albumin, etc. These substances cannot pass through the cell membrane, and their function is to protect cells by maintaining the stability of the cell membrane. Non-permeable cryoprotectants can increase the extracellular osmotic pressure, so that the water in the cells quickly exudes to the outside of the cells during the freezing process, reducing the water content in the cells, thereby reducing the formation of ice crystals in the cells and protecting the cells from mechanical damage caused by the formation of ice crystals. In addition, when the frozen cells are rewarmed, due to the high osmotic concentration formed by the non-permeable protectant in the extracellular fluid, it can effectively prevent the swelling caused by the rapid entry of water into the cells from damaging the cryopreservation of the cells.

[0006] Cell cryopreservation is one of the most critical steps in the cell storage and transportation links during cell immunotherapy. The cryopreservation quality determines the survival quality and functional status of the cells after resuscitation, thus affecting the effect of cell therapy. The composition of different cryopreservation solutions and the formed osmotic pressure are one of the main factors affecting the viability and function of cells during the cryopreservation and resuscitation process. Most CAR-T products approved by the FDA usually use cryopreservation solutions containing 5-10% DMSO, serum, and human serum albumin for cryopreservation, and then resuscitate in a 37°C water bath. Compared with CAR-T products, NK cells are more sensitive to cryopreservation and resuscitation than T cells. NK cells are more difficult to cryopreserve in a highly active state (or the resuscitation effect after cryopreservation is worse). Using conventional cryopreservation solutions makes it difficult to ensure the viability, yield, and function of NK cells after resuscitation. Using a simple, efficient and clinically compliant cryopreservation solution to maintain high activity and killing effect after cryopreservation and resuscitation has always 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] The present invention provides a cell cryopreservation solution, and the components of the cryopreservation solution are 5-10% DMSO, 50%-75% dextran 40 glucose injection, 15%-45% human 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; the human albumin is human albumin injection with an initial mass concentration of 20%.

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

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

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

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

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

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

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

[0017] The term "cell cryopreservation" used in this application is one of the main methods for cell preservation. By using cryopreservation technology to place cells in low temperature or ultra-low temperature for preservation, the cells can be temporarily separated from the growth state and their cell characteristics can be preserved. In this way, the cells can be resuscitated and used when needed. In addition, appropriately preserving a certain amount of cells can prevent the loss of cell strains due to contamination of the cells being cultured or other unexpected events, thus playing a role in cell strain preservation.

[0018] In some embodiments, the dextran 40 glucose injection is a commonly used plasma substitute, mainly used for treating hypovolemia caused by blood loss, trauma, burns, etc. Dextran 40 is a commonly used blood volume expander, and clinically, the two are often combined for treating diseases such as cerebral infarction, osteoarticular soft tissue injury, intractable ascites in liver cirrhosis, and vascular vertigo.

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

[0020] In some embodiments, the compound electrolyte solution generally contains components such as sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride. In the examples of the present invention, the compound electrolyte solution used is from a commercial source.

[0021] The cell cryopreservation solution of the present 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 liver cells, and human-derived cell lines (such as human tumor cell lines like HepG11, A549, SK-BR-3, etc.). The cells can be cells directly isolated from the human body or cells amplified in vitro. The cells recovered after cryopreservation, such as activated lymphocytes, can continue to be cultured and amplified or directly transfused clinically for disease treatment, such as tumor treatment.

[0022] In some embodiments, the concentration of DMSO is 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 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%, 75%. In some embodiments, the concentration of human 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%, 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, 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, 40 ng / mL.

[0023] The present invention provides a method for cryopreserving cells. The steps of the cryopreservation method are as follows: centrifugally washing the cells with 0.9% sodium chloride injection, appropriately pre-cooling the aforementioned cryopreservation solution, resuspending the cells with the pre-cooled cryopreservation solution, and adjusting the density of the cell suspension to 5×10 6 / mL - 1×108 / mL, the cell suspension is filled into a cell cryopreservation bag, subjected to programmed cooling, and then stored in gaseous nitrogen.

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

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

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

[0027] The term "NK cell" used in this application usually originates from bone marrow lymphoid stem cells, and its differentiation and development depend on the bone marrow and thymus microenvironment. It is mainly distributed in the bone marrow, peripheral blood, liver, spleen, lung, and lymph nodes, and can also be induced from PBMC (Peripheral Blood Mononuclear Cells). NK cells are different from T cells and B cells and are a type of lymphocyte that can non-specifically kill tumor cells and virus-infected cells without prior sensitization.

[0028] The term "PBMC" used in this application refers to peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC), which are cells with a single nucleus in peripheral blood and include lymphocytes and monocytes.

[0029] The present invention provides a method for resuscitating and inoculating cryopreserved cells obtained based on the aforementioned cryopreservation method. The method is as follows: Resuscitate the cells in a 37°C water bath, inoculate the cells with a cell density of 1.0 - 3.0×10 6 cells / mL into the activation medium. When culturing for 3 days, replenish the liquid with expansion medium 1, and the replenishment volume is 1 times the volume at the time of inoculation. When culturing for 2 days after replenishment, replenish the liquid with expansion medium 2 for the second time, and the replenishment volume is 1 times the volume at the time of inoculation. When culturing for 1 day after the second replenishment, replenish the liquid with expansion medium 2 for the third time, and the replenishment volume is 1 times the volume at the time of inoculation.

[0030] When culturing for 1 day after the third replenishment, replenish the liquid with expansion medium 2 for the fourth time. After the fourth replenishment, adjust the cell density to 1.0 - 2.0×10 6 cells / mL.

[0031] When culturing for 2 days after the fourth replenishment, replenish the liquid with expansion medium 3 for the fifth time. After the fifth replenishment, adjust the cell density to 1.0 - 2.0×10 6 cells / mL. When culturing for 2 days after the fifth replenishment, replenish the liquid with expansion medium 3 for the sixth time. After the sixth replenishment, adjust the cell density to 1.0 - 2.0×10 6cells / mL. After the sixth fluid replenishment, the amplification medium 3 was used for seven fluid replenishments on the 2nd day of culture. After the seven fluid replenishments, the cell density was adjusted to 1.0 - 2.0×10 6 cells / mL. After the seventh fluid replenishment, the amplification medium 3 was used for eight fluid replenishments on the 2nd day of culture. After the eight fluid replenishments, the cell density was adjusted to 1.0 - 2.0×10 6 cells / mL. After the eight fluid replenishments, the cells were collected after 2 days of culture; the cells are NK cells and PBMC cells.

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

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

[0034] Furthermore, 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 the vitamin is 0 - 1000 ng / mL. At the same time, 3 - 10% of serum substitute is added.

[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 substitute.

[0037] Furthermore, the amplification medium 1 is an NK basal medium containing 3 - 10% of 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.

[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 substitute.

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

[0041] Further, the amplification medium 2 is an NK basal medium containing 3-10% serum substitute, IL-2 1000-10000 IU / mL, IL-15 50-2000 IU / mL, and vitamins 0-1000 ng / mL.

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

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

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

[0045] Further, the amplification medium 3 is an NK basal medium containing 3-10% serum substitute, IL-2 1000-10000 IU / mL, IL-15 50-2000 IU / mL, and vitamins 0-1000 ng / mL.

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

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

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

[0049] The term "serum substitute" used in the present invention refers to a reagent that is used as a substitute for serum (such as FBS) in cell culture to maintain the undifferentiated state of cells and their culture. Examples of serum substitutes include KNOCKOUTTM SR (KnockOutTM Serum Replacement or KSR; Gibco), StemSure Serum Replacement (SSR; Wako Pure Chemical Industries, Ltd.), N-2 Supplement (Wako Pure Chemical Industries, Ltd.), and CTSTM Immune Cell Serum Substitute. In a specific embodiment of the present invention, the serum substitute is CTSTM Immune Cell Serum Substitute.

[0050] The present invention provides the use of the aforementioned cryopreservation method or the aforementioned resuscitation and inoculation method in the preparation of cells that can be stored for a long time and whose functions are completely preserved, and the cells are NK cells and PBMC cells.

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

[0052] The present invention provides a ready-to-use cryopreservation solution without animal-derived components that can be used for cell therapy. This cryopreservation solution uses human albumin without animal-derived proteins as a nutrient protectant, solving the problem of immune responses caused by the activation of immune cells by animal-derived substances. The high-molecular cryoprotectant dextran is used to reduce the concentration of low-molecular solutes in the cryopreservation solution and alleviate salt damage. Vitamin C is added to effectively ensure the survival rate, recovery rate, in vitro activation and amplification ability, and in vitro killing activity and other biological activities of cells after thawing. Description of the Drawings

[0053] Figure 1 It is a graph showing the results of the cell recovery rate of PBMC after resuscitation in different cryopreservation solution groups.

[0054] Figure 2 It is a graph showing the results of the cell viability of PBMC after resuscitation in different cryopreservation solution groups.

[0055] Figure 3 It is a graph showing the results of the purity of NK cells in PBMC after resuscitation in different cryopreservation solution groups.

[0056] Figure 4 It is a graph showing the results of the amplification ability of NK cells in PBMC after resuscitation in different cryopreservation solution groups.

[0057] Figure 5 It is a graph showing the results of the purity of NK cells after 9 days of amplification in PBMC after resuscitation in different cryopreservation solution groups.

[0058] Figure 6 It is a graph showing the results of the purity of NK cells after 13 days of amplification in PBMC after resuscitation in different cryopreservation solution groups.

[0059] Figure 7 It is a graph showing the purity of NK cells after 17 days of amplification of PBMCs in different cryopreservation solution groups after resuscitation.

[0060] Figure 8 It is a graph showing the expression of NKG2D on the surface of NK cells after 17 days of amplification culture of PBMCs in different cryopreservation solution groups after resuscitation.

[0061] Figure 9 It is a graph showing the expression of NKp46 on the surface of NK cells after 17 days of amplification culture of PBMCs in different cryopreservation solution groups after resuscitation.

[0062] Figure 10 It is a graph showing the killing activity of NK cells against K562 cells after 17 days of amplification culture of PBMCs in different cryopreservation solution groups after resuscitation.

[0063] Figure 11 It is a graph showing the cell recovery rate of NK cells after resuscitation in different cryopreservation solution groups.

[0064] Figure 12 It is a graph showing the cell viability of NK cells after resuscitation in different cryopreservation solution groups.

[0065] Figure 13 It is a graph showing the purity of NK cells after resuscitation in different cryopreservation solution groups.

[0066] Figure 14 It is a graph showing the apoptosis of NK cells after resuscitation in different cryopreservation solution groups.

[0067] Figure 15 It is a graph showing the killing activity of NK cells against K562 cells after resuscitation in different cryopreservation solution groups.

[0068] Figure 16 It is a graph showing the in vitro proliferation ability of NK cells after resuscitation in different cryopreservation solution groups.

[0069] Figure 17 It is a graph showing the apoptosis of NK cells cryopreserved for 1 month and 6 months.

[0070] Figure 18 It is a graph showing the in vitro amplification ability of NK cells after resuscitation after being cryopreserved for 1 month and 6 months. Detailed implementation manners

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

[0072] Preparation of Cell Cryopreservation Medium in Example 1

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

[0074] Specifically, the NK cell cryopreservation medium shown in Table 1 was used for experiments.

[0075] Table 1 Cryopreservation Medium Formulation

[0076]

[0077] Example 2: Study on the Applicability of Cryopreservation Medium to PBMC

[0078] 1. Isolation of Peripheral Blood Mononuclear Cells (PBMC)

[0079] (1) Centrifuge 40 mL of the patient's peripheral blood at low speed in a differential centrifugation horizontal centrifuge at room temperature for 30 minutes to separate plasma and blood cells.

[0080] (2) Mix the blood cell precipitate with an equal volume of normal saline and separate peripheral blood mononuclear cells (PBMC) 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 by differential centrifugation for 20 minutes. Aspirate the PBMC layer, try to aspirate as much as possible the cell layer at the interface of the two liquid surfaces, add normal saline and pipette to mix evenly, and centrifuge at 1500 rpm at room temperature for 10 minutes.

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

[0083] (5) Take 1×10 6 cells and detect the purity of NK cells by flow cytometry.

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

[0085] 2. Cryopreservation of PBMC

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

[0087] (2) Take a 100 μL sample for counting. According to the counting result, adjust the density of the cell suspension to 2×10 7 / mL.

[0088] (3) Aliquot the PBMC into cryotubes at 1.5 mL per tube.

[0089] (4) Place the cryotubes in a programmable freezing container, leave them at -80 °C overnight, and then transfer them to a liquid nitrogen tank for storage.

[0090] 3. PBMC Recovery and Seeding

[0091] (1) One month after PBMC cryopreservation, take out the cryopreserved PBMC from the liquid nitrogen tank.

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

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

[0094] (4) After centrifugation, discard the supernatant, and resuspend the cells with 5 mL of NK cell activation medium (containing 5% CTS TM immunocyte serum replacement, IL-2 1000 IU / mL, IL-15 300 IU / mL, IL-2 10.2 IU / mL, CD137 5 μg / mL, CD3 5 μg / mL, vitamin C 100 ng / mL of NK MACS, Miltenyi).

[0095] (5) Take a 100 μL sample of the cell suspension for counting, take 1×10 6 cells, and detect the purity of NK cells by flow cytometry.

[0096] (6) Seed the cells at a density of 1.0×10 6 cells / mL into a T75 cm 2 cell culture flask, with a final volume of 15 mL, and place it in a 37 °C, 5% CO 2 cell culture incubator for culture.

[0097] 4. First Medium Supplementation

[0098] Perform medium supplementation on the 3rd day after cell seeding, and supplement the expansion medium 1 (containing 5% CTS TMNK MACS with immune cell serum replacement, IL-2 at 1000 IU / mL, IL-15 at 300 IU / mL, IL-21 at 0.2 IU / mL, and vitamin C at 100 ng / mL (Miltenyi). The volume of the replenishing fluid is 15 mL.

[0099] 5. Second replenishment

[0100] On the 5th day of culture, perform the second replenishment by adding expansion medium 2 (containing 5% CTS TM NK MACS with immune cell serum replacement, IL-2 at 1000 IU / mL, IL-15 at 300 IU / mL, vitamin C at 100 ng / mL (Miltenyi). The volume of the replenishing fluid is 15 mL.

[0101] 6. Third replenishment

[0102] On the 6th day of culture, perform the third replenishment by adding expansion medium 2 (containing 5% CTS TM NK MACS with immune cell serum replacement, IL-2 at 1000 IU / mL, IL-15 at 300 IU / mL, vitamin C at 100 ng / mL (Miltenyi). The volume of the replenishing fluid is 15 mL.

[0103] 7. Fourth replenishment

[0104] On the 7th day, measure the cell density and supplement expansion medium 2 (containing 5% CTS 6 at a cell density of 1×10 TM NK MACS with immune cell serum replacement, IL-2 at 1000 IU / mL, IL-15 at 300 IU / mL, vitamin C at 100 ng / mL (Miltenyi)), and place the cell culture flask back into the incubator for continued culture.

[0105] 8. Fifth replenishment

[0106] On the 9th day, measure the cell density, take 1×10 6 cells, and detect the expression of NK and CD16 by flow cytometry.

[0107] Supplement expansion medium 3 (containing 5% CTS 6 at a cell density of 1×10 TM NK MACS with immune cell serum replacement, IL-2 at 1000 IU / mL, IL-15 at 300 IU / mL, vitamin C at 100 ng / mL (Tonglihaiyuan AS01-2)), and place the cell culture flask back into the incubator for continued culture.

[0108] 9. Sixth replenishment

[0109] On day 11, the cell density was measured and the cell density was calculated as 1×10 6 / mL cell density supplemented with expansion medium 3 (containing 5% CTS TM Immune cell serum replacement, IL-2 1000IU / mL, IL-15 300IU / mL, Vitamin C 100ng / mL Tongli Haiyuan AS01-2), and place the cell culture flask in the incubator for continued culture.

[0110] 10. Seventh fluid replenishment

[0111] On day 13, the cell density was measured and 1×10 6 Cells were detected by flow cytometry. Flow cytometry was used to detect the expression of NK and CD16.

[0112] Press 1×10 6 / mL cell density supplemented with expansion medium 3 (containing 5% CTS TM Immune cell serum replacement, IL-2 1000IU / mL, IL-15 300IU / mL, and vitamin C 100ng / mL of Tongli Haiyuan AS01-2), and the cell culture flask was placed in the incubator for continued culture.

[0113] 11. Eighth fluid replenishment

[0114] On day 15, the cell density was measured and the cell density was calculated as 1×10 6 / mL cell density supplemented with expansion medium 3 (containing 5% CTS TM Immune cell serum replacement, IL-2 1000IU / mL, IL-15 300IU / mL, Vitamin C 100ng / mL Tongli Haiyuan AS01-2), and place the cell culture flask in the incubator for continued culture.

[0115] 12.NK cell collection and functional testing

[0116] On day 17, the cell density was measured and 1×10 6 The cells were analyzed by flow cytometry to detect the expression of NK, CD16, and activation receptors NKp46 and NKG2D.

[0117] Take 5×10 6 Calcein method was used to detect the cytotoxic activity of NK cells against K562 cells in vitro.

[0118] Experimental results:

[0119] Table 2 PBMC cell recovery rate (%) test results of different cryopreservation solution groups

[0120] Grouping KA119 KA120 Mean ± Standard Deviation 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 the PBMCs in different cryopreservation solution groups were thawed, the number of cells decreased in all groups. There was no significant difference in cell recovery rate and cell viability between JDNK001-1 group and JDNK001-2 group, and both were higher than those in CS5 group. (As shown in Table 2, Table 3 Figure 1 and Figure 2 ).

[0122] Table 3 Detection results of PBMC cell viability (%) in different cryopreservation solution groups

[0123]

[0124]

[0125] After the PBMCs in different cryopreservation solution groups were thawed, there was no significant difference in the purity of NK cells in PBMC among groups (as shown in Table 4 and Figure 3 ).

[0126] Table 4 Detection results of NK cell purity (%) in PBMC in different cryopreservation solution groups

[0127] Grouping KA119 KA120 Mean ± Standard Deviation 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 the PBMCs in each cryopreservation solution group were thawed, NK cells were activated and amplified, and the amplification ability of PBMCs and the purity of NK cells were detected on the 9th day, 13th day, and 17th day respectively.

[0129] Table 5 Detection results of NK cell amplification ability of PBMC in different cryopreservation solution groups

[0130]

[0131] The results showed that there was no significant difference in the amplification ability of PBMCs in each group on the 9th day; there were significant differences in the amplification ability on the 13th day and 9th day, and JDNK001-1 group > JDNK001-2 group > CS5 group. However, there was no significant difference in the purity of NK cells among groups (as shown in Table 5, Table 6 and Figure 4 , Figure 5 , Figure 6 , Figure 7 ).

[0132] Table 6 Detection results of NK cell purity after PBMC amplification in different cryopreservation solution groups (% viable cells)

[0133]

[0134]

[0135] On the 17th day after cultivation, the cells were collected to evaluate the activity of NK cells. First, there was no significant change in the expression of the activating receptor NKG2D on the surface of NK cells, and the expression rate could reach over 95% (as shown in Table 7 and Figure 8 ). Table 7 Detection of NKG2D expression (%) on the surface of NK cells after 17 days of PBMC amplification culture in different cryopreservation solution groups

[0136] Grouping KA119 KA120 Mean ± Standard Deviation 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 the activating receptor NKp46 on the surface of NK cells in the JDNK001-1 group and the JDNK001-2 group was higher than that in the CS5 group (as shown in Table 8 and Figure 9 ).

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

[0139] Grouping KA119 KA120 Mean ± Standard Deviation 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] The in vitro killing activity showed no significant difference among the groups (as shown in Table 9 and Figure 10 ).

[0141] Table 9 Detection of the killing activity (%) of NK cells against K562 cells after 17 days of PBMC amplification culture in different cryopreservation solution groups

[0142]

[0143]

[0144] The above results indicate that considering the cryopreservation applicability to PBMC, the protective effect of CS5 is significantly weaker than that of the JDNK001-1 group and the JDNK001-2 group; the activation and amplification ability of NK cells in the PBMC cryopreserved in the JDNK001-1 group is better than that in the JDNK001-2 group after resuscitation.

[0145] Example 3: Study on the applicability of cryopreservation solution to NK cells

[0146] 1. Cryopreservation of NK cells

[0147] (1) Collection of cells:

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

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

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

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

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

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

[0154] ③ According to the counting results, add pre-cooled (2 - 8°C) cryopreservation solution to adjust the density to 3×10 7 cells / mL.

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

[0156] (3) Programmed cooling:

[0157] ① Put the cryopreservation bag into a fixed box and quickly place it in a programmed freezer. Fix the temperature probe below the cryopreservation bag.

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

[0159] ③ After the cooling program ends, immediately take out the cryopreserved cells from the programmed freezer and quickly transfer them to a vapor-phase tank for storage.

[0160] 2. Resuscitation of NK cells

[0161] (1) Start a constant temperature water bath and keep its temperature constant at 37°C.

[0162] (2) Locate the cryopreservation position, take out the cells of each group from the vapor-phase liquid nitrogen tank, quickly put 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 are completely thawed, wipe the outside of the cryopreservation bag with dust-free paper moistened with alcohol.

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

[0165] 2. Functional evaluation of cells after resuscitation

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

[0167] (2) Take 1×10 6Cells were used to detect the expression of NK and T cells, the expression of CD16, and the apoptosis of NK cells by flow cytometry.

[0168] Experimental results:

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

[0170] Grouping WM086 WM088 Mean ± Standard Deviation 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 the NK cells in different cryopreservation solution groups were thawed, the number of cells decreased. There was no significant difference in the cell recovery rate between JDNK001-1 group and JDNK001-2 group, and both groups were higher than CS5 group. The cell viability of JDNK001-1 group was higher than that of JDNK001-2 group and CS5 group (as shown in Table 10, Table 11 and Figure 11 、 Figure 12 shown).

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

[0173] Grouping WM086 WM088 Mean ± Standard Deviation 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 the NK cells in different cryopreservation solution groups were thawed, there was no significant difference in the purity of NK cells (as shown in Table 12 and Figure 13 shown).

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

[0176] Grouping WM086 WM088 Mean ± Standard Deviation 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] The apoptosis data showed that the apoptosis rate of JDNK001-1 group was significantly lower than that of JDNK001-2 group and CS5 group (as shown in Table 13 and Figure 14 shown).

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

[0179] Grouping WM086 WM088 Mean ± Standard Deviation 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] In this example, the killing efficiency of NK cells in different cryopreservation solution groups against target cell K562 was detected.

[0182] ① Preparation of target cells:

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

[0184] ② Preparation of effector cells:

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

[0186] ③ Seeding:

[0187] Spontaneous release well (100 μL medium + 100 μL target cells)

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

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

[0190] Set up 3 replicate wells, incubate in the CO2 incubator in the dark for 4 h.

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

[0192] Experimental results:

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

[0194]

[0195]

[0196] The killing ability of NK cells against target cells is an important indicator reflecting the activity of NK cells. The detection results of the killing activity after resuscitation show that the killing activity of the JDNK001-1 group is higher than that of the JDNK001-2 group and the CS5 group (as shown in Table 14 and Figure 15 shown).

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

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

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

[0200] ③ According to the counting results, inoculate into T75 cell culture flasks at a cell density of 2.0×10 6 cells / mL.

[0201] ④ Take samples on day 3 of cell culture, stain with AOPI and count, and perform expansion culture according to the counting results at a cell density of 1.0×10 6 cells / mL.

[0202] ⑤ Subsequently, take samples and count every other day. According to the counting results, replenish the liquid at a cell density of 1.0×10 6 cells / mL. Draw a proliferation curve.

[0203] Experimental results:

[0204] Table 15 Detection of in vitro expansion ability of NK cells after resuscitation in different cryopreservation solution groups

[0205]

[0206]

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

[0208] According to the research data on the applicability of cryopreservation solutions to PBMC and NK cells, JDNK001-1 was preferably selected as the cryopreservation solution formula in the NK cell culture process.

[0209] Example 4: Stability study of cryopreserving NK cells with the preferred JDNK001-1

[0210] The NK cells cryopreserved with JDNK001-1 were resuscitated after being stored for 1 month and 6 months, and the viable cell density, cell viability, NK cell purity, NK cell apoptosis, cytotoxic activity, and in vitro expansion ability after resuscitation were detected.

[0211] 1. Resuscitation of NK cells

[0212] (1) Start the constant temperature water bath and set its temperature to 37 °C.

[0213] (2) Locate the cryopreservation position, take out the cell groups from the vapor-phase liquid nitrogen tank, quickly put the cryopreservation bag into the 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 are completely thawed, wipe the outside of the cryopreservation bag with dust-free paper moistened with alcohol.

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

[0216] 2. Functional evaluation of cells after resuscitation

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

[0218] (2) Take 1×10 6 cells, and detect the expression of NK and T cells, the expression of CD16, and the apoptosis of NK cells by flow cytometry.

[0219] (3) Killing efficiency against target cells

[0220] ① Preparation of target cells:

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

[0222] ② Preparation of effector cells:

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

[0224] ③ Plating:

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

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

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

[0228] Set up 3 replicate wells, and incubate in the CO 2 incubator in the dark for 4 h.

[0229] ④ Loading onto the machine: Add 50 μL of Triton-X-100 with a final concentration of 0.1% to the maximum release pore, centrifuge at 400 g for 5 min, transfer 150 μL of the supernatant to an opaque microplate, and detect on the machine (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 of WM086

[0232]

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

[0234] Table 17 Stability data of WM088

[0235]

[0236] The 6-month stability results showed that the cryopreserved NK cells of JDNK001-1 were stored in liquid nitrogen for 6 months, and there was no significant change in the cell viability, which was all greater than 80%; there were no significant changes in the purity of NK cells and the in vitro killing activity of NK cells (as shown in Table 16 and Table 17).

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

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

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

[0240] ③ According to the counting results, inoculate into a T75 cell culture flask at a cell density of 2.0×10 6 cells / mL.

[0241] ④Sample on day 3 of cell culture, stain with AOPI and count, and perform amplification culture according to the counting results at a cell density of 1.0×10 6 cells / mL.

[0242] ⑤Subsequently, sample and count every other day, and replenish the liquid according to the counting results at a cell density of 1.0×10 6 cells / mL. Plot the proliferation curve.

[0243] Experimental results: The detection of the in vitro amplification ability of WM086 after resuscitation is shown in Table 18.

[0244] Table 18 Detection of the in vitro amplification ability of WM086 after resuscitation

[0245]

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

[0247] Table 19 Detection of the in vitro amplification ability of WM088 after resuscitation

[0248]

[0249]

[0250] As the cryopreservation time prolongs, the apoptosis of cells shows an increasing trend (as Figure 17 shown); while the in vitro amplification ability after resuscitation is not affected by the cryopreservation time, and it still maintains good in vitro amplification ability after 6 months of cryopreservation (as shown in Table 18, Table 19 and Figure 18 shown).

Claims

1. A cell freezing solution, the freezing solution components are DMSO 5-10%, dextran 40 glucose injection 50%-75%, human serum 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.

2. The cryopreservation solution as claimed in claim 1, wherein DMSO is USP grade high purity dimethyl sulfoxide; dextran 40 injection is dextran 40 glucose injection; and human albumin is human albumin injection with an initial mass concentration of 20%; Preferably, the dextran 40 glucose injection comprises 30 g dextran 40 and 25 g glucose; Preferably, the DMSO concentration is 5%, 7.5%; Preferably, the concentration of dextran 40 glucose injection is 75%, 60%; Preferably, the vitamin C concentration is 40 ng / mL; Preferably, the vitamin B6 concentration is 20 ng / mL; Preferably, the concentration of the composite electrolyte solution is 2.5%, 5%; Preferably, the cells are NK cells or PBMC cells.

3. A cell freezing method, the freezing method comprising the steps of: washing the cells by centrifugation with 0.9% sodium chloride injection, precooling the freezing solution of claim 1 or 2 in an appropriate amount, resuspending the cells in the precooled freezing solution, and adjusting the cell suspension density to 5×10 6 / mL-1×10 8 / mL, fill the cell suspension into a cell freezing bag, perform programmed cooling, and then store it in vapor phase liquid nitrogen.

4. The cryopreservation method according to claim 3, wherein the cell density needs to be counted after the cells are resuspended in the precooled cryopreservation solution; Preferably, the precooling temperature is 2-8°C; Preferably, the cells are NK cells or PBMC cells.

5. A method for reviving and inoculating frozen cells obtained by the freezing method according to claim 3 or 4, the method comprising: reviving the cells in a 37°C water bath, inoculating 1.0-3.0×10 6 Cells with a cell density of 100 / mL were inoculated into activation medium. After culturing for 3 days, the cells were replenished with expansion medium 1, and the replenishment volume was 1 times the volume of the inoculation. After the second rehydration, the cells were rehydrated for a second time using expansion medium 2 on day 2 of culture. The volume of the rehydration was 1 times the volume at the time of inoculation. After the second rehydration, the cells were rehydrated for a third time using expansion medium 2 on day 1 of culture. The volume of the rehydration was 1 times the volume at the time of inoculation. After three rehydration, the cell density was adjusted to 1.0-2.0×10 cells / mL after 1 day of culture using expansion medium 2. 6 Pieces / mL, After four rehydration, the cell density was adjusted to 1.0-2.0×10 cells / mL after five rehydration. 6 Pieces / mL, After five rehydration, the cell density was adjusted to 1.0-2.0×10 cells / mL after 2 days of culture using expansion medium 3. 6 Pieces / mL, After six rehydration, the cell density was adjusted to 1.0-2.0×10 cells / mL after seven rehydration with expansion medium 3 for 2 days. 6 Pieces / mL, After seven rehydration, the cell density was adjusted to 1.0-2.0×10 cells / mL after eight rehydration. 6 / mL, and the cells were collected after culturing for 2 days after eight times of rehydration; the cells were NK cells and PBMC cells.

6. The resuscitation inoculation method according to claim 5, wherein the activation medium is a NK basal medium containing cytokines IL-2, IL-15, IL-21, humanized CD137 monoclonal antibody, humanized CD3 monoclonal antibody, and vitamins; Preferably, the NK basal medium is NK MACS, Miltenyi or Tongli Haiyuan AS01-2 or X-VIVO15, Lonza or GT-T551H3, TAKARA or cellgrowSCGM, Cellgenix or AIM-V, GIBCO; Preferably, the IL-2 1000-10000 IU / mL, IL-15 50-2000 IU / mL, IL-2 1 0.1-5 IU / mL, CD13 7 5-15 μg / mL, CD3 5-15 μg / mL, vitamin 0-1000 ng / mL, and 3-10% serum replacement are added at the same time; Preferably, the vitamins include one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, vitamin C / L-ascorbic acid; Preferably, the serum replacement is CTS TM Immune cell serum replacement.

7. The resuscitation inoculation method according to claim 5, wherein the expansion medium 1 is a NK basal medium containing 3-10% serum replacement, IL-2 1000-10000 IU / mL, IL-1 550-2000 IU / mL, IL-2 10.1-5 IU / mL, and vitamin 0-1000 ng / mL; Preferably, the vitamins include one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, vitamin C / L-ascorbic acid; Preferably, the serum replacement is CTS TM Immune cell serum replacement; Preferably, 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, GIBCO.

8. The resuscitation inoculation method according to claim 5, wherein the expansion medium 2 is a NK basal medium containing 3-10% serum replacement, IL-2 1000-10000 IU / mL, IL-15 50-2000 IU / mL, and vitamin 0-1000 ng / mL; Preferably, the vitamins include one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, vitamin C / L-ascorbic acid; Preferably, the serum replacement is CTS TM Immune cell serum replacement; Preferably, 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, GIBCO.

9. The resuscitation inoculation method according to claim 5, wherein the expansion medium 3 is a NK basal medium containing 3-10% serum replacement, IL-2 1000-10000 IU / mL, IL-15 50-2000 IU / mL, and vitamin 0-1000 ng / mL; Preferably, the vitamins include one or more of vitamin A / retinol, vitamin B2 / vitamin B6 / vitamin B12, vitamin C / L-ascorbic acid; Preferably, the serum replacement is CTS TM Immune cell serum replacement; Preferably, 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, GIBCO.

10. Use of the freezing method according to claim 3 or 4, or the resuscitation inoculation method according to any one of claims 5 to 9, in the preparation of cells that can be stored for a long time and whose functions are preserved intact, wherein the cells are NK cells or PBMC cells.

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