Cryopreservation resuscitation method and cryopreservation resuscitation kit for NK (Natural Killer) cells

Abstract

The invention provides an NK cell cryopreservation resuscitation method and an NK cell cryopreservation resuscitation kit, and relates to the technical field of biology. The cryopreservation recovery method of the NK cells, provided by the invention, comprises the following steps: pre-treating the NK cells to be cryopreserved in an NK cell in-vitro amplification culture medium containing 0.1-50mM of NAM and 1-1000ng / mL of recombinant human IL-18; the pretreated NK cells are subjected to cryopreservation in an NK cell cryopreservation solution containing 1-50 mM of NAM; and resuscitating the cryopreserved NK cells to obtain resuscitated NK cells. According to the cryopreservation recovery method, the survival rate, the recovery rate and the functional activity of NK cell recovery for 24 hours can be remarkably improved, and possibility is provided for NK cell seed bank construction, gene editing and spot type product development.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a NK cell cryopreservation and resuscitation method and a NK cell cryopreservation and resuscitation kit. Background Art

[0002] Natural killer (NK) cells are members of the innate lymphoid cell family. They are key effector lymphocytes that mediate tumor immune surveillance and clearance. They are also a key component of the anti-tumor defense system and can produce effective immune responses to virus-infected and malignantly transformed cells. NK cells do not express T cell receptors and cannot recognize self and non-self through major histocompatibility complex (MHC) antigen binding, so the risk of graft-versus-host disease (GVHD) of allogeneic NK cell infusion is low. Activated NK cells produce cytokines and do not proliferate in vivo for several weeks, reducing the risk of cytokine release syndrome (CRS) and neurotoxicity. Mature NK cells have a limited lifespan in the circulation, reducing the possibility of long-term adverse events. These characteristics of NK cells make it a promising candidate for "off-the-shelf" immune cell therapy for cancer. The ultimate commercialization of NK cell therapy will depend on the ability to provide effective cryopreserved preparations. However, cryopreserved NK cells have severe functional defects, reduced cytotoxicity and impaired migration ability compared with non-frozen NK cells.

[0003] In order to solve the above technical problems, the existing technology currently focuses on improving the viability, recovery rate and functional activity of NK cells after recovery by optimizing NK cell cryoprotectant. However, the characteristics of NK cells as large granular cells make their large granules easy to rupture during the cryopreservation recovery process, resulting in significant damage to the viability and functional activity of NK cells. Simply changing the composition of the cell cryoprotectant cannot significantly improve the viability and functional activity of NK cells after recovery. In particular, if the recovered NK cells continue to be expanded and cultured, their viability, recovery rate and killing activity will drop significantly, resulting in unsatisfactory results of continued expansion and culture.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The first purpose of the present invention is to provide a method for cryopreservation and resuscitation of NK cells to solve the above-mentioned technical problems.

[0006] The second purpose of the present invention is to provide a NK cell cryopreservation and resuscitation kit.

[0007] In order to achieve the above objectives, the following technical solutions are adopted: In a first aspect, the present invention provides a method for cryopreservation and resuscitation of NK cells, comprising the following steps: a. The NK cells to be frozen are pretreated in an NK cell in vitro expansion medium containing 0.1-50 mM NAM and 1-1000 ng / mL recombinant human IL-18; b. The NK cells pretreated in step a are cryopreserved in a NK cell cryoprotectant containing 1-50 mM NAM; c. Resuscitate the NK cells cryopreserved in step b to obtain resuscitated NK cells.

[0008] As a further technical solution, in step a, the NK cell in vitro expansion culture medium includes GT-T551 H3 cell culture medium, NK MACS®Basal culture medium, CellGenix® GMP SCGM, CTS™ NK-Xpander™ culture medium, X-VIVOTM-15, Nobimpex NK Xeno-free culture medium or lymphocyte HIPP-T009 serum-free culture medium.

[0009] As a further technical solution, the pretreatment time is 6-48h.

[0010] As a further technical solution, the NK cell cryoprotectant solution includes, by weight, 20-80 parts of CryoStor® CS10 cell freezing solution, 10-40 parts of 20% human albumin injection solution, and 10-40 parts of compound electrolyte injection solution.

[0011] As a further technical solution, the steps of resuscitation are as follows: The NK cells cryopreserved in step b are thawed and then placed in NK cell resuscitation solution at 2-8°C for 5-20 minutes.

[0012] As a further technical solution, the NK cell resuscitation solution includes the NK cell in vitro expansion culture medium.

[0013] As a further technical solution, the following steps are also included: d. The NK cells revived in step c were expanded and cultured in NK cell recovery medium.

[0014] As a further technical solution, the NK cell recovery medium is an NK cell in vitro expansion medium containing 50-1000 IU / mL recombinant human IL-2 and 1-20 mM NAM.

[0015] In a second aspect, the present invention provides a NK cell cryopreservation and resuscitation kit, comprising a pretreatment solution, a NK cell cryoprotectant solution, and a NK cell resuscitation solution; The pretreatment solution is an NK cell in vitro expansion medium containing 0.1-50 mM NAM and 1-1000 ng / mL recombinant human IL-18, which is used to pretreat the NK cells to be frozen; The NK cell cryoprotective solution is a NK cell cryoprotective solution containing 1-50 mM NAM.

[0016] As a further technical solution, it also includes NK cell recovery medium; The NK cell recovery medium is an NK cell in vitro expansion medium containing 50-1000 IU / mL recombinant human IL-2 and 1-20 mM NAM.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The NK cell cryopreservation and resuscitation method provided by the present invention can significantly improve the viability, recovery rate and functional activity of NK cells after 24 hours of recovery by pretreating the NK cells with an NK cell in vitro expansion culture medium containing NAM and recombinant human IL-18 before cryopreservation, and cryopreserving the pretreated NK cells with a cryoprotectant containing NAM, thereby providing possibilities for the construction of NK cell seed banks, gene editing and the development of "off-the-shelf" products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The cell morphology after 24h recovery in Example 1 and Comparative Example 7; Figure 2 The cell viability of Example 1 and Comparative Examples 2, 4, 6, and 7 after 24 hours of recovery; Figure 3 The NK cell recovery rate after 24 hours of recovery in Example 1 and Comparative Examples 2, 4, 6, and 7; Figure 4 The killing activity of K562 in Example 1 and Comparative Examples 2, 4, 6 and 7 after 24 hours of recovery. DETAILED DESCRIPTION

[0020] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0021] In a first aspect, the present invention provides a method for cryopreservation and resuscitation of NK cells, comprising the following steps: a. The NK cells to be frozen are pretreated in an NK cell in vitro expansion medium containing 0.1-50mM NAM and 1-1000ng / mL recombinant human IL-18; during the pretreatment process, the concentration of NAM in the NK cell in vitro expansion medium may be, for example, but not limited to 0.1mM, 1mM, 10mM, 25mM or 50mM, preferably 5mM, and the concentration of recombinant human IL-18 may be, for example, but not limited to 1ng / mL, 10ng / mL, 100ng / mL or 1000ng / mL, preferably 250ng / mL; b. The NK cells pretreated in step a are cryopreserved in a NK cell cryoprotectant containing 1-50 mM NAM; in this process, the concentration of NAM in the NK cell cryoprotectant may be, for example, but not limited to, 1 mM, 10 mM, 25 mM or 50 mM, preferably 5 mM; c. Resuscitate the NK cells cryopreserved in step b to obtain resuscitated NK cells.

[0022] The NK cell cryopreservation and resuscitation method provided by the present invention can significantly improve the viability, recovery rate and functional activity of NK cells after 24 hours of recovery by pretreating the NK cells with an NK cell in vitro expansion culture medium containing NAM and recombinant human IL-18 before cryopreservation, and cryopreserving the pretreated NK cells with a cryoprotectant containing NAM, thereby providing possibilities for the construction of NK cell seed banks, gene editing and the development of "off-the-shelf" products.

[0023] In some optional embodiments, in step a, the NK cell in vitro expansion medium includes but is not limited to GT-T551 H3 cell culture medium, NK MACS®Basal culture medium, CellGenix® GMP SCGM, CTS™ NK-Xpander™ culture medium, X-VIVOTM-15, Nobimpex NK Xeno-free culture medium or lymphocyte HIPP-T009 serum-free culture medium, or other NK cell in vitro expansion culture medium known to those skilled in the art.

[0024] In some optional embodiments, in order to better improve the culture effect of NK cells, the NK cell in vitro expansion medium further contains cytokines; The cytokines include at least one of IL-2, IL-15, IL-12, IL-21 or IL-27, preferably IL-2 and IL-15.

[0025] In some optional embodiments, the pretreatment time may be, for example, but not limited to, 6 h, 12 h, 24 h, 36 h or 48 h, preferably 24 h.

[0026] In some optional embodiments, the NK cell cryoprotectant includes CryoStor®CS10 cell cryopreservation solution, 20% human albumin injection and compound electrolyte injection. Among them, CryoStor®CS10 cell cryopreservation solution can be, for example, Stemcell Technology, with a product number of 7930; 20% human albumin injection can be, for example, CSL Behring AG, Switzerland, with an import drug registration certificate number of S20120070; compound electrolyte injection can be, for example, Shanghai Baxter Medical Supplies Co., Ltd., with a product number of A6E2543.

[0027] In terms of mass fractions, the mass fractions of CryoStor®CS10 cell freezing solution can be, for example, but not limited to 20 parts, 40 parts, 60 parts or 80 parts, preferably 40 parts; the mass fractions of 20% human albumin injection can be, for example, but not limited to 10 parts, 20 parts, 30 parts or 40 parts, preferably 30 parts; the mass fractions of compound electrolyte injection can be, for example, but not limited to 10 parts, 20 parts, 30 parts or 40 parts, preferably 30 parts.

[0028] In some optional embodiments, the resuscitation steps are as follows: The NK cells cryopreserved in step b are thawed and then placed in NK cell resuscitation solution at 2-8°C for 5-20 minutes.

[0029] In some optional embodiments, the thawing method includes placing the NK cells in a 37° C. water bath for thawing.

[0030] In some optional embodiments, the NK cell resuscitation solution includes NK cell in vitro expansion culture medium.

[0031] The NK cell in vitro expansion medium includes but is not limited to GT-T551 H3 cell culture medium, NK MACS®Basal culture medium, CellGenix® GMP SCGM, CTS™ NK-Xpander™ culture medium, X-VIVOTM-15, Nobimpex NK Xeno-free culture medium or lymphocyte HIPP-T009 serum-free culture medium, or other NK cell in vitro expansion culture medium known to those skilled in the art.

[0032] In some optional embodiments, the method further comprises the following steps: d. The NK cells revived in step c were expanded and cultured in NK cell recovery medium.

[0033] In some optional embodiments, the NK cell recovery medium contains recombinant human IL-2 and NK cell in vitro expansion medium, wherein the concentration of recombinant human IL-2 can be, for example, but not limited to 50 IU / mL, 100 IU / mL, 500 IU / mL or 1000 IU / mL, preferably 500 IU / mL, and the concentration of NAM can be, for example, but not limited to 1 mM, 5 mM, 10 mM, 15 mM or 20 mM, preferably 5 mM.

[0034] The inventors have found that the viability, recovery rate and functional activity of the NK cells can be further improved by amplifying and culturing the NK cells recovered above using an NK cell in vitro expansion medium containing 50-1000 IU / mL recombinant human IL-2 and 1-20 mM NAM.

[0035] In some optional embodiments, the NK cells are peripheral blood NK cells or umbilical cord blood NK cells.

[0036] In a second aspect, the present invention provides a NK cell cryopreservation and resuscitation kit, comprising a pretreatment solution, a NK cell cryoprotectant solution, and a NK cell resuscitation solution; The pretreatment solution is an NK cell in vitro expansion medium containing 0.1-50 mM NAM and 1-1000 ng / mL recombinant human IL-18, which is used to pretreat the NK cells to be frozen; The NK cell cryoprotective solution is a NK cell cryoprotective solution containing 1-50 mM NAM.

[0037] The culture medium is obtained based on the above-mentioned NK cell cryopreservation and resuscitation method and can be used for cryopreservation and resuscitation of NK cells.

[0038] In some optional embodiments, it further comprises NK cell recovery medium; The NK cell recovery medium is an NK cell in vitro expansion medium containing 50-1000 IU / mL recombinant human IL-2 and 1-20 mM NAM.

[0039] The present invention is further described below by means of specific examples and comparative examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0040] The reagents used in the following examples and comparative examples are as follows: GT-T551 H3 cell culture medium (liquid form): Takara, product number GT-T551 H3; NK MACS® Basal Medium (NK MACS® Medium, human, liquid form): Miltenyi Biotec, catalog number 130-114-429; CellGenix® GMP SCGM (Serum-free Stem Cell Growth Medium, liquid form): CellGenix, catalog number 20802-0500; CTS™ NK-Xpander™ Medium (liquid): Gibco, product number A49161-01; X-VIVOTM-15 (liquid): Lonza, product number BEBP02-054Q; Nobimpex NK Xeno-free Medium (product form is liquid): Nobimpex, product number is N115-500; Lymphocyte HIPP-T009 serum-free culture medium (liquid form): Shanghai Bio-Gene Biotechnology Co., Ltd., product number FG0103801; Recombinant human IL-2 (solid form): Jinan Protein Technology Co., Ltd., product number GMP-CD66; Recombinant human IL-18 (solid form): Jinan Protein Technology Co., Ltd., product number GMP-CD72; NAM (niacinamide, solid form): Stemcell Technology, catalog number 07154; 20% human albumin injection (20% human albumin intravenous infusion solution): CSL Behring AG, Switzerland, imported drug registration certificate number S20120070; CryoStor®CS10 cell freezing medium (defined cryopreservation medium with 10% DMSO, the product form is liquid): Stemcell Technology, catalog number 7930; Bomel A (compound electrolyte injection): Shanghai Baxter Medical Products Co., Ltd., product number A6E2543; Sodium chloride injection (0.9%): Shijiazhuang No. 4 Pharmaceutical Co., Ltd., product number: National Medicine Standard H20066533; CFSE: BD Biosciences, catalog number: 565082; FITC anti-human CD45: Biolegend, catalog number: 304006; APC anti-human CD3: Biolegend, catalog number: 300412; PE anti-human CD56 (NCAM): Biolegend, Catalog No.: 318306; 7-AAD: BD Biosciences, Catalog No.: 559925; PE Mouse IgG1: Biolegend, Catalog No.: 400114; FITC Mouse IgG1: Biolegend, Catalog No.: 400108; APC Mouse IgG1: Biolegend, Cat. No.: 400120.

[0041] Example 1 A method for cryopreservation and resuscitation of umbilical cord blood NK cells, comprising the following steps: 1) Pre-treating the umbilical cord blood NK cells that have been expanded in vitro for 7-28 days and are to be cryopreserved to obtain cells that have been pre-treated before cryopreservation; Among them, the pretreatment method is to culture the umbilical cord blood NK cells to be frozen in NK cell in vitro expansion medium (GT-T551 H3 cell culture medium) containing 5mM NAM and 250ng / mL IL-18 for 24h.

[0042] 2) Enriching the umbilical cord blood NK cells pretreated before cryopreservation and then cryopreserving them; Transfer the umbilical cord blood NK cell suspension that has been pre-treated before freezing to a 50 mL sterile centrifuge tube and centrifuge at 350 g for 10 minutes at room temperature. After pouring off the supernatant, wash three times with saline containing 0.1% HSA. After the last wash, pour off the supernatant and use NK cell cryoprotectant at 0.5 × 10 8 cells / mL-2×10 8 Resuspend the cells at a density of 1 cell / mL, then transfer the cell suspension to 1 mL of a 2 mL cell cryovial, place the cryovial in a program-controlled cooling box, place the program-controlled cooling box in a -80 °C refrigerator for 24 h, and then place in liquid nitrogen for long-term storage.

[0043] Among them, the NK cell cryoprotectant is prepared by 40% CryoStor® CS10 cell freezing solution, 30% human albumin injection (20%) and 30% compound electrolyte injection, and contains 5mM NAM.

[0044] 3) Resuscitate frozen cord blood NK cells; Thaw the umbilical cord blood NK cell cryopreservation tube in a 37°C water bath, then slowly transfer the thawed cell suspension into the NK cell resuscitation solution precooled to 2-8°C and let it stand for 10 minutes.

[0045] The NK cell resuscitation fluid is NK cell basal culture medium (GT-T551 H3 cell culture medium).

[0046] 4) Inoculate the revived umbilical cord blood NK cells into a culture bottle and culture them in a carbon dioxide incubator; The umbilical cord blood NK cells placed in the resuscitation solution were centrifuged to remove the cryopreserved solution and 3-8×10 6 After resuspending at a density of 10 cells / mL, the cells were inoculated into a T75 cell culture flask and placed in a carbon dioxide incubator for static culture. After 24 hours of culture, the quality of umbilical cord blood NK cells was tested.

[0047] Among them, the NK cell recovery medium is an NK cell in vitro expansion medium (GT-T551 H3 cell culture medium) containing 500 IU / mL recombinant human IL-2 and 5 mM NAM.

[0048] Example 2 A method for cryopreservation and resuscitation of umbilical cord blood NK cells, comprising the following steps: 1) Pre-treating the umbilical cord blood NK cells that have been expanded in vitro for 7-28 days and are to be cryopreserved to obtain cells that have been pre-treated before cryopreservation; Among them, the pretreatment method is to culture the umbilical cord blood NK cells to be frozen in NK cell in vitro expansion medium (NK MACS®Basal medium) containing 0.1mM NAM and 1ng / mL IL-18 for 48h.

[0049] 2) Enriching the umbilical cord blood NK cells pretreated before cryopreservation and then cryopreserving them; Transfer the umbilical cord blood NK cell suspension that has been pre-treated before freezing to a 50 mL sterile centrifuge tube and centrifuge at 350 g for 10 minutes at room temperature. After pouring off the supernatant, wash three times with saline containing 0.1% HSA. After the last wash, pour off the supernatant and use NK cell cryoprotectant at 0.5 × 10 8 cells / mL-2×10 8 Resuspend the cells at a density of 1 cell / mL, then transfer the cell suspension to 1 mL of a 2 mL cell cryovial, place the cryovial in a program-controlled cooling box, place the program-controlled cooling box in a -80 °C refrigerator for 24 h, and then place in liquid nitrogen for long-term storage.

[0050] Among them, the NK cell cryoprotectant is prepared by 20% CryoStor® CS10 cell freezing solution, 40% human albumin injection (20%) and 40% compound electrolyte injection, and contains 1mM NAM.

[0051] 3) Resuscitate frozen cord blood NK cells; Thaw the umbilical cord blood NK cell cryopreservation tube in a 37°C water bath, then slowly transfer the thawed cell suspension into the NK cell resuscitation solution precooled to 2-8°C and let it stand for 10 minutes.

[0052] The NK cell resuscitation fluid is NK cell basal culture medium (NK MACS®Basal culture medium).

[0053] 4) Inoculate the revived NK cells into culture bottles and culture them in a carbon dioxide incubator; The NK cells in the resuscitation solution were centrifuged to remove the cryopreserved solution and 3-8×10 6 After resuspending at a density of 10 cells / mL, the cells were inoculated into a T75 cell culture flask and placed in a carbon dioxide incubator for static culture. After 24 hours of culture, the quality of umbilical cord blood NK cells was tested.

[0054] Among them, the NK cell recovery medium is an NK cell in vitro expansion medium (NK MACS®Basal medium) containing 50 IU / mL recombinant human IL-2 and 1 mM NAM.

[0055] Example 3 A method for cryopreservation and resuscitation of umbilical cord blood NK cells, comprising the following steps: 1) Pre-treating the umbilical cord blood NK cells that have been expanded in vitro for 7-28 days and are to be cryopreserved to obtain cells that have been pre-treated before cryopreservation; Among them, the pretreatment method is to culture the umbilical cord blood NK cells to be frozen in NK cell in vitro expansion medium (CellGenix® GMP SCGM) containing 50mM NAM and 1000ng / mL IL-18 for 6h.

[0056] 2) Enriching the umbilical cord blood NK cells pretreated before cryopreservation and then cryopreserving them; Transfer the umbilical cord blood NK cell suspension that has been pre-treated before freezing to a 50 mL sterile centrifuge tube and centrifuge at 350 g for 10 minutes at room temperature. After pouring off the supernatant, wash three times with saline containing 0.1% HSA. After the last wash, pour off the supernatant and use NK cell cryoprotectant at 0.5 × 10 8 cells / mL-2×10 8 Resuspend the cells at a density of 1 cell / mL, then transfer the cell suspension to 1 mL of a 2 mL cell cryovial, place the cryovial in a program-controlled cooling box, place the program-controlled cooling box in a -80 °C refrigerator for 24 h, and then place in liquid nitrogen for long-term storage.

[0057] Among them, the NK cell cryoprotectant is prepared by 80% CryoStor® CS10 cell freezing solution, 10% human albumin injection (20%) and 10% compound electrolyte injection, and contains 50mM NAM.

[0058] 3) Resuscitate frozen cord blood NK cells; Thaw the NK cell cryopreservation tube in a 37°C water bath, then slowly transfer the thawed cell suspension into the NK cell resuscitation solution precooled to 2-8°C and let stand for 10 minutes.

[0059] The NK cell resuscitation fluid is NK cell basal culture medium (CellGenix® GMP SCGM).

[0060] 4) Inoculate the revived umbilical cord blood NK cells into a culture bottle and culture them in a carbon dioxide incubator; The umbilical cord blood NK cells placed in the resuscitation solution were centrifuged to remove the cryopreserved solution and 3-8×10 6 After resuspending at a density of 10 cells / mL, the cells were inoculated into a T75 cell culture flask and placed in a carbon dioxide incubator for static culture. After 24 hours of culture, the quality of umbilical cord blood NK cells was tested.

[0061] Among them, the NK cell recovery medium is an NK cell in vitro expansion medium (CellGenix® GMP SCGM) containing 1000IU / mL recombinant human IL-2 and 20mM NAM.

[0062] Comparative Example 1 The difference from Example 1 is that in step 4), the NK cell recovery medium is an NK cell in vitro expansion medium containing 500 IU / mL recombinant human IL-2.

[0063] Comparative Example 2 The difference from Example 1 is that in step 2), NAM is not added to the NK cell cryoprotectant solution.

[0064] Comparative Example 3 The difference from Example 1 is that in step 2), NAM is not added to the NK cell cryoprotectant; in step 4), the NK cell recovery medium is an NK cell in vitro expansion medium containing 500 IU / mL recombinant human IL-2.

[0065] Comparative Example 4 The difference from Example 1 is that no pretreatment is performed.

[0066] Comparative Example 5 The difference from Example 1 is that no pretreatment is performed; in step 4), the NK cell recovery medium is an NK cell in vitro expansion medium containing 500 IU / mL recombinant human IL-2.

[0067] Comparative Example 6 The difference from Example 1 is that no pretreatment is performed; in step 2), NAM is not added to the NK cell cryoprotective solution; Comparative Example 7 The difference from Example 1 is that no pretreatment is performed; in step 2), no NAM is added to the NK cell cryoprotectant; in step 4), the NK cell recovery medium is an NK cell in vitro expansion medium containing 500 IU / mL recombinant human IL-2.

[0068] Test Example 1 The cell treatment conditions of Examples 1-3 and Comparative Examples 2-7 are shown in Table 1.

[0069] Table 1 .

[0070] The cord blood NK cells before cryopreservation, the cord blood NK cells obtained by resuscitation, and the cord blood NK cells cultured for 24 hours after resuscitation in Examples 1-3 and Comparative Examples 2-7 were tested (cell viability, NK cell recovery rate, and K562 cytotoxicity), and the testing method was as follows: 1. Cell viability counting: Mix 20uL of cell suspension and 20uL of AO / PI dye solution evenly, pipette 20uL of suspension and slowly add it to the fully automatic fluorescent cell counting plate, let it stand horizontally for 1-2 minutes, put the fully automatic fluorescent cell counting plate into the cell counter, and use the PBMC program to count the cell viability.

[0071] 2. Detection of purity and CD16 expression of umbilical cord blood NK cells: After mixing the cell suspension, take 1×10 6 Cells were collected by centrifugation (300g for 6 minutes). After discarding the supernatant, the cells were resuspended in 100uL of 1×PBS per test tube, and the corresponding antibodies were added. Cord blood NK cell test tube: FITC anti-human CD45, BV421 anti-human CD16, APC anti-human CD3, PE anti-human CD56 (NCAM), 7-AAD; cord blood NK cell isotype test tube: FITC Mouse IgG1, BV421 MouseIgG1, APC Mouse IgG1, PE Mouse IgG1. The cells were incubated with antibodies at room temperature for 20 minutes, and 2mL 1×PBS was added to each tube to terminate the staining, and centrifuged (400g for 5 minutes). The supernatant was discarded by turning the test tube upside down, and 200-300μL 1×PBS was added to each tube. After vortexing, the cells were detected by BD FACSCelesta flow cytometer.

[0072] 3. Detection of NK cell killing activity in umbilical cord blood: Umbilical cord blood NK cells were co-cultured with CFSE-stained K562 cells at a ratio of 1:1, 2.5:1, 5:1, 10:1, and 20:1 for 4 h, and the cells were collected for 7-AAD staining. The cytotoxic activity of umbilical cord blood NK cells was then detected using a BD FACSCelesta flow cytometer.

[0073] The results are shown in Table 2, Table 3 and Table 4. The cell morphology of Examples 1-3 and Comparative Example 7 after 24 hours of recovery is as follows Figure 1 The cell viability, NK cell recovery rate and K562 cytotoxicity of Examples 1-3 and Comparative Examples 2, 4, 6 and 7 after 24 hours of recovery are shown in the table below. Figure 2 , Figure 3 and Figure 4 shown.

[0074] Table 2 Characteristics of NK cells in umbilical cord blood before cryopreservation .

[0075] Table 3 Characteristics of NK cells from resuscitated cord blood .

[0076] Table 4 Characteristics of NK cells in cord blood after 24 hours of resuscitation .

[0077] The results showed that compared with no treatment and only adding IL-2 to the recovery medium (A8), the group pretreated with IL-18 and NAM (A4) and the group added with NAM to the cell cryoprotective solution (A6) could improve the viability of cells and the recovery rate of NK cells after 24 hours of recovery, and the NK cell recovery rate of group A4 was better than that of group A6. Pretreatment with IL-18 and NAM and adding NAM to the cell cryoprotective solution (A2) could synergistically improve cell viability and NK cell recovery rate. Pretreatment with IL-18 and NAM and adding NAM to the cell cryoprotective solution and the recovery medium containing IL-2 and NAM (A1) could synergistically improve cell viability, NK cell recovery rate and NK cell killing activity.

[0078] Comparative Example 8 The difference from Example 1 is that in step 1), the pretreatment method is to culture the cord blood NK cells to be frozen in an NK cell in vitro expansion medium (GT-T551 H3 cell culture medium) containing 5 mM NAM for 24 hours.

[0079] Comparative Example 9 The difference from Example 1 is that in step 1), the pretreatment method is to culture the umbilical cord blood NK cells to be frozen in an NK cell in vitro expansion medium (GT-T551 H3 cell culture medium) containing 250 ng / mL IL-18 for 24 hours.

[0080] Test Example 2 The umbilical cord blood NK cells of Example 1 and Comparative Examples 8-9 were tested after being resuscitated and cultured for 24 hours. The results are shown in Table 5.

[0081] Table 5 Characteristics of NK cells in cord blood after 24 hours of resuscitation .

[0082] The results showed that during the pretreatment phase, IL-18 and NAM synergistically improved the cell viability, NK cell recovery rate and NK cell functional activity after 24 hours of recovery.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for cryopreservation and resuscitation of NK cells, characterized in that: The following steps are involved: a. The NK cells to be frozen are pretreated in an NK cell in vitro expansion medium containing 0.1-50 mM NAM and 1-1000 ng / mL recombinant human IL-18; b. The NK cells pretreated in step a are cryopreserved in a NK cell cryoprotectant containing 1-50 mM NAM; c. Resuscitate the NK cells cryopreserved in step b to obtain resuscitated NK cells.

2. The cryopreservation and resuscitation method according to claim 1, characterized in that: In step a, the NK cell in vitro expansion medium includes GT-T551 H3 cell culture medium, NK MACS®Basal culture medium, CellGenix® GMP SCGM, CTS™NK-Xpander™ culture medium, X-VIVOTM-15, Nobimpex NK Xeno-free culture medium or lymphocyte HIPP-T009 serum-free culture medium.

3. The cryopreservation and resuscitation method according to claim 1, characterized in that: The pretreatment time is 6-48h.

4. The cryopreservation and resuscitation method according to claim 1, characterized in that: Calculated by weight, the NK cell cryoprotectant solution includes 20-80 parts of CryoStor® CS10 cell freezing solution, 10-40 parts of 20% human albumin injection solution and 10-40 parts of compound electrolyte injection solution.

5. The cryopreservation and resuscitation method according to claim 1, characterized in that: The steps of resuscitation are as follows: The NK cells cryopreserved in step b are thawed and then placed in NK cell resuscitation solution at 2-8°C for 5-20 minutes.

6. The cryopreservation and resuscitation method according to claim 5, characterized in that: The NK cell resuscitation solution includes the NK cell in vitro expansion culture medium.

7. The cryopreservation and resuscitation method according to claim 1, characterized in that: The following steps are also included: d. The NK cells revived in step c were expanded and cultured in NK cell recovery medium.

8. The cryopreservation and resuscitation method according to claim 7, characterized in that: The NK cell recovery medium is an NK cell in vitro expansion medium containing 50-1000 IU / mL recombinant human IL-2 and 1-20 mM NAM.

9. A NK cell cryopreservation and resuscitation kit, characterized in that: Including pretreatment solution, NK cell cryoprotection solution and NK cell resuscitation solution; The pretreatment solution is an NK cell in vitro expansion medium containing 0.1-50 mM NAM and 1-1000 ng / mL recombinant human IL-18, which is used to pretreat the NK cells to be frozen; The NK cell cryoprotective solution is a NK cell cryoprotective solution containing 1-50 mM NAM.

10. The NK cell cryopreservation and resuscitation kit according to claim 9, characterized in that: Also included is NK cell recovery medium; The NK cell recovery medium is an NK cell in vitro expansion medium containing 50-1000 IU / mL recombinant human IL-2 and 1-20 mM NAM.

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