Clinical CAR-NK (Chimeric Antigen Receptor-Natural Killer) cell freezing medium as well as preparation method and application thereof

Abstract

The invention relates to a CAR-NK (chimeric antigen receptor-natural killer) cell cryopreservation solution for clinical use as well as a preparation method and application thereof. The CAR-NK cell freezing medium disclosed by the invention is prepared from a washing solution, a dextran 40 glucose injection and Cryostat CS10, or is prepared from the washing solution, human serum albumin and Cryostat CS10; wherein the washing liquid is prepared from sodium chloride injection and human serum albumin. The cryopreservation liquid disclosed by the invention is used for cryopreserving CAR-NK cells, does not have the problem of cell agglomeration, and can effectively maintain the viability and vitality of the cells; the cryopreservation liquid is simple in component, is in a GMP level or a medicine level, can be directly infused back to a human body, is convenient to prepare and easy to prepare in a large scale, and saves the production and manufacturing cost.

Description

Technical Field

[0001] This invention relates to the field of biology, specifically to a CAR-NK cell cryopreservation solution that can be used clinically, its preparation method, and its applications. Background Technology

[0002] In recent years, cell immunotherapy has emerged as a novel treatment option for certain types of hematologic malignancies. With several CAR-T therapies entering clinical trials, most CAR-T cell therapies utilize cells derived from the patient's own body. The preparation process is complex and time-consuming, highlighting the limitations of autologous cell products and the challenges of CAR-T cell toxicity. Therefore, more convenient, safe, and effective novel cell therapy products have become an important research direction.

[0003] NK cells belong to the lymphocyte family and are found in human peripheral blood, bone marrow, and tissues and organs. NK cells do not require human leukocyte antigen (HLA) matching and primarily kill target cells through three mechanisms: directly killing target cells by releasing cytoplasmic granules containing perforin and granzymes; releasing cytokines, such as IFN-γ and TNF-α, which induce tumor cell apoptosis through interaction with corresponding receptors on the surface of tumor cells; and binding of the Fc receptor CD16 to the Fc fragment of antibodies, triggering antibody-dependent cell-mediated cytotoxicity (ADCC) to kill cells.

[0004] Compared to CAR-T cells, allogeneic NK cells do not express individual-specific TCRs. Current clinical studies show that CAR-NK cells do not produce GvHD (graft-versus-host disease), and the risk of developing GvHD in clinical treatment is far lower than that of allogeneic T cell therapy. Therefore, they have a higher safety profile and have become a strong candidate for targeted therapy. NK cells have a wide range of sources, such as peripheral blood, umbilical cord blood, embryonic stem cells, human induced pluripotent stem cells, and NK cell lines. Therefore, CAR-NK cells are more easily developed into universal, large-scale, "off-the-shelf products."

[0005] In recent years, the number of clinical studies on CAR-NK immunotherapy has been increasing year by year, and there are currently several CAR-NK cell products undergoing clinical trials. CAR-NK production also faces many challenges, such as amplification and transduction efficiency, and cryopreservation.

[0006] CAR-NK cells are sensitive to the freeze-thaw process and tend to clump together. After cryopreservation, the survival rate and cytotoxicity of NK cells are significantly reduced, increasing the difficulty of clinical use.

[0007] While some cryopreservation formulations for NK cells have been disclosed in existing technologies, they still fail to adequately address the aforementioned issues. Extensive and in-depth research is needed to truly advance CAR-NK therapy towards large-scale clinical use. Summary of the Invention

[0008] To address at least one of the many problems associated with CAR-NK cell cryopreservation, this invention provides a CAR-NK cell cryopreservation solution, its preparation method, and its uses.

[0009] In a first aspect of the present invention, an NK cell cryopreservation solution is provided, wherein the formulation of the NK cell cryopreservation solution is selected from any of the following:

[0010] a)

[0011] Components Final concentration (v / v) detergent 20％-55％ Dextran 40 glucose injection 0.1％-25％ Cryostor CS10 40％-75％

[0012] b)

[0013] Components Final concentration (v / v) detergent 20％-55％ Human serum albumin (20% (v / v)) 0.1％-25％ Cryostor CS10 40％-75％

[0014] The detergent formula is as follows:

[0015] Components Final concentration (v / v) Sodium chloride injection 95％-99.99％ Human serum albumin (20%) 0.01％-5％ .

[0016] In one embodiment of the present invention, the formulation of the NK cell cryopreservation solution is selected from any one of the following:

[0017] a)

[0018] Components Final concentration (v / v) detergent 30％-35％ Dextran 40 glucose injection 15％-20％ Cryostor CS10 50％-55％

[0019] b)

[0020] Components Final concentration (v / v) detergent 20％-30％ Human serum albumin (20% (v / v)) 20％-25％ Cryostor CS10 50％-55％

[0021] The detergent formula is as follows:

[0022] Components Final concentration (v / v) Sodium chloride injection 97％-99.99％ Human serum albumin (20%) 0.01％-3％ .

[0023] In a preferred embodiment of the present invention, the formulation of the NK cell cryopreservation solution is selected from any one of the following:

[0024] a)

[0025]

[0026]

[0027] b)

[0028] Components Final concentration (v / v) detergent 30％ Human serum albumin (20% (v / v)) 20％ Cryostor CS10 50％

[0029] The detergent formula is as follows:

[0030] Components Final concentration (v / v) Sodium chloride injection 97.5％ Human serum albumin (20%) 2.5％ .

[0031] In another embodiment of the present invention, the NK cells are derived from NK cell lines, peripheral blood cells, umbilical cord blood, induced pluripotent stem cells, or embryonic stem cells.

[0032] In another embodiment of the invention, the NK cells are genetically engineered NK cells. In a preferred embodiment of the invention, the NK cells are NK cells expressing chimeric antigen receptors.

[0033] In a second aspect of the invention, a method for cryopreserving NK cells is provided, comprising:

[0034] (1) Add the NK cell cryopreservation solution as described in any of the embodiments herein to the NK cells and transfer them into a cryopreservation bag;

[0035] (2) Cool the cryopreservation bag to -90°C or below and freeze it.

[0036] In a third aspect of the invention, the use of NK cell cryopreservation solutions as described in any embodiment herein in cryopreservation of NK cells is provided.

[0037] In one embodiment of the present invention, the NK cells are derived from NK cell lines, peripheral blood cells, umbilical cord blood, induced pluripotent stem cells, or embryonic stem cells.

[0038] In another embodiment of the invention, the NK cells are genetically engineered NK cells. In a preferred embodiment of the invention, the NK cells are NK cells expressing chimeric antigen receptors.

[0039] In a fourth aspect of the invention, a method for preparing an NK cell cryopreservation solution as described in any embodiment herein is provided, comprising:

[0040] (1) Provide each component separately;

[0041] (2) Mix the components to obtain a mixture;

[0042] (3) The mixture is sterilized or not sterilized to obtain the NK cell cryopreservation solution.

[0043] The advantages of this invention are as follows: This invention provides a cryopreservation solution and method for the production and cryopreservation of CAR-NK cells suitable for clinical use. The CAR-NK cells produced by this method can be expanded tens of thousands of times. The provided CAR-NK cell cryopreservation solution: ① No cell clumping problem; ② After one week of liquid nitrogen cryopreservation, the cell viability is basically the same as before cryopreservation, reaching over 83%, with a decrease of no more than 2.5% compared to before cryopreservation; ③ After one week of liquid nitrogen cryopreservation, cell activity and CAR positivity rate recover to pre-cryopreservation levels within 48 hours of thawing, with no significant decrease in cell purity and CAR positivity rate; ④ After one week of cryopreservation, the cells have continuous in vitro proliferation capacity, continuously proliferating over 100 times for 11 consecutive days; ⑤ After one week of cryopreservation, the CAR-NK cells thawing have excellent in vitro cell-killing function, with a killing efficiency of over 97%, showing no significant difference from fresh CAR-NK cells; ⑥ The cryopreservation solution has a simple composition, does not contain NK cell culture medium, and is all GMP or pharmaceutical grade, allowing for direct reinfusion; it is easy to prepare, can be prepared in batches, and saves production costs. Based on extensive experience in CAR-NK cell research and a thorough understanding of the factors affecting cryopreservation, this invention has yielded an NK cell cryopreservation solution that surpasses existing cryopreservation solutions in many aspects. This invention lays the foundation for the large-scale clinical production of heterologous commercial CAR-NK cell therapy products. Attached Figure Description

[0044] Figure 1 CAR-NK cell production and proliferation curves.

[0045] Figure 2 Flow cytometry analysis of CAR-NK cells at harvest. (A) Batch 1; (B) Batch 2.

[0046] Figure 3 The effect of different cryopreservation solutions on CAR-NK cell clumping.

[0047] Figure 4 Changes in cell viability before and after cryopreservation of CAR-NK cells.

[0048] Figure 5 CAR-NK cell resuscitation and in vitro cell killing.

[0049] Figure 6 CAR-NK cell resuscitation and in vitro culture. Detailed Implementation

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0051] The terminology used in this article

[0052] The "NK cells" described in this application are natural killer cells, a subset of lymphocytes. NK cells primarily attack tumor cells, virus-infected cells (malignant and inflammatory cells), larger pathogens (such as fungi and parasites), and allogeneic transplanted organs and tissues. They play an effector role in anti-tumor innate and specific immunity, directly killing various tumor cells and virus-infected cells, serving as the body's first line of defense in innate immunity.

[0053] The NK cells in this article can be NK cells from any of the following sources: NK cells derived from peripheral blood mononuclear cells (PBMCs), which are usually obtained by collecting anticoagulated human peripheral whole blood and centrifuging it according to standard procedures; NK cells derived from NK cell lines, which are obtained by culturing NK cell lines from leukemia or lymphoma patients, such as NK-92, NKG, NKL, KHYG-1, YT, NKYS, SNK-6, or IMC-1; NK cells derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), which are induced to differentiate from iPSCs or ESCs; and NK cells derived from umbilical cord blood, which are directly expanded from umbilical cord blood or differentiated in vitro from umbilical cord blood hematopoietic stem cells.

[0054] The “Cryostor CS10” described in this application is a serum-free, animal-free commercial cryopreservation solution containing 10% dimethyl sulfoxide (DMSO).

[0055] The "sodium chloride injection (washing solution)" described in this application is a 0.9% sodium chloride solution, and 250 ml of the aqueous solution contains 2.25 g of sodium chloride.

[0056] The “dextran 40 glucose injection” described in this application refers to a sterile aqueous solution of dextran 40 and 5% glucose, specifically 30g of dextran 40 and 25g of glucose in 500ml of solution.

[0057] The "human serum albumin" described in this application is a blood product extracted, separated, and prepared from the blood of healthy individuals, which can be directly injected into the human body and has albumin as its main component.

[0058] NK cell cryopreservation solution

[0059] In one aspect of the invention, an NK cell cryopreservation solution is provided. According to one embodiment of the invention, the NK cell cryopreservation solution is prepared from a washing buffer, dextran 40 glucose injection, and Cryptor CS10, wherein the washing buffer is prepared from sodium chloride injection and human serum albumin. In a preferred embodiment, the washing buffer consists of sodium chloride injection at a final concentration of 95%-99.99% (v / v) and human serum albumin (20%, v / v) at a final concentration of 0.01%-5% (v / v), and the NK cell cryopreservation solution consists of a washing buffer at a final concentration of 20%-55% (v / v), 0.1%-25% (v / v) dextran 40 glucose injection, and 40%-75% (v / v) Cryptor CS10. More preferably, the washing solution consists of sodium chloride injection with a final concentration of 97%-99.99% (v / v) and human serum albumin (20%, v / v) with a final concentration of 0.01%-3% (v / v), and the NK cell cryopreservation solution consists of washing solution with a final concentration of 30%-35% (v / v), 15%-20% (v / v) dextran 40 glucose injection, and 50%-55% (v / v) Cryptor CS10. More preferably, the washing solution consists of sodium chloride injection with a final concentration of 97.5% (v / v) and human serum albumin (20%, v / v) with a final concentration of 2.5% (v / v), and the NK cell cryopreservation solution consists of washing solution with a final concentration of 30% (v / v), 20% (v / v) dextran 40 glucose injection, and 50% (v / v) Cryptor S10. According to another embodiment of the present invention, the NK cell cryopreservation solution is prepared from a washing solution, human serum albumin, and Cryptor CS10, wherein the washing solution is prepared from sodium chloride injection and human serum albumin. In a preferred embodiment, the washing solution consists of sodium chloride injection with a final concentration of 95%-99.99% (v / v) and human serum albumin (20%, v / v) with a final concentration of 0.01%-5% (v / v), and the NK cell cryopreservation solution consists of a washing solution with a final concentration of 20%-55% (v / v), 0.1%-25% (v / v) human serum albumin (20%, v / v), and 40%-75% (v / v) Cryptor CS10. More preferably, the washing solution consists of sodium chloride injection solution with a final concentration of 97%-99.99% (v / v) and human serum albumin (20%, v / v) with a final concentration of 0.01%-3% (v / v), and the NK cell cryopreservation solution consists of washing solution with a final concentration of 20%-30% (v / v), human serum albumin (20%, v / v) with a final concentration of 20%-25% (v / v), and Cryptotor CS10 with a final concentration of 50%-55% (v / v).More preferably, the washing solution consists of sodium chloride injection solution with a final concentration of 97.5% (v / v) and human serum albumin (20%, v / v) with a final concentration of 2.5% (v / v), and the NK cell cryopreservation solution consists of washing solution with a final concentration of 30% (v / v), human serum albumin (20%, v / v) and Cryptotor S10 with a final concentration of 50% (v / v).

[0060] According to embodiments of the present invention, the cryopreserved NK cells can be from various sources, including NK cell lines, peripheral blood cells, umbilical cord blood, and NK cells derived from induced pluripotent stem cells and embryonic stem cells.

[0061] According to an embodiment of the present invention, the CAR-NK cells are NK cells expressing chimeric antigen receptors. The "chimeric antigen receptor," abbreviated as CAR, is a type of fusion protein prepared by fusing the antigen-binding portion (variable region) of a monoclonal antibody with an intracellular signaling site derived from a lymphocyte-activated receptor. Typically, CARs include molecules that recognize peptides derived from tumor antigens presented by major histocompatibility (MHC) molecules; or antibodies or fragments thereof (such as Fab, scFv, Fv) expressed on the surface of CAR cells targeting cancer antigens. Typically, cryopreserved CAR-NK cells are off-the-shelf products, providing rapid access to therapeutic cell products for clinical patients. Currently, numerous clinical studies of CAR-NK cells in hematological malignancies, solid tumors, and autoimmune diseases are underway.

[0062] Methods for cryopreserving NK cells

[0063] In another aspect of the invention, a method for cryopreserving NK cells is provided. According to an embodiment of the invention, the NK cell cryopreservation protocol involves using any of the NK cell cryopreservation solutions described herein. Cryopreservation of NK cells typically includes the following steps: (1) mixing the cells with the cell cryopreservation composition described herein, and (2) gradually cooling the mixture to -90°C or below. The gradual cooling is any programmed cooling known in the art for freezing cells (e.g., immune cells), such as using a programmed cooling device. An exemplary programmed cooling process is performed using a programmed cooling instrument (ThermoFisher, 7451TF), running program 4. The specific cooling process is as follows: 1) Reach 20°C and wait; 2) The sample temperature is cooled to -6°C at a rate of 1.0°C / min; 3) The chamber temperature is cooled to -50°C at a rate of 25.0°C / min; 4) The chamber temperature is heated to -14°C at a rate of 10.0°C / min; 5) The chamber temperature is cooled to -45°C at a rate of 1.0°C / min; 6) The chamber temperature is cooled to -90°C at a rate of 10.0°C / min.

[0064] Uses of NK cell cryopreservation solution

[0065] The NK cell cryopreservation composition of this invention can be used for cryopreservation of cells and preparation of cell cryopreservation formulations. It has the following advantages: ① No cell clumping problem; ② After one week of liquid nitrogen cryopreservation, cell viability remains essentially the same as before cryopreservation, reaching over 83%, with a decrease of no more than 2.5% compared to before cryopreservation; ③ After one week of thawing from liquid nitrogen cryopreservation, cell activity and CAR positivity rate recover to pre-cryopreservation levels within 48 hours, with no significant decrease in cell purity and CAR positivity rate; ④ After one week of cryopreservation, cells exhibit continuous in vitro proliferation capacity, continuously proliferating more than 100-fold for 11 consecutive days; ⑤ After one week of cryopreservation and thawing, CAR-NK cells have excellent in vitro cell-killing function, with a killing efficiency of over 97%, showing no significant difference from fresh CAR-NK cells; ⑥ The cryopreservation solution has a simple composition, does not contain NK cell culture medium, and is entirely GMP or pharmaceutical grade, allowing for direct reinfusion; it is easy to prepare, can be prepared in batches, and saves production costs.

[0066] Preparation method of NK cell cryopreservation solution

[0067] In another aspect of the present invention, the present invention also provides a method for preparing any of the NK cell cryopreservation solutions described herein, comprising the following steps: (1) providing each component separately; (2) mixing the components to obtain a mixture; and (3) sterilizing the mixture to obtain the NK cell cryopreservation solution. The components of the NK cell cryopreservation solution can be obtained commercially or prepared by the individual. After obtaining each component, after obtaining the specified amount through conventional measurement steps, these specific amounts of components are mixed to obtain the NK cell cryopreservation solution. Typically, a filtration and sterilization step can also be added, which is a common practice for those skilled in the art. The obtained NK cell cryopreservation solution is usually stored at low temperature for future use if it is not needed immediately. For convenience, it can also be aliquoted and stored.

[0068] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and materials used in the embodiments are conventional materials and methods in the art.

[0069] Example

[0070] Example 1

[0071] Prepare the washing solution and cryopreservation solutions CSR-01, CSR-02 and CSR-03 according to the following formulas and preparation methods.

[0072] The detergent formula is as follows:

[0073] Components Manufacturers Item number Final concentration (v / v) Sodium chloride injection Hunan Kelun Pharmaceutical National Drug Approval Number H43020455 97.5％ Human serum albumin (20%) Jet Belin S20170005 2.5％

[0074] Cryopreservation solution CSR-01, the formula is as follows:

[0075]

[0076] Cryopreservation solution CSR-02, the formula is as follows:

[0077] Components Manufacturers Item number Final concentration (v / v) detergent self made 30％ Human serum albumin (20%) Jet Belin S20170005 20％ Cryostor CS10 STEMCELL 100-1061 50％

[0078] Cryopreservation solution CSR-03, the formula is as follows:

[0079] Components Manufacturers Item number Final concentration (v / v) detergent self made 30％ Dextran 40 glucose injection Sichuan Kelun H51020230 20％ Cryostor CS10 STEMCELL 100-1061 50％

[0080] The main reagent preparation methods are as follows:

[0081] Preparation of detergent:

[0082] Add 12.8 ml of human serum albumin (20%) to the sodium chloride injection bag using a 20 ml syringe, invert and mix well to prepare as a washing solution.

[0083] Element Sodium chloride injection 20% human serum albumin Ratio (v / v) 97.5％ 2.5％

[0084] Preparation of cryopreservation solution:

[0085] Prepare 40ml of each cryopreservation solution according to the table below. Add each cryopreservation solution component to a 50ml centrifuge tube in sequence, mix well, and store in a 4℃ refrigerator for later use.

[0086]

[0087] Example 2

[0088] 1. NK cell sorting

[0089] Take approximately 1×10 8 One CBMC (Cord Blood Mononuclear Cell) was revived in a 37°C water bath. The revived cells were then transferred to a 50mL centrifuge tube containing NK medium, centrifuged at 300g for 10 minutes, and the supernatant was discarded. The cells were then transferred to NK medium (…). NK cells were expanded using serum-free culture medium P01 (P01), and the cells were resuspended in Excel Bio to a density of 1–2.5 × 10⁻⁶ cells / mL. 6 1 live cells / ml, incubated overnight at 37°C with 5% CO2.

[0090] CBMC cells that had been relieved for 24 hours were centrifuged at 300g for 10 min, and resuspended in pre-cooled sorting buffer (97.5% DPBS + 2.5% (20% human serum albumin)) to adjust the cell density to 1×10⁻⁶ cells / mL. 8Live cells / ml. Following the CD3 bead addition ratio: 200 μl CD3 beads / ml cell suspension, add the corresponding CD3 beads (CD3 Microbeads, human, Miltenyi), gently mix, and incubate at 4°C for 15 minutes. Separate the cells using an LS sorting column, collecting the filtrate into new centrifuge tubes. Centrifuge the filtered cell suspension at 300g for 10 minutes, resuspend the cells in pre-chilled sorting buffer, and adjust the cell density to 1×10⁻⁶ cells / ml. 8 Live cells / ml. Add the corresponding CD56 magnetic beads (CD56Mircrobeads, human, Miltenyi) to a cell suspension of 100 μl CD56 magnetic beads / ml, mix gently, and incubate at 4°C for 15 minutes.

[0091] After incubation, resuspend the cells in sorting buffer at a ratio of 20 ml sorting buffer / ml cell suspension, and centrifuge at 300 g for 10 minutes at 4°C. Discard the supernatant, resuspend the cells in 2 mL of sorting buffer, and sort them through an LS sorting column. After sorting, remove the LS sorting column, add 5 mL of sorting buffer, and use a syringe to eject the cells from the sorting column into a new centrifuge tube; these are NK cells.

[0092] 2. Preparation and culture of CAR-NK cells

[0093] The sorted NK cells were mixed with feeder cells at a ratio of NK:FC of 1:1 to 1:2, and the cell density was adjusted to 1–2.0 × 10⁶ cells / year using NK culture medium. 6 Cells were cultured at 100 viable cells / ml in a 37°C, 5% CO2 incubator. On day 3, half the culture volume was replenished with fresh NK medium, and cultured until day 6, at which point the cell density was adjusted to 0.5 × 10⁶ cells / ml. 6 Calculate the required virus volume based on 1 live cells / mL and MOI=3. Transfer the cell suspension and virus solution into T25 cell culture flasks coated with 15μg / mL RetroNectin (TAKARA) one day in advance, and incubate at 37℃ and 5% CO2 for 48-72 hours for virus transduction.

[0094] After viral transduction, centrifuge at 300g for 10 min, discard the supernatant, add feeder cells at an NK:FC ratio of 1:1 to 1:2, mix well, and adjust the cell density to 1–2.0 × 10⁶ cells / year using NK medium. 6 Cells were cultured at 100 viable cells / ml in a 37°C, 5% CO2 incubator. Cell counts were performed every 3 days, and fresh NK medium was added to adjust the cell density to 1–2.0 × 10⁶ cells / ml. 6 1 live cells / ml, cultured for 7 days. Add feeder cells at an NK:FC ratio of 1:1 to 1:2 for a new round of culture for 7 days, then harvest the cells. Figure 1After 24 days of continuous culture, CAR-NK cells proliferated more than 20,000 times, and 1–5.0 × 10⁻⁶ cells could be isolated and purified from one CBMC cell. 7 This process can produce 1,000 NK cells, thus potentially enabling large-scale production of CAR-NK cells.

[0095] 3. Flow cytometry detection of CAR-NK cells at harvest

[0096] Cell samples were taken before harvest for flow cytometry analysis to detect CAR positivity rate, CD56 positivity, and CD3 positivity. 1×10⁻⁶ cells were collected. 6 Cell suspensions containing a total viable cell count were transferred to labeled flow cytometry tubes. Each tube was washed with approximately 3-5 ml of PBS buffer, centrifuged at 450 g for 5 min, the supernatant was discarded, and 95 μL of mixed antibody working solution was added per tube. The tubes were mixed thoroughly and incubated at 2-8°C in the dark for 30 min. After incubation, each tube was washed with approximately 3-5 ml of PBS buffer, centrifuged at 450 g for 5 min, the supernatant was discarded, and 95 μL of the corresponding secondary antibody working solution was added per tube. The tubes were mixed thoroughly and incubated at 2-8°C in the dark for 30 min. After incubation, each tube was washed with approximately 3-5 ml of PBS buffer, centrifuged at 450 g for 5 min, the supernatant was discarded, and approximately 200 μL of PBS buffer was added. The results were analyzed by flow cytometry. Figure 2 At harvest, CD3-CD56+ cells had a purity of over 99%, while CD3+ cells had a purity of less than 0.5%.

[0097] 4. Washing and cryopreservation of CAR-NK cells

[0098] Approximately 2 L of cultured cells were centrifuged at 300 g for 10 min, resuspended in washing buffer, and then divided into three aliquots. Each aliquot was centrifuged at 300 g for 10 min, and the cells were resuspended in cryopreservation solutions CSR-01, CSR-02, and CSR-03, respectively. Cell samples were taken for counting, and the cell density was adjusted to 2.5–5 × 10⁻⁶ cells / mL using cryopreservation solution. 7 1 live cells / ml, mix well, and dispense into cryopreservation bags, 10-20ml per bag. Observe the appearance of each bag. Figure 3 The cells in the CSR-01 cryopreservation solution were severely clumped and were discarded. The cells in the CSR-02 and CSR-03 cryopreservation solutions were not clumped. These two cryopreservation bags were placed in a programmed cooling system (ThermoFisher, 7451TF) and programmed cooling was run. After the programmed cooling was completed, the cryopreservation bags were transferred to a liquid nitrogen tank for storage via a liquid nitrogen transport tank.

[0099] 5. Cell resuscitation viability detection

[0100] After freezing in liquid nitrogen for 7 days, one bag of cells was removed, thawed in a 37°C water bath, and transferred to a 50mL centrifuge tube containing NK medium B (PO1 + 200 IU IL2). The cells were centrifuged at 300g for 10 min, the supernatant was discarded, and the cells were resuspended in NK medium. Cell viability was measured using an NC200 cell counter. Figure 4 After thawing, the cell viability was over 83%, basically the same as before cryopreservation, with no significant decline.

[0101] 6. In vitro killing effect of CAR-NK cells after resuscitation

[0102] On day 3 of cell resuscitation after 7 days of cryopreservation, CAR-NK cell suspension was seeded into an opaque microplate, 100 μl of 3×10⁻⁶ microplate. 4 Add 50 μL of L363 cells per well at an effector-to-target ratio of 3:1, mix gently, and incubate overnight at 37°C with 5% CO2. After incubation, remove the cells from the CO2 incubator and gently aspirate 50 μL of culture medium. Add 100 μL of luciferase substrate to each well, react for 3-5 minutes, and calculate the killing efficiency using a microplate reader. Figure 5 The NK cell killing efficiency of the control group was about 33%, while the cell killing efficiency of cells frozen in the two cryopreservation solutions CSR-02 and CSR-03 after thawing was over 97%. There was no significant difference in the killing effect compared with freshly prepared CAR-NK cells, indicating that the two cryopreservation solutions have a good protective effect on CAR-NK cells.

[0103] 7. Cell resuscitation and in vitro culture

[0104] After cell resuscitation, the cell density was adjusted to 1×10⁶ cells / year using NK medium. 6 Cells were cultured at 100 viable cells / ml, and IL2 (Beijing Shuanglu) was added to a final concentration of 200 IU / ml. The cells were then incubated at 37°C in a 5% CO2 incubator. Cell counts were performed every 3 days based on cell growth rate, and NK medium was added to adjust the cell density to 1×10⁶ cells / ml. 6 1 live cells / ml, add IL2 to a final concentration of 200 IU / ml, and calculate the cell proliferation fold. Figure 6 Each group of cells can be cultured in vitro for 11 days, expanding approximately 100-fold.

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An NK cell cryopreservation solution, characterized in that, The formulation of the NK cell cryopreservation solution is selected from any one of the following: a) b) The detergent formula is as follows: 。 2. The NK cell cryopreservation solution according to claim 1, characterized in that, The formulation of the NK cell cryopreservation solution is selected from any one of the following: a) b) The detergent formula is as follows: 。 3. The NK cell cryopreservation solution according to claim 2, characterized in that, The formulation of the NK cell cryopreservation solution is selected from any one of the following: a) b) The detergent formula is as follows: 。 4. The NK cell cryopreservation solution according to claim 1, characterized in that, The NK cells are derived from NK cell lines, peripheral blood cells, umbilical cord blood, induced pluripotent stem cells, or embryonic stem cells.

5. The NK cell cryopreservation solution according to claim 1, characterized in that, The NK cells mentioned are genetically engineered NK cells.

6. The NK cell cryopreservation solution according to claim 5, characterized in that, The NK cells mentioned are NK cells that express chimeric antigen receptors.

7. A method for cryopreserving NK cells, comprising: (1) Add the NK cell cryopreservation solution as described in any one of claims 1-6 to the NK cells and transfer them into a cryopreservation bag; (2) Cool the cryopreservation bag to -90°C or below and freeze it.

8. Use of the NK cell cryopreservation solution according to any one of claims 1-6 in cryopreserving NK cells.

9. The use according to claim 8, characterized in that, The NK cells are derived from NK cell lines, peripheral blood cells, umbilical cord blood, induced pluripotent stem cells, or embryonic stem cells; or, the NK cells are genetically engineered NK cells, preferably, the NK cells are NK cells expressing chimeric antigen receptors.

10. A method for preparing NK cell cryopreservation solution as described in any one of claims 1-6, comprising: (1) Provide each component separately; (2) Mix the components to obtain a mixture; (3) The mixture is sterilized or not sterilized to obtain the NK cell cryopreservation solution.

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