Cryopreservation method of NK cell cryopreservation liquid and application of cryopreservation method

Abstract

The invention discloses a cryopreservation method and application of an NK cell cryopreservation solution, and belongs to the technical field of cell cryopreservation. The NK cell cryopreservation solution comprises DMSO, a PBS buffer solution and a hydroxyethyl starch solution; the volume fraction of the DMSO in the NK cell freezing medium is 2.8 to 3.2 percent; the cryopreservation method of the NK cell cryopreservation liquid comprises the following steps: culturing activated NK cells by using a complete culture medium added with IL-15 and IL-18, centrifuging, cleaning, preparing the NK cell cryopreservation liquid, re-suspending to a preset cell concentration by using the NK cell cryopreservation liquid, cooling, freezing, and preserving in liquid nitrogen. The NK cell cryopreservation liquid used in the invention is a universal NK cell cryopreservation liquid, and the cryopreservation method provided by the invention has a good cryopreservation effect and can be used instantly.

Description

Technical Field

[0001] The present invention relates to the technical field of cell cryopreservation, and in particular to a cryopreservation method of a NK cell cryopreservation solution and an application thereof. Background Art

[0002] NK cells (natural killer cells) are a key innate immune cell in the human immune system. They possess powerful immune surveillance and cytotoxic functions, capable of recognizing and killing abnormal cells such as tumor cells and virus-infected cells. They do not require prior sensitization and can rapidly respond to pathogen invasion. Currently, NK cell applications primarily include autologous NK cell and allogeneic NK cell therapies. Numerous clinical trials have demonstrated that autologous NK cell therapy has demonstrated efficacy in cancer treatment without significant side effects. However, with aging, the number and activity of NK cells in the body decrease, making them particularly inadequate for treatment in the elderly or immunocompromised patients. Furthermore, the preparation of autologous NK cells requires isolation and expansion, which is time-consuming and may not be optimal for patients requiring emergency treatment. Allogeneic NK cells are primarily used in clinical practice as fresh preparations. While fresh preparations offer higher cell activity, their viable nature results in a short shelf life, making them inaccessible for immediate use, and they are subject to delays in product testing. Therefore, the development of a cryopreservative for allogeneic NK cells that allows for better cryopreservation and immediate use is urgent.

[0003] However, the basic components of existing NK cell freezing solutions include permeability protectants (common permeability protectants include DMSO, glycerol, etc.), non-permeability protectants (common non-permeability protectants include dextran 40, etc.), and basal culture media (common basal culture media include DMEM, RPMI-1640, etc.). There are two problems in their use: The first problem is that the effective ingredients of existing NK cell freezing solutions mostly contain scientific research-grade cell culture medium and fetal bovine serum and other ingredients, which cannot be directly used for human infusion. For example, Chinese patent CN111248192A discloses an NK cell freezing solution, whose ingredients include dextran, CPLL and basal culture medium. Among them, because it contains basal culture medium, it cannot be used for clinical and direct infusion. Chinese patent CN104862275A discloses a method for cell freezing and recovery and the cell preparation prepared therefrom. In this patent, dimethyl sulfoxide, 1640 cell basal culture medium, fetal bovine serum, dextran, and ddH2O are used as components of the freezing solution. However, because the freezing solution of this patent contains animal-derived serum, there is a potential risk of introducing animal-derived antigenic substances and it cannot be used for direct reinfusion into the human body.

[0004] The second issue is low cryopreservation efficiency. For example, Chinese patent CN114258910B discloses an infusible cell cryopreservation solution and its application. Although the patent aims to improve the cryopreservation efficiency of CAR-NK cells, the survival rate of CAR-NK cells after 30 days of cryopreservation is 82.75%, and the killing efficiency against K562 cells is only 45.90%. This relatively low cryopreservation efficiency limits its clinical use. Summary of the Invention

[0005] In response to the deficiencies in the prior art, the present invention discloses a freezing method for NK cell freezing fluid and its application. The NK cell freezing fluid used in the present invention is a universal NK cell freezing fluid. The freezing method of the present invention has good freezing effect and can be used immediately.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows: A method for freezing NK cell cryopreservation solution, comprising culturing activated NK cells using complete culture medium supplemented with IL-15 and IL-18, centrifuging, washing, resuspending the cells in NK cell cryopreservation solution to a predetermined cell concentration, cooling and freezing, and storing the cells in liquid nitrogen; The NK cell freezing solution includes DMSO, PBS buffer, hydroxyethyl starch solution, human serum albumin, dextran 40, glucose, trehalose, and vitamin C; Preferably, the volume fraction of DMSO in the NK cell freezing solution is 2.8-3.2%; Preferably, the PBS buffer is 1×PBS buffer with a pH of 7.2-7.6; Preferably, the volume fraction of PBS buffer in the NK cell freezing solution is 26.8-27.2%; Preferably, the volume fraction of hydroxyethyl starch solution in NK cell freezing solution is 69.7-70.3%; Furthermore, the preparation method of the hydroxyethyl starch solution is as follows: dissolving hydroxyethyl starch in sodium chloride injection; In the preparation of the hydroxyethyl starch solution, the hydroxyethyl starch is hydroxyethyl starch 200 / 0.5; The sodium chloride injection is 0.9% sodium chloride injection; The dosage ratio of hydroxyethyl starch to sodium chloride injection is 29-31 g:500 mL; Preferably, the concentration of human serum albumin in the NK cell freezing solution is 38-42 mg / mL; Preferably, the concentration of dextran 40 in the NK cell freezing solution is 24-26 mg / mL; Preferably, the concentration of glucose in the NK cell freezing solution is 12-13 mg / mL; Preferably, the concentration of trehalose in the NK cell freezing solution is 48-52 mM; Preferably, the concentration of vitamin C in the NK cell freezing solution is 3.8-4.2 mg / mL.

[0007] Preferably, the concentration of IL-15 in the complete culture medium supplemented with IL-15 and IL-18 is 9.5-10.5 ng / mL, and the concentration of IL-18 is 48-52 ng / mL; Preferably, when the activated NK cells are cultured using a complete medium supplemented with IL-15 and IL-18, the culture time is 23-25 ​​hours; Preferably, the centrifugal force of the centrifugation is 1200-1300g, and the centrifugation time is 5-6min; Preferably, the cleaning is performed using 0.9% sodium chloride injection for two to five times; Preferably, the cooling rate during cooling and freezing is -1.1°C / min to -0.9°C / min; Preferably, the preset cell concentration is 0.8×10 7 cells / mL-1.2×10 7 cells / mL.

[0008] An application of the aforementioned cryopreservation method in cryopreserving NK cells.

[0009] Compared with the prior art, the present invention has the following beneficial effects: (1) The NK cell freezing solution used in the present invention is a universal NK cell freezing solution with good freezing effect, immediate use, high cell activity after recovery, high target cell killing rate, and good safety.

[0010] (2) The NK cell cryopreservation solution used in the present invention is a NK cell cryopreservation solution specifically for NK cells that can be directly injected intravenously. The volume content of dimethyl sulfoxide (DMSO) in the cryopreservation solution is only 3%, which greatly reduces the toxicity to both the human body and the cryopreserved cells, making it safer. In addition, the NK cell cryopreservation solution of the present invention combines a permeable cryoprotectant with a non-permeable cryoprotectant, and pre-treats the frozen cells with IL-15 and IL-18, which can protect the cells both internally and externally, reduce the mechanical damage of ice crystals during the cryopreservation process, and protect the cells from swelling and rupture damage caused by the rapid entry of water into the cells during cell recovery.

[0011] (3) The NK cell cryopreservation solution used in the present invention can better maintain the activity and killing ability of frozen NK cells. After 12 months of cryopreservation and thawing, the cell viability of NK cells can reach 90.3%. When the effector-target ratio is 5:1, the killing efficiency of NK cells against target cells K562 is 86.39%. The prepared cryopreservation solution was injected into the tail vein of C57BL6 mice. 45 days after injection, the weight gain of C57BL6 mice was 27.9 g. There was no significant difference in weight gain compared with the control C57BL6 mice. The C57BL6 mice were dissected and the main tissues were observed for pathological examination, and no tissue lesions were found. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Graph showing the cell viability of NK cells after recovery from cryopreservation in each group of cryopreserved solutions in Example 3; Figure 2 The graph shows the killing rate of K562 cells after NK cells frozen in each group of cryopreservation solutions were revived in Example 4; Figure 3 Figure 2 is the weight gain change diagram of the two groups of mice; Figure 4 HE staining images of brain tissues of two groups of mice; Figure 5 HE staining images of heart tissues of two groups of mice; Figure 6 HE staining images of liver tissues of two groups of mice; Figure 7 HE staining images of kidney tissues of two groups of mice. DETAILED DESCRIPTION

[0013] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described.

[0014] Example 1 Preparation of NK cell bank 1) Plasma extraction Take 50 mL of peripheral blood and divide it evenly into two 50 mL centrifuge tubes. Centrifuge at 650 g for 15 min at room temperature. Take the upper light yellow plasma and transfer it to a new 50 mL centrifuge tube (the lower red liquid is used to extract mononuclear cells). Inactivate it in a 56°C water bath for 30 min. Centrifuge it at 900 g for 10 min. Take the supernatant, let it stand at -20°C for 15 min, centrifuge it again at 900 g for 10 min, take the supernatant, and store it at 4°C to obtain inactivated plasma.

[0015] 2) Isolation of monocytes A. Take the lower red liquid obtained from the plasma extraction in step 1) and dilute it with an equal volume of physiological saline to a total volume of 20 mL. Mix well to obtain diluted blood for later use.

[0016] B. Take two new 50 mL centrifuge tubes and add 20 mL of lymphocyte separation solution (purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.) to each tube.

[0017] C. Slowly add 20 mL of diluted blood to a centrifuge tube containing 20 mL of lymphocyte separation medium, allowing the blood and lymphocyte separation medium to form a distinct layer. Do not mix the diluted blood with the lymphocyte separation medium during addition. Centrifuge at 650g for 30 minutes at room temperature.

[0018] D. Gently aspirate the mononuclear cells (buffy coat) and half of the lymphocyte separation solution below it and transfer them to a new 50 mL centrifuge tube. Add an equal volume of normal saline and centrifuge at 650 g for 10 minutes at room temperature. Discard the supernatant and repeat this washing step once. Resuspend the cells in normal saline and take a small amount of the cell suspension for trypan blue staining and counting. After counting, centrifuge at 260 g for 10 minutes, collect the cells, and adjust the cell density to 2 × 10 cells / mL using NK serum-free medium containing a final concentration of 1000 IU / mL TNF-α. 6 / mL, the volume was 22mL, and a mononuclear cell suspension was obtained.

[0019] 3) Inoculation and activation of monocytes 1. Day 0 6-well plate coating: Add 2 mL of DPBS (Beijing Solebow Technology Co., Ltd., D1040) and 100 μL of YCOOA to a 6-well plate, mix thoroughly, incubate at 37°C for 2 h, discard the supernatant, and wash once with 2 mL of DPBS. Be careful not to flush the bottom of the well plate. Discard the washing solution and set aside.

[0020] Monocyte inoculation: Add activation medium, 100uL induction factor YC00B, 10% autologous plasma (0.4mL) and monocyte suspension to a 6-well plate to ensure that the number of monocytes is 8×10 6 The total volume is 4 mL, that is, the seeding density of peripheral blood mononuclear cells is 2×10 6 cells / mL.

[0021] 2. Day 3 Add 8 mL of activation medium and 5% autologous plasma (0.4 mL) to the 6-well plate. Be gentle and do not shake the adherent cells.

[0022] 3. Day 5 28 mL of activation medium and 5% autologous plasma (1.4 mL) were added, and the medium and cells in the 6-well plate were transferred to a T175 cell culture flask.

[0023] 4. Day 7 The cell density was greater than 1×10 6 cells / mL, supplemented with 80 mL of expansion medium and 1% plasma (0.8 mL).

[0024] 5. Day 9 Add 180 mL of expansion medium and transfer the medium and cells to a FEP-1L cell culture bag.

[0025] 6. Day 11 Add 300 mL of expansion medium.

[0026] 7. Day 13 Add 440 mL of expansion medium.

[0027] 8. Day 16 The density was determined and the cells were harvested.

[0028] Activation medium: Take the YCO0C factor melted at room temperature and add it to the immune cell repair medium 2.0 at a ratio of 1:1000 and mix well for later use.

[0029] Expansion medium: Dissolve each vial of YC005 in 1 mL of NK cell serum-free medium, add to NK cell serum-free medium at a ratio of 1:1000, mix well and set aside.

[0030] The culture medium used in this example was purchased from Beijing Youkang Biotechnology Co., Ltd., and the name and product number are shown in Table 1 below: Table 1 Product number of each culture medium

[0031] Example 2 Preparation of different cryopreservation solutions and treatment of NK cells Cryopreservation medium 1: 10% (V / V) Dextran 40 (purchased from Wuhan Yuanxing Biopharmaceutical Technology Co., Ltd., 9004-54-0), 10% (V / V) DMSO (purchased from Merck, 67-68-5), 80% (V / V) NK cell serum-free medium (Beijing Youkang Biotechnology Co., Ltd., NC0102).

[0032] Cryopreservation solution 2: Human albumin (human serum albumin, purchased from JetBelin, P100225458) 40 mg / mL, Dextran 4025 mg / mL, glucose (purchased from Macklin, 50-99-7) 12.5 mg / mL, 3% (v / v) DMSO (permeable cryoprotectant) + trehalose (non-permeable cryoprotectant, Beijing Solebold Technology Co., Ltd., 99-20-7) 50 mM, 27% (v / v) 1× PBS buffer (pH 7.2-7.6, Merck, P5493), vitamin C (Yisheng Biological, 60374ES) 4 mg / mL, 70% (v / v) hydroxyethyl starch solution; The hydroxyethyl starch solution was prepared by dissolving 30 g of hydroxyethyl starch 200 / 0.5 (Beijing Solebaugh Technology Co., Ltd., H7620) in 500 mL of 0.9% sodium chloride injection (Qidu Pharmaceutical Co., Ltd.).

[0033] Experimental group A: 1 mL of activated NK cells prepared in Example 1 was centrifuged at 1200 g for 5 min, the supernatant was discarded, and the cells were washed twice with 0.9% sodium chloride injection and resuspended in freezing solution 1 to a cell concentration of 1×10 7 cells / mL, and then programmed freezing at a cooling rate of -1℃ / min and stored in liquid nitrogen, and repeated freezing for 30 times.

[0034] Experimental group B: 1 mL of activated NK cells prepared in Example 1 was taken and centrifuged at 1200 g for 5 min. The supernatant was discarded, washed twice with 0.9% sodium chloride injection, and then resuspended in freezing solution to a cell concentration of 1×10 7 cells / mL, programmed freezing at a cooling rate of -1°C / min and stored in liquid nitrogen, repeated freezing for 30 times.

[0035] Experimental group C: 1 mL of activated NK cells prepared in Example 1 was taken. 24 hours before freezing, 10 ng / mL IL-15 and 50 ng / mL IL-18 were added to the complete culture medium. The NK cells were cultured. After 24 hours, the cells were centrifuged at 1200 g for 5 minutes, the supernatant was discarded, and the cells were washed twice with 0.9% sodium chloride injection and resuspended in freezing medium to a cell concentration of 1×10 7 cells / mL, programmed freezing at a cooling rate of -1°C / min and stored in liquid nitrogen, repeated freezing for 30 times.

[0036] Control group: NK cell cryopreservation solution purchased from the market (purchased from Saicun Biotechnology Co., Ltd., CS-NK-D1) Take 1 mL of activated NK cells prepared in Example 1, centrifuge at 1200 g for 5 min, discard the supernatant, wash twice with 0.9% sodium chloride injection, and resuspend in the control group NK cell freezing solution to a cell concentration of 1×10 7 cells / mL, programmed freezing at a cooling rate of -1°C / min and stored in liquid nitrogen, repeated freezing for 30 times.

[0037] Example 3 Effects of different freezing solutions on cell viability The frozen NK cells of Experimental Group A, Experimental Group B, Experimental Group C, and Control Group in Example 2 were removed from the liquid nitrogen tank after 1 week, 1, 3, 6, and 12 months, thawed in a 37°C water bath, and a small amount of cells was taken to detect cell viability using trypan blue staining.

[0038] The cell viability of each group after cell recovery is shown in Table 2: Table 2 Cell viability of NK cells after thawing in each group of cryopreservation solutions (%)

[0039] The results are shown in Table 2 and Figure 1 As shown, it can be seen that the NK cells revived after being preserved in the NK cell cryopreservative solution of experimental group C had higher cell survival rates at 1 week, 1 month, 3 months, 6 months, and 12 months than those of other groups, indicating that the NK cells obtained after resuscitation in the cryopreservative solution dedicated to NK cells in experimental group C had higher activity and had a better protective effect on the cells.

[0040] Example 4 Killing efficiency of NK cells in different cryopreservation groups against target cells K562 1. Target cell preparation: Take enough target cells K562, centrifuge at 500g for 10min, discard the supernatant, resuspend in 1640 basal medium and count the viability. Adjust the density of target cells to 1×10 6 Cells / mL, add 1mM Calcein-AM solution (7.5μL / 1×10 6 cells / mL) and stained for 30 min. After staining, wash twice with PBS and resuspend in 1640 basal medium (Sigma, R8758) to count the viability. The target cell density was adjusted to 2×10 5 Cells / mL, set aside.

[0041] 2. Effector cell preparation: Sufficient amounts of NK cells from Experimental Groups A, B, C, and the control group (from Example 3) frozen for 1 week, 1 month, 3 months, 6 months, and 12 months were collected, thawed, centrifuged at 500 g for 10 minutes, and the supernatant discarded. The cells were resuspended in 1640 basal medium and viable cells were counted. Effector cells were diluted to the appropriate concentration at an effector-target ratio of 5:1.

[0042] 3. Killing activity determination: The experimental groups and control groups are as follows: Each experimental group: target cells + resuscitated NK cells in each group (experimental groups AC and control group); Control group 1: To maximize the release of LDH from target cells, a certain volume of cell lysis buffer (Biyuntian Biotechnology Co., Ltd., P0013) was added; Control group 2: target cells spontaneously released LDH; Control group 3: effector cells spontaneously released LDH; Control group 4: blank culture medium background; Control group 5: volume calibration background, blank culture medium was added with a certain volume of cell lysate.

[0043] Assay Method: The CytoTox96® Non-Radioactive Cytotoxicity Assay Kit (Promega) is used to assess the cytotoxicity of effector cells against target cells. This method is based on a colorimetric assay and is an alternative to the 51Cr release assay. The CytoTox assay quantitatively measures lactate dehydrogenase (LDH). LDH is a stable cytosolic enzyme that is released upon cell lysis in a manner similar to that of 51Cr in radioactive assays. Released LDH is detected in the culture supernatant via a 30-minute coupled enzymatic reaction in which LDH converts a tetrazolium salt (INT) into a red formazan. The amount of red product generated is proportional to the number of cells lysed. For details, refer to the CytoTox96® Non-Radioactive Cytotoxicity Assay Kit instructions.

[0044] The cytotoxicity calculation formula is:

[0045] Table 3 Killing rate of NK cells against K562 cells after thawing in each group of cryopreservation solutions

[0046] The results plotted based on the above results are shown in Figure 2 The results are as follows Figure 2As shown in Table 3, compared with traditional cell freezing solution, the NK cells obtained after formula optimization and pre-treatment with IL-15 and IL-18 have significant advantages in killing activity. After 12 months of cryopreservation and thawing, the killing rate of NK cells against K562 can still reach 82.39%.

[0047] Example 5 Safety Verification of Cryopreservation Solution C57BL6 mice aged 6-8 weeks (purchased from Nanjing Junke Biotechnology Co., Ltd.) were divided into two groups, 10 mice in each group, to verify the safety of the cryopreservation solution. The experimental groups were: a. Control group, 200 μL of PBS was injected into the tail vein; b. Experimental group, 200 μL of cryopreservation solution II was injected into the tail vein; After injection, the mice's behavior and condition were observed daily and weighed weekly. After 45 days, the mice were dissected and pathological examinations were performed on major tissues, including the brain, heart, lungs, liver, colon, and kidneys.

[0048] During the experiment, no abnormal behavior of mice was observed. Figure 3 As shown in Figure 2, there was no significant difference in the weight gain of mice. Figure 4-Figure 7 As shown, mice in the experimental group did not develop lesions.

Claims

1. A method for freezing NK cell cryopreservation solution, characterized in that: The activated NK cells were cultured in complete medium supplemented with IL-15 and IL-18, centrifuged, washed, resuspended in NK cell freezing solution to a preset cell concentration, cooled and frozen, and stored in liquid nitrogen; The NK cell freezing solution includes DMSO, PBS buffer, hydroxyethyl starch solution, human serum albumin, dextran 40, glucose, trehalose, and vitamin C; The volume fraction of the DMSO in the NK cell freezing solution is 2.8-3.2%.

2. The freezing method of NK cell freezing solution according to claim 1, characterized in that: The PBS buffer is 1×PBS buffer with a pH of 7.2-7.6; The volume fraction of PBS buffer in NK cell cryopreservation solution is 26.8-27.2%; The volume fraction of hydroxyethyl starch solution in NK cell freezing medium is 69.7-70.3%.

3. The freezing method of the NK cell freezing solution according to claim 1, characterized in that: The preparation method of the hydroxyethyl starch solution is as follows: dissolving hydroxyethyl starch in sodium chloride injection; In the preparation of the hydroxyethyl starch solution, the hydroxyethyl starch is hydroxyethyl starch 200 / 0.5; The sodium chloride injection is 0.9% sodium chloride injection; The dosage ratio of hydroxyethyl starch to sodium chloride injection is 29-31g:500mL.

4. The freezing method of NK cell freezing solution according to claim 1, characterized in that: The concentration of human serum albumin in NK cell cryopreservation medium is 38-42 mg / mL; The concentration of dextran 40 in NK cell cryopreservation medium is 24-26 mg / mL; The concentration of glucose in NK cell cryopreservation medium is 12-13 mg / mL; The concentration of trehalose in NK cell cryopreservation solution is 48-52 mM; The concentration of vitamin C in NK cell freezing medium is 3.8-4.2 mg / mL.

5. The freezing method of NK cell freezing solution according to claim 1, characterized in that: In the complete culture medium supplemented with IL-15 and IL-18, the concentration of IL-15 is 9.5-10.5 ng / mL, and the concentration of IL-18 is 48-52 ng / mL.

6. The freezing method of NK cell freezing solution according to claim 1, characterized in that: When activated NK cells are cultured in complete medium supplemented with IL-15 and IL-18, the culture time is 23-25 ​​hours.

7. The freezing method of NK cell freezing solution according to claim 1, characterized in that: The centrifugal force of the centrifugation is 1200-1300g, and the centrifugation time is 5-6min; The cleaning is performed by using 0.9% sodium chloride injection for two to five times.

8. The method for freezing NK cell cryopreservation solution according to claim 1, characterized in that: The cooling rate during cooling and freezing is -1.1°C / min to -0.9°C / min.

9. The method for freezing NK cell cryopreservation solution according to claim 1, characterized in that: The preset cell concentration is 0.8×10 7 cells / mL-1.2×10 7 cells / mL.

10. Use of the cryopreservation method according to any one of claims 1 to 9 in cryopreserving NK cells.

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