Cell freezing medium as well as preparation method and application thereof

Abstract

The invention provides a cell freezing medium as well as a preparation method and application thereof. The cell cryopreservation liquid provided by the invention comprises an electrolyte solution and a cryopreservation protective agent, wherein the cryopreservation protective agent comprises at least one of trehalose, ethylene glycol, glucose, L-proline, whey protein and human serum albumin; the cell freezing medium does not comprise DMSO and fetal calf serum. The cell cryopreservation liquid provided by the invention is a cell cryopreservation liquid without DMSO and fetal calf serum, can be used for cryopreservation of immune cells, and is beneficial to improving the cryopreservation survival rate and recovery rate of the immune cells and maintaining the multiplication capacity of the immune cells and the killing activity of the immune cells to target cells on the basis of avoiding the influence of DMSO and fetal calf serum on cell cryopreservation.

Description

Technical Field

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

[0002] At present, the focus of the biomedical field is mainly on areas such as stem cells and immune cells. Among them, the research on immune cells has important social benefits and development potential. In recent years, the registration and application of innovative cell-based drugs and clinical research projects have increased significantly. Among them, immune cells involved in the body's immune response or related to the immune response play an extremely important role in the prevention and treatment of various diseases. In order to maximize the efficacy of cell products, for spot cell products, only good cell freezing and preservation technology can maximize the survival rate of cells during long-term freezing and the efficacy of products after recovery, so as to ensure the efficacy of clinical application of cell therapy products. In view of the role of cell cryopreservation technology in promoting the clinical application of cell therapy products, it is urgent to develop a cryopreservation solution suitable for immune cells with clear ingredients and safety guarantees.

[0003] Commonly used cryopreservation fluids are fetal bovine serum and dimethyl sulfoxide (DMSO). However, studies have shown that DMSO may be toxic or have adverse reactions to cells in certain circumstances, and there is a potential risk of carcinogenesis. Fetal bovine serum, as an animal-derived ingredient, is not only complex in composition, but also varies from batch to batch, with great uncertainty, which affects the recovery rate after cryopreservation. Therefore, it is necessary to develop a cryopreservation fluid with clear composition and safety suitable for immune cell cryopreservation to achieve standardized, large-scale cryopreservation effects and the success rate of post-cryopreservation recovery. Summary of the invention

[0004] The invention provides a cell freezing solution and a preparation method and application thereof.

[0005] In a first aspect, the present invention provides a cell freezing solution, comprising an electrolyte solution and a cryoprotectant; wherein the cryoprotectant comprises at least one of trehalose, ethylene glycol, glucose, L-proline, whey protein, and human serum albumin;

[0006] The cell freezing solution does not include DMSO and fetal bovine serum.

[0007] In the cell freezing solution as described above, the electrolyte solution refers to a mineral component that maintains the balance between inside and outside the cell. Specifically, the electrolyte solution includes at least one of sodium chloride, potassium chloride, calcium chloride, sodium bicarbonate, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride.

[0008] Further, the electrolyte solution can be a sodium chloride injection, including 0.85 g / L sodium chloride, 30 mg / L potassium chloride and 33 mg / L calcium chloride. The electrolyte solution can be a sodium bicarbonate injection, including 50 g / L sodium bicarbonate. The electrolyte solution can be a compound electrolyte solution, including sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride.

[0009] Furthermore, the electrolyte solution is a compound electrolyte solution, wherein the concentration of sodium chloride is 5.26 g / L, the concentration of sodium gluconate is 5.02 g / L, the concentration of sodium acetate is 3.68 g / L, the concentration of potassium chloride is 0.37 g / L, and the concentration of magnesium chloride is 0.30 g / L.

[0010] In the cell freezing solution as described above, the solvent used in the electrolyte solution can be water for injection.

[0011] In the cell freezing solution as described above, the cryoprotectant comprises trehalose, ethylene glycol, L-proline and human albumin. The CAS number of trehalose is 6138-23-4, the CAS number of L-proline is 147-85-3, and the CAS number of human albumin is 70024-90-7.

[0012] Furthermore, in the cell freezing solution, the concentration of trehalose is 0.1-1 mol / L, the concentration of ethylene glycol is 0.5-1 mol / L, the concentration of L-proline is 0.5-1 mol / L, and the concentration of human albumin is 10-50 g / L.

[0013] Furthermore, in the freezing solution, the concentration of trehalose is 0.5 mol / L, the concentration of ethylene glycol is 0.5 mol / L, the concentration of L-proline is 0.5 mol / L, and the concentration of human albumin is 10 g / L.

[0014] In a second aspect, the present invention provides a method for preparing any of the above-mentioned cell freezing solutions, comprising: mixing the electrolyte solution and a cryoprotectant to obtain the cell freezing solution.

[0015] In a third aspect, the present invention provides use of any of the above-mentioned cell freezing solutions in the cryopreservation of immune cells.

[0016] In a fourth aspect, the present invention provides a method for freezing immune cells, comprising mixing the above-mentioned cell freezing solution with immune cells to obtain a mixed system, and cryopreserving the mixed system comprising the cell freezing solution and immune cells.

[0017] In the method as described above, the immune cells can be at least one of T cells (T lymphocytes), B cells (Blymphocytes), natural killer cells (Natural Killer cells, NK cells), macrophages (Macrophages), dendritic cells (Dendritic cells), neutrophils (Neutrophils), eosinophils (Eosinophils), basophils (Basophils), and mast cells (Mast cells).

[0018] In the method as described above, the immune cells may also be peripheral blood mononuclear cells (PBMC).

[0019] As described above, the immune cells can be derived from mammals, which can be selected from bovines, equines, felines, canines, lagomorphs, porcines, camelids, rodents and primates, including but not limited to cattle, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, gorillas) and humans, preferably cattle, horses, dogs, goats, sheep, pigs, camels, rats, mice, monkeys and humans.

[0020] In the above method, the freezing process can adopt a programmed cooling method. Specifically, the immune cells to be frozen are mixed with the above cell freezing solution, and the density of the immune cells is adjusted to 1*10 7 -5*10 7 / mL, the immune cells were stored in a 4°C refrigerator for equilibration for 10 min, then stored in a programmed cooling box and placed in a -80°C refrigerator for freezing, and finally transferred to liquid nitrogen for long-term cryopreservation.

[0021] The cell freezing solution provided by the present invention is a cell freezing solution free of DMSO and fetal bovine serum, which can be used for the freezing of immune cells. On the basis of avoiding the influence of DMSO and fetal bovine serum on cell freezing, it helps to improve the freezing survival rate and recovery rate of immune cells, maintain the proliferation ability of immune cells and the killing activity on target cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The statistical results of cell survival rate after PBMCs were cryopreserved and revived using cell cryopreservation solutions containing different concentrations of cell cryopreservation agents;

[0023] Figure 2The statistical results of cell recovery rate after PBMCs were cryopreserved and revived using cell cryopreservation solutions containing different concentrations of cell cryopreservation agents;

[0024] Figure 3 The statistical results of immune cell survival rate after PBMCs were cryopreserved and revived using the cell cryopreservation solutions of the examples and control examples;

[0025] Figure 4 The statistical results of the killing rate of K562 cells by immune cells after PBMCs were cryopreserved and revived using the cell cryopreservation solutions of the examples and control examples.

[0026] Figure 1-2 In the table, different lowercase letters indicate that the data of the three groups in the same group are significantly different (P < 0.05), and the same lowercase letters indicate that the data of the three groups in the same group are not significantly different (P > 0.05). Figure 3-4 In the table, different lowercase letters indicate significant differences between the two groups (P < 0.05), and the same lowercase letters indicate no significant differences between the two groups (P > 0.05). DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0028] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0029] The compound electrolyte injection involved in the following examples (containing 5.26g sodium chloride, 5.02g sodium gluconate, 3.68g sodium acetate, 0.37g potassium chloride, and 0.30g magnesium chloride per 1000ml) was purchased from Shanxi Zhendong Taisheng Pharmaceutical, with the national medicine standard H20113035. Trehalose was purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd., with the CAS number of 6138-23-4. Ethylene glycol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with the product number E103321. L-proline was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., with the CAS number of 147-85-3. Human albumin was purchased from Hunan Yunbang Biotechnology Co., Ltd., with the CAS number of 70024-90-7.

[0030] Example 1 Determination of Cell Cryopreservation Protectant Concentration

[0031] 1. Preparation of cell freezing solutions containing different concentrations of cryoprotectants: trehalose, ethylene glycol, L-proline and human albumin were added to the compound electrolyte injection. According to the different concentrations, the cell freezing solutions were set up in 4 groups. Among them, the first group set up three cell freezing solutions containing different concentrations of trehalose for single factor comparison, namely group 1-1, group 1-2 and group 1-3. The cell freezing solution of group 1-1 included 0.1 mol / L trehalose, 0.5 mol / L ethylene glycol, 0.5 mol / L L-proline and 10 g / L human albumin; the cell freezing solution of group 1-2 included 0.5 mol / L trehalose, 0.5 mol / L ethylene glycol, 0.5 mol / L L-proline and 10 g / L human albumin; the cell freezing solution of group 1-3 included 1 mol / L trehalose, 0. 5mol / L ethylene glycol, 0.5mol / L L-proline, 10g / L human albumin; Group 2 set up three cell freezing solutions containing different concentrations of ethylene glycol for single factor comparison, namely Group 2-1, Group 2-2 and Group 2-3. The cell freezing solution of Group 2-1 included 0.5mol / L trehalose, 0.5mol / L ethylene glycol, 0.5mol / L L-proline, and 10g / L human albumin; the cell freezing solution of Group 2-2 included 0.5mol / L trehalose, 0.75mol / L ethylene glycol, 0.5mol / L L-proline, and 10g / L human albumin; the cell freezing solution of Group 2-3 included The first group had three cell freezing solutions containing different concentrations of L-proline for single factor comparison, namely group 3-1, group 3-2 and group 3-3. The cell freezing solution of group 3-1 included 0.5 mol / L trehalose, 0.5 mol / L ethylene glycol, 0.5 mol / L L-proline and 10 g / L human albumin; the cell freezing solution of group 3-2 included 0.5 mol / L trehalose, 0.5 mol / L ethylene glycol, 0.75 mol / L L-proline and 10 g / L human albumin. L of human albumin; the cell cryopreservation solution of group 3-3 included 0.5mol / L trehalose, 0.5mol / L ethylene glycol, 1mol / L L-proline, and 10g / L human albumin; three cell cryopreservation solutions containing different concentrations of human albumin were set up in group 4 for single factor comparison, namely group 4-1, group 4-2 and group 4-3. The cell cryopreservation solution of group 4-1 included 0.5mol / L trehalose, 0.5mol / L ethylene glycol, 0.5mol / L L-proline, and 10g / L human albumin; the cell cryopreservation solution of group 4-2 included 0.5mol / L trehalose, 0.5mol / L ethylene glycol, 0.5mol / L L-proline, 25g / L human albumin; the cell freezing solution of group 4-3 includes 0.5mol / L trehalose, 0.5mol / L ethylene glycol, 0.5mol / L L-proline, and 50g / L human albumin; the cell freezing solutions of each group are evenly mixed and stored at 2-8℃.

[0032] 2. Add the above-mentioned different cell freezing solutions to human peripheral blood mononuclear cells (PBMC) and adjust the cell density to 1*10 7 -5*10 7 / mL, mix thoroughly and transfer into a cryopreservation tube; put the cryopreservation tube into a 4℃ refrigerator for equilibration for 10 min, after equilibration, place the cryopreservation tube in a programmed cooling box and put it into a -80℃ refrigerator for 12-15 h, and then transfer it into a liquid nitrogen tank for storage for 30 days.

[0033] The cryovials were taken out from the liquid nitrogen tank and quickly placed in a 37°C constant temperature water bath for 1.5-2.5 min. The cells were shaken until the cell suspension was completely melted. The revived PBMCs were then eluted three times with 9-fold volume of PBS. The survival rate and recovery rate of the revived cells were statistically analyzed within the group.

[0034] Statistical method for cell survival rate: The revived PBMCs were stained with dual fluorescence AO / PI method, and the stained PBMCs were added to the cell counting plate. The survival rate was calculated as the number of surviving cells / total number of cells*100%, and the recovery rate was calculated as the number of immune cells / number of surviving cells*100%.

[0035] The experimental results are as follows Figure 1-2 As shown, the survival rate and recovery rate of Group 1-2, Group 2-1, Group 3-1 and Group 4-1 were significantly higher than those of other experimental groups. Therefore, the final concentration of each component in the freezing solution was preferably determined to be: 0.5 mol / L trehalose; 0.5 mol / L ethylene glycol; 0.5 mol / L L-proline; 10 g / L human albumin.

[0036] Example 2

[0037] According to the above optimal concentrations, 0.5 mol / L of trehalose, 0.5 mol / L of ethylene glycol, 0.5 mol / L of L-proline and 10 g / L of human albumin were added to the compound electrolyte injection to obtain the cell freezing solution of the embodiment.

[0038] 10% DMSO (purchased from Beijing Solebow Technology Co., Ltd., catalog number: D8371) and 20% fetal bovine serum FBS (purchased from Beyotime Biotechnology, catalog number: C0235) were added to RPMI-1640 cell culture medium (purchased from Thermo Fisher Scientific, catalog number: 11875119) to obtain a control cell freezing solution.

[0039] The same cell freezing method as in Example 1 was used to cryopreserve PBMCs using the experimental example cell freezing solution and the control example cell freezing solution. The PBMCs were taken out after being frozen in the liquid nitrogen tank for 30 days, 60 days, and 90 days and resuscitated. The survival rate and cell killing rate of the immune cells after resuscitation were calculated.

[0040] The statistical method of cell survival rate is the same as that of Example 1. The statistical method of cell killing rate includes: mixing the resuscitated PBMC with the K562 cells in the logarithmic phase at an effector-target ratio of 20:1, setting separate target cell and effector cell wells, and using the LDH release method to measure the absorbance (A) value at 490nm, and calculating the killing rate (%) of each group of PBMC on K562 cells.

[0041] The experimental results are as follows Figure 3-4 As shown, after PBMCs were cryopreserved for a certain number of days using the cell cryopreservation solution of the example, the survival rate and cell activity of PBMCs were significantly higher than those of the cell cryopreservation solution of the control example, and the cryopreservation effect was significantly better than that of the cryopreservation solution of the control example.

[0042] 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 make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell freezing solution, characterized in that: It comprises an electrolyte solution and a cryoprotectant; wherein the cryoprotectant comprises at least one of trehalose, ethylene glycol, glucose, L-proline, whey protein, and human serum albumin; and the cell freezing solution does not comprise DMSO and fetal bovine serum.

2. The cell freezing solution according to claim 1, characterized in that The electrolyte solution includes at least one of sodium chloride, potassium chloride, calcium chloride, sodium bicarbonate, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride.

3. The cell freezing solution according to claim 1 or 2, characterized in that: The electrolyte solution includes sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride.

4. The cell freezing solution according to claim 3, characterized in that The electrolyte solution has a sodium chloride concentration of 5.26 g / L, a sodium gluconate concentration of 5.02 g / L, a sodium acetate concentration of 3.68 g / L, a potassium chloride concentration of 0.37 g / L, and a magnesium chloride concentration of 0.30 g / L.

5. The cell freezing solution according to any one of claims 1 to 4, characterized in that: The cryoprotectant comprises trehalose, ethylene glycol, L-proline and human albumin.

6. The cell freezing solution according to claim 5, characterized in that: In the cell freezing solution, the concentration of trehalose is 0.1-1 mol / L, the concentration of ethylene glycol is 0.5-1 mol / L, the concentration of L-proline is 0.5-1 mol / L, and the concentration of human serum albumin is 10-50 g / L.

7. The cell freezing solution according to claim 5, characterized in that In the cell freezing solution, the concentration of trehalose is 0.5 mol / L, the concentration of ethylene glycol is 0.5 mol / L, the concentration of L-proline is 0.5 mol / L, and the concentration of human serum albumin is 10 g / L.

8. The method for preparing a cell freezing solution according to any one of claims 1 to 7, characterized in that: include: The electrolyte solution and the cryoprotectant are mixed to obtain the cell freezing solution.

9. Use of the cell freezing solution according to any one of claims 1 to 7 in the freezing of immune cells.

10. A method for freezing immune cells, characterized in that: include: The cell freezing solution according to any one of claims 1 to 7 is mixed with immune cells to obtain a mixed system, and the mixed system including the cell freezing solution and immune cells is cryopreserved.

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