Cell cryopreservation protection solution and use thereof

Abstract

Disclosed are a cell cryopreservation protection solution and a use thereof. The cell cryopreservation protection solution comprises the following components: polyethylene glycol, hydroxyethyl starch, human serum albumin, and trehalose. The molecular weight of polyethylene glycol is 200-600. In the cell cryopreservation protection solution, a safe and stable cryopreservation environment is provided for cells by means of the combined use of polyethylene glycol, hydroxyethyl starch, human serum albumin, and trehalose having proper molecular weights, and the damage of a cryoprotectant to the cells is reduced, so that the cells subjected to cryopreservation and resuscitation have high cell viability and stability. The cell cryopreservation protection solution is especially suitable for cryopreservation of mesenchymal stem cells. In addition, the cell cryopreservation protection solution provided by the present invention does not contain DMSO, glycerol, animal serum and other reagents that are limited in clinical application, and can be directly injected into patients without requiring the process of centrifugation and washing to obtain cells, thereby providing feasibility for the clinical application of mesenchymal stem cells.

Description

A cell cryopreservation solution and its application Technical Field

[0001] This invention relates to the field of cell cryopreservation solution technology, specifically to a cell cryopreservation protection solution and its application. Background Technology

[0002] Mesenchymal stem cells are a type of poorly differentiated cell with the potential for multi-lineage differentiation and self-replication. Once dormant MSCs are activated, they will regain active proliferation and differentiation capabilities. Freshly isolated mesenchymal stem cells are limited in number and cannot generate enough cells in a short period of time. Slow freezing technology and liquid nitrogen storage methods can be used to store a sufficient amount of mesenchymal stem cells for long-term use [Zhang R, Duan X, Liu Y, et al. The Application of Mesenchymal Stem Cells in Future Vaccine Synthesis[J]. Vaccines(Basel),2023,11(11)].

[0003] Commonly used cryoprotectants include dimethyl sulfoxide (DMSO) and glycerol. Their protective mechanism involves binding water molecules in the solution, causing hydration, increasing the solution viscosity, and inhibiting the formation of ice crystals. Currently, mesenchymal stem cell cryoprotectants typically contain 5%-10% DMSO, glycerol, and animal serum. However, DMSO can cause changes in cellular and genetic characteristics after freezing / thawing. Glycerol can lead to increased plasma osmotic pressure, resulting in extravascular dehydration, circulatory overload, pulmonary edema, and heart failure [Oh SJ, Jo CH, Kim TS, et al. Sphingosine-1-phosphate Treatment Improves Cryopreservation Efficiency in Human Mesenchymal Stem Cells[J]. Life (Basel), 2023, 13(6)]. Animal serum carries the potential risk of carrying foreign antigens and pathogens, thus limiting the clinical application of cell cryopreservation solutions containing DMSO, glycerol, and animal serum.

[0004] Therefore, there is an urgent need to develop a cell cryopreservation solution that does not contain DMSO, glycerol, or animal serum to overcome the above-mentioned shortcomings and provide feasibility for the clinical application of mesenchymal stem cells. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a cell cryopreservation solution and its applications. This cell cryopreservation solution is free of DMSO, glycerol, and animal serum. Instead, it provides a safe and stable cryopreservation environment for cells through the synergistic effect of polyethylene glycol, hydroxyethyl starch, human serum albumin, and trehalose of appropriate molecular weights. This reduces damage to cells from cryoprotectants, resulting in high cell viability and good stability after cryopreservation and thawing, making it particularly suitable for the cryopreservation of mesenchymal stem cells. The components of this cell cryopreservation solution have high clinical safety and can be used as a cryopreservation solution for the long-term storage of clinical-grade mesenchymal stem cells. Furthermore, after thawing, the cell cryopreservation solution containing cells can be directly used for intravenous reinfusion.

[0006] This invention provides the following technical solutions:

[0007] The first aspect of the present invention provides a cell cryopreservation solution comprising the following components: polyethylene glycol, hydroxyethyl starch, human serum albumin and trehalose; wherein the molecular weight of the polyethylene glycol is 200-600.

[0008] This invention utilizes low molecular weight polyethylene glycol and hydroxyethyl starch in synergy to effectively increase the osmotic pressure of the extracellular solution, promoting water expulsion from the cell and reducing intracellular water content. This reduces the formation of intracellular ice crystals and thus avoids freeze-drying damage to cells. The preferred polyethylene glycol is polyethylene glycol 400, a pharmaceutical excipient for injection as specified in the Pharmacopoeia of the People's Republic of China (2020 Edition). Simultaneously, the introduction of human serum albumin maintains plasma colloid osmotic pressure and utilizes the antioxidants in human serum albumin to reduce oxygen free radicals generated by cellular oxidation, thereby reducing cell damage caused by oxygen free radicals and further improving cell survival rate after cryopreservation. Furthermore, the presence of human serum albumin facilitates cell dispersion and improves the survival rate of frozen cells. Furthermore, this invention introduces trehalose into the cell cryopreservation solution. During cell freeze-thaw cycles, trehalose can bind to the cell membrane and stabilize its structure, preventing membrane lipid dissolution and protein denaturation caused by freezing. It can also inhibit the rise in intracellular calcium ions caused by freezing, reducing calcium ion damage to cells and helping to maintain cell integrity and function. In addition, trehalose has good osmotic adaptability, regulating the osmotic balance between the inside and outside of cells during cryopreservation. The synergistic effect of these components can effectively improve the cell survival rate of mesenchymal stem cells after cryopreservation and thawing.

[0009] Furthermore, in every 100 mL of cell cryopreservation solution: the content of polyethylene glycol is preferably 0.5-2 g, the content of hydroxyethyl starch is preferably 6-12 g, the content of human serum albumin is preferably 0.4-1.2 g, and the content of trehalose is preferably 0.1-2 g.

[0010] Furthermore, the content of polyethylene glycol in each 100 mL of cell cryopreservation solution may be 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1.0 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2.0 g, etc., including but not limited to the contents listed above, and more preferably 0.8-1.2 g, for example 1 g.

[0011] Furthermore, the content of hydroxyethyl starch in each 100 mL of cell cryopreservation solution can be 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, etc., including but not limited to the contents listed above, and more preferably 9-12 g. Preferably, the hydroxyethyl starch content in each 100 mL of cell cryopreservation solution is controlled to be within 12 g to meet the safety requirements for direct intravenous reinfusion of the cell cryopreservation solution.

[0012] Furthermore, human serum albumin in the cell cryopreservation solution can be introduced by adding 20% ​​human serum albumin; each 50 mL of 20% human serum albumin contains 10 g of human serum albumin. Preferably, the content of 20% human serum albumin in each 100 mL of cell cryopreservation solution can be 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, etc., including but not limited to the contents listed above, and more preferably 4 mL. In addition to the above-mentioned effects, the present invention introduces human serum albumin into the cell cryopreservation solution, and by controlling the amount added, the pH of the cell cryopreservation solution can be stabilized at 6.0-8.0, which is consistent with the pH range of the mesenchymal stem cell product formulation.

[0013] Furthermore, the trehalose content in each 100mL of cell cryopreservation solution can be 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.85g, 0.9g, 0.95g, 1.0g, 1.1g, 1.2g, 1.3g, 1.4g, 1.5g, 1.6g, 1.7g, 1.8g, 1.9g, 2.0g, etc., including but not limited to the contents listed above.

[0014] Furthermore, the polyethylene glycol is preferably PEG400.

[0015] Furthermore, the hydroxyethyl starch is hydroxyethyl starch 200 / 0.5.

[0016] Furthermore, the cell cryopreservation solution also includes sodium chloride injection and / or compound electrolyte injection; wherein each 1000 mL of compound electrolyte injection contains 5.26 g of sodium chloride, 5.02 g of sodium gluconate, 3.68 g of sodium acetate, 0.37 g of potassium chloride, and 0.30 g of magnesium chloride.

[0017] Preferably, each 100 mL of cell cryopreservation solution contains: 1 g of polyethylene glycol 400, 12 g of hydroxyethyl starch 200 / 0.5, 4 mL of human serum albumin 20%, 0.85 g of trehalose, and the remainder is sodium chloride injection and / or compound electrolyte injection.

[0018] Furthermore, the cell cryopreservation solution also contains dextran. Preferably, the content of dextran in the cell cryopreservation solution is 1-10g, such as 1g, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10g, etc., including but not limited to the mass percentage content listed above, and more preferably 2-5g.

[0019] Furthermore, the dextran is preferably dextran 40.

[0020] Furthermore, the preferred mass ratio of hydroxyethyl starch to dextran and trehalose is 1:(0.17-0.41):(0.07-0.14). In cell cryopreservation solutions containing a relatively low concentration of hydroxyethyl starch, for example, when the concentration of hydroxyethyl starch in the cell cryopreservation solution is 6%-9%, the simultaneous addition of dextran and trehalose, while controlling the amount added, can effectively improve the cryopreservation effect of the cell cryopreservation solution.

[0021] Further, in every 100 mL of cell cryopreservation solution: polyethylene glycol 400 contains 1 g, hydroxyethyl starch 200 / 0.5 contains 9-12 g, human serum albumin 20% contains 4 mL, trehalose contains 0.85-1.7 g, dextran 40 contains 2-5 g, and the remainder is sodium chloride injection and / or compound electrolyte injection; more preferably,

[0022] Per 100 mL of cell cryopreservation solution: 1 g of polyethylene glycol 400, 9 g of hydroxyethyl starch 200 / 0.5, 4 mL of human serum albumin 20%, 0.85 g of trehalose, 2 g of dextran 40, and the remainder being sodium chloride injection and / or compound electrolyte injection; or,

[0023] Per 100 mL of cell cryopreservation solution: 1 g of polyethylene glycol 400, 9 g of hydroxyethyl starch 200 / 0.5, 4 mL of human serum albumin 20%, 1.7 g of trehalose, 5 g of dextran 40, with the remainder being sodium chloride injection and / or compound electrolyte injection; or,

[0024] Each 100mL of cell cryopreservation solution contains: 1g of polyethylene glycol 400, 12g of hydroxyethyl starch 200 / 0.5, 4mL of human serum albumin 20%, 1.7g of trehalose, 2-5g of dextran 40, and the remainder is sodium chloride injection and / or compound electrolyte injection.

[0025] Furthermore, the cell cryopreservation solution also contains glucose. Preferably, the glucose content in the cell cryopreservation solution is 0.5-1.5g, such as 0.5g, 0.6g, 0.7g, 0.8g, 0.85g, 0.9g, 0.95g, 1.0g, 1.1g, 1.2g, 1.3g, 1.4g, 1.5g, etc., including but not limited to the contents listed above.

[0026] In some preferred embodiments of the present invention, each 100 mL of cell cryopreservation solution contains: 1 g of polyethylene glycol 400, 12 g of hydroxyethyl starch 200 / 0.5, 4 mL of human serum albumin 20%, 1.7 g of trehalose, 2 g of dextran 40, 0.9 g of glucose, and the remainder is sodium chloride injection and / or compound electrolyte injection.

[0027] Furthermore, the preparation method of the cell cryopreservation solution includes the following steps:

[0028] S1: Weigh each component according to the formula and mix them evenly to obtain a mixed solution with a pH of 6.0-8.0;

[0029] S2: The mixed solution prepared in S1 is filtered and sterilized in a sterile environment to obtain the cell cryopreservation solution.

[0030] The second aspect of this invention provides the application of the cell cryopreservation solution described in the first aspect in the cryopreservation of mesenchymal stem cells.

[0031] Furthermore, the method for cryopreserving mesenchymal stem cells using the cell cryopreservation protectant includes the following steps: resuspending the mesenchymal stem cells in the cell cryopreservation protectant, then filling them into cryopreservation tubes, and transferring them to liquid nitrogen for cryopreservation after programmed cooling.

[0032] Furthermore, the cryopreservation density of the mesenchymal stem cells in the cell cryopreservation solution is 1.50 × 10⁻⁶. 6 Total cells / mL.

[0033] The beneficial effects of this invention are:

[0034] 1. This invention provides a cell cryopreservation solution that provides a safe and stable cryopreservation environment for cells through the synergistic effect of low molecular weight polyethylene glycol, hydroxyethyl starch, human serum albumin and trehalose, reducing the damage of cryoprotectants to cells and resulting in high cell viability and stability after cryopreservation and thawing.

[0035] 2. This invention effectively improves the cell survival rate of mesenchymal stem cells after cryopreservation and thawing by introducing dextran into a cell cryopreservation solution with a low hydroxyethyl starch content. Furthermore, introducing an appropriate amount of glucose into a cell cryopreservation solution containing both high trehalose and low dextran content can further improve the cell survival rate of mesenchymal stem cells after cryopreservation.

[0036] 3. Using the cell cryopreservation solution provided by this invention for cryopreservation of mesenchymal stem cells can maintain a high cell viability rate after cryopreservation and thawing. Furthermore, by adjusting the component content of the cell cryopreservation solution to fully leverage the synergistic effects of the components, the decrease in cell viability before and after freezing is only 1%-8%. Compared to commercially available DMSO-free and glycerol-free cryopreservation solutions, the cell viability rate of cryopreserved mesenchymal stem cells is increased by nearly 15%. Moreover, the components of this cell cryopreservation solution have high clinical safety and can be used as a cryopreservation solution for long-term storage of clinical-grade mesenchymal stem cells. After thawing, the cell cryopreservation solution containing cells can be directly used for intravenous reinfusion.

[0037] 4. The cell cryopreservation solution provided by this invention is selective for cell types and is more suitable for the cryopreservation of mesenchymal stem cells than 293T cells. Detailed Implementation

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items. "Comprising" or "containing" as used herein means that it may include or contain other components in addition to the stated components. "Comprising" or "containing" as used herein may also be replaced with the closed form "is" or "consisting of".

[0039] The present invention will be further described below with reference to embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0040] The reagents used in the following examples and comparative examples are all commercially available, and specific information is as follows:

[0041] Polyethylene glycol 400 was purchased from Shandong Ruisheng Pharmaceutical Excipients Co., Ltd.; polyethylene glycol 800 and polyethylene glycol 2000 were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; polyethylene glycol 8000 was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number P274350; hydroxyethyl starch 130 / 0.4 was purchased from Shaanxi Shengrui Pharmaceutical Technology Co., Ltd.; hydroxyethyl starch 200 / 0.5 was purchased from Shaanxi Shengrui Pharmaceutical Technology Co., Ltd.; dextran 40 was purchased from Xi'an Wanlong Pharmaceutical Co., Ltd.; trehalose was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number D110019; 20% human serum albumin solution (20% HSA, specification: 10g human serum albumin per 50mL solution) was purchased from Grifols, Inc., USA; anhydrous glucose was purchased from Weifang Shengtai Pharmaceutical Co., Ltd.; sodium chloride injection was purchased from Shijiazhuang No. 4 Pharmaceutical Co., Ltd.; compound electrolyte injection was purchased from Shanghai Baxter Medical Supplies Co., Ltd.

[0042] Example 1

[0043] This example investigated the effects of the molecular weight and degree of substitution of hydroxyethyl starch on the survival rate of mesenchymal stem cells after cryopreservation and thawing. The specific procedures are as follows:

[0044] Preparation of cell cryoprotection solution: 1g polyethylene glycol 400 was mixed with 6g hydroxyethyl starch 130 / 0.4, 9g hydroxyethyl starch 130 / 0.4, 12g hydroxyethyl starch 130 / 0.4, 6g hydroxyethyl starch 200 / 0.5, 9g hydroxyethyl starch 200 / 0.5, and 12g hydroxyethyl starch 200 / 0.5, respectively. The pH was adjusted to 6.0-8.0, and sodium chloride injection solution was added to bring the volume to 100mL. Sterilization was achieved by filtration through a 0.22 μm filter in a sterile environment to obtain cryoprotective solutions for mesenchymal stem cells: A1 (6g hydroxyethyl starch 130 / 0.4g), A2 (9g hydroxyethyl starch 130 / 0.4g), A3 (12g hydroxyethyl starch 130 / 0.4g), B1 (6g hydroxyethyl starch 200 / 0.5g), B2 (9g hydroxyethyl starch 200 / 0.5g), and B3 (12g hydroxyethyl starch 200 / 0.5g). The cell cryoprotective solutions were stored in a medical refrigerator at 2-8°C.

[0045] Cryopreservation: After harvesting mesenchymal stem cells, wash the cells with sodium chloride injection containing 0.2% HSA, centrifuge at 1200 rpm for 5 min, and discard the supernatant; add the above-mentioned mesenchymal stem cell cryopreservation solution, resuspend and mix the cells, and the cell cryopreservation density is 1.50 × 10⁶ cells / year. 6 Total cells / mL; place cryovials in a programmed cooling box and store at -80°C for 24 hours, then transfer to a liquid nitrogen tank and freeze for 5 days.

[0046] Resuscitation of mesenchymal stem cells: Remove mesenchymal stem cells from the liquid nitrogen tank and quickly place them in a 37°C constant temperature water bath to thaw. Once only a small amount of ice remains, transfer them to a biosafety cabinet, mix well, count the cells, and calculate the cell viability.

[0047] The cryopreservation results are shown in Table 1 below:

[0048] Table 1

[0049] As shown in Table 1, the cell survival rate of mesenchymal stem cells after cryopreservation and thawing in Group B cell cryopreservation solution containing 200 / 0.5% hydroxyethyl starch was significantly higher than that of cells after cryopreservation and thawing in Group A cell cryopreservation solution containing 130 / 0.4% hydroxyethyl starch. Therefore, 200 / 0.5% hydroxyethyl starch is the preferred cryopreservation solution for mesenchymal stem cells.

[0050] Example 2

[0051] This example investigated the effect of PEG molecular weight on the survival rate of mesenchymal stem cells after cryopreservation and thawing. The specific procedures are as follows:

[0052] Preparation of cell cryoprotective solutions: 1g of polyethylene glycol of different molecular weights (PEG400, PEG800, PEG2000, PEG8000) was mixed with 12g of hydroxyethyl starch 200 / 0.5, and the pH was adjusted to 6.0-8.0. Sodium chloride injection solution was added to bring the volume to 100mL, and then the mixture was filtered through a 0.22μm filter membrane for sterilization in a sterile environment to obtain cryoprotective solutions for mesenchymal stem cells C1 (PEG400), C2 (PEG800), C3 (PEG2000), and C4 (PEG8000). The cell cryoprotective solutions were stored in a medical refrigerator at 2-8℃.

[0053] Cryopreservation: After harvesting mesenchymal stem cells, wash the cells with sodium chloride injection containing 0.2% HSA, centrifuge at 1200 rpm for 5 min, and discard the supernatant; add the above-mentioned mesenchymal stem cell cryopreservation solution, resuspend and mix the cells, and the cell cryopreservation density is 1.50 × 10⁶ cells / year. 6 Total cells / mL; place cryovials in a programmed cooling box and store at -80°C for 24 hours, then transfer to liquid nitrogen and freeze for 5 days.

[0054] Resuscitation of mesenchymal stem cells: Remove mesenchymal stem cells from the liquid nitrogen tank and quickly place them in a 37°C constant temperature water bath to thaw. Once only a small amount of ice remains, transfer them to a biosafety cabinet, mix well, count the cells, and calculate the cell viability.

[0055] The cryopreservation results are shown in Table 2 below:

[0056] Table 2

[0057] As shown in Table 2, the cell survival rate of mesenchymal stem cells after cryopreservation and thawing in cell cryopreservation solution containing PEG400 was as high as 87.15%, which was 7.7%-21.8% higher than that of cell cryopreservation solutions containing PEG800, PEG2000 or PEG8000. This also shows that using low molecular weight polyethylene glycol is more conducive to the cryopreservation of mesenchymal stem cells.

[0058] Example 3

[0059] This embodiment provides a cell cryoprotectant solution, prepared from PEG400, hydroxyethyl starch 200 / 0.5, 20% HSA, trehalose, and sodium chloride injection. The effects of the hydroxyethyl starch 200 / 0.5 and trehalose content on the cryoprotectant solution's freezing effect were investigated, as detailed below:

[0060] Preparation of cell cryoprotection solution: Mix the components in the formula separately, add sodium chloride injection solution to make up to 100 mL, and then filter sterilize in a sterile environment using a 0.22 μm filter membrane to obtain mesenchymal stem cell cryoprotection solution D1~D10 with a pH value of 6.0-8.0.

[0061] The formulations of different cell cryoprotection solutions are shown in Table 3 below:

[0062] Table 3

[0063] Cryopreservation: After harvesting mesenchymal stem cells, wash the cells with sodium chloride injection containing 0.2% HSA, centrifuge at 1200 rpm for 5 min, and discard the supernatant; add the mesenchymal stem cell cryopreservation solutions listed in Table 3 above, resuspend and mix the cells, and the cell cryopreservation density is 1.50 × 10⁻⁶. 6 Total cells / mL; place cryovials in a programmed cooling box and store at -80°C for 24 hours, then transfer to a liquid nitrogen tank and freeze for 5 days.

[0064] Resuscitation of mesenchymal stem cells: Remove mesenchymal stem cells from the liquid nitrogen tank and quickly place them in a 37°C constant temperature water bath to thaw. Once only a small amount of ice remains, transfer them to a biosafety cabinet, mix well, count the cells, and calculate the cell viability.

[0065] The cryopreservation results are shown in Table 4 below:

[0066] Table 4

[0067] As shown in D1 / D4 / D8, D2 / D5 / D9, D3 / D7, and D6 / D10, when the cell cryoprotectant solution contains only PEG400, hydroxyethyl starch 200 / 0.5, 20% HSA, trehalose, and sodium chloride injection, the cell viability increases with increasing hydroxyethyl starch content in the cryoprotectant solution, assuming other component contents are constant. However, as shown in D3-D6 and D7-D10, the cell viability initially increases and then decreases with increasing trehalose content in the cryoprotectant solution, assuming other component contents are constant, which differs from the expected results before experimental verification. Specifically, when 100 mL of cell cryoprotectant solution contains 1 g of PEG400, 12 g of hydroxyethyl starch 200 / 0.5, 4 mL of 20% HSA, 0.85 g of trehalose, and the remainder is sodium chloride injection (formulation D8), the cell viability of mesenchymal stem cells after cryopreservation and thawing only decreases by 3.4%.

[0068] Example 4

[0069] This embodiment provides a cell cryoprotectant solution, prepared from PEG400, hydroxyethyl starch 200 / 0.5, 20% HSA, trehalose, dextran 40, and sodium chloride injection. The effects of the contents of hydroxyethyl starch 200 / 0.5, trehalose, and dextran on the cryoprotectant solution's freezing effect are investigated, as detailed below:

[0070] Preparation of cell cryoprotection solution: Mix the components of the formula separately, add sodium chloride injection solution to make up to 100 mL, and then filter sterilize in a sterile environment using a 0.22 μm filter membrane to obtain mesenchymal stem cell cryoprotection solution E1~E20 with a pH value of 6.0-8.0.

[0071] The formulations of different cell cryoprotection solutions are shown in Table 5 below:

[0072] Table 5

[0073] Cryopreservation: After harvesting mesenchymal stem cells, wash the cells with sodium chloride injection containing 0.2% HSA, centrifuge at 1200 rpm for 5 min, and discard the supernatant; add the mesenchymal stem cell cryopreservation solutions listed in Table 3 above, resuspend and mix the cells, and the cell cryopreservation density is 1.50 × 10⁻⁶. 6 Total cells / mL; place cryovials in a programmed cooling box and store at -80°C for 24 hours, then transfer to a liquid nitrogen tank and freeze for 5 days.

[0074] Resuscitation of mesenchymal stem cells: Remove mesenchymal stem cells from the liquid nitrogen tank and quickly place them in a 37°C constant temperature water bath to thaw. Once only a small amount of ice remains, transfer them to a biosafety cabinet, mix well, count the cells, and calculate the cell viability.

[0075] The cryopreservation results are shown in Table 6 below:

[0076] Table 6

[0077] As shown by D1 / E1 / E2, D2 / E3 / E4, D4 / E7 / E8, and D5 / E9 / E10, adding dextran to cell cryopreservation solutions with low hydroxyethyl starch content (6%-9%) can effectively improve cell viability.

[0078] As shown in E19 / E3 / E9 / E15 and E20 / E4 / E10 / E16, when the trehalose content is 1.7g, cell viability increases with increasing hydroxyethyl starch content. However, the cell viability after cryopreservation and thawing in E19 / E20 without added hydroxyethyl starch is only 14.86% / 35.65%, significantly lower than that after cryopreservation and thawing in cell cryoprotectant containing hydroxyethyl starch. As shown in E1 / E7 / E13, when the trehalose content is 0.85g and dextran 40 is 2g, cell viability first increases and then decreases with increasing hydroxyethyl starch content. As shown in E2 / E8 / E14, when the trehalose content is 0.85g and dextran 40 is 5g, cell viability decreases with increasing hydroxyethyl starch content. Therefore, hydroxyethyl starch is a key component in cell cryopreservation solutions, and trehalose, dextran 40, and hydroxyethyl starch are interconnected. When PEG400 and HSA levels are constant, simultaneously regulating the levels of trehalose, dextran 40, and hydroxyethyl starch can effectively improve the survival rate of cryopreserved mesenchymal stem cells after thawing. For example:

[0079] When 100 mL of cell cryoprotectant solution contains 1 g of PEG400, 9 g of hydroxyethyl starch 200 / 0.5, 4 mL of 20% HSA, 0.85 g of trehalose, 2 g of dextran 40, and the remainder is sodium chloride injection solution (formula E7), the cell survival rate of mesenchymal stem cells after cryopreservation and thawing only decreased by 6.87%.

[0080] When 100 mL of cell cryoprotectant solution contains 1 g of PEG400, 9 g of hydroxyethyl starch 200 / 0.5, 4 mL of 20% HSA, 1.7 g of trehalose, 5 g of dextran 40, and the remainder is sodium chloride injection solution (formula E10), the cell survival rate of mesenchymal stem cells after cryopreservation and thawing only decreased by 9.17%.

[0081] When 100 mL of cell cryoprotectant solution contains 1 g of PEG400, 12 g of hydroxyethyl starch 200 / 0.5, 4 mL of 20% HSA, 1.7 g of trehalose, 2 g of dextran 40, and the remainder is sodium chloride injection solution (formula E15), the cell survival rate of mesenchymal stem cells after cryopreservation and thawing only decreased by 7.61%.

[0082] When 100 mL of cell cryoprotectant solution contains 1 g of PEG400, 12 g of hydroxyethyl starch 200 / 0.5, 4 mL of 20% HSA, 1.7 g of trehalose, 5 g of dextran 40, and the remainder is sodium chloride injection solution (formula E16), the cell survival rate of mesenchymal stem cells after cryopreservation and thawing only decreased by 5.13%.

[0083] Example 5

[0084] This embodiment uses the preferred cell cryoprotectant formulations D8, E15, and E16 from Examples 3 and 4 as examples to study the effect of glucose addition on the survival rate of mesenchymal stem cells after cryopreservation and thawing. The specific operation is as follows:

[0085] Preparation of cell cryoprotection solutions: Mix the components in the prescribed amounts separately, adjust the pH value to 6.0-8.0, add sodium chloride injection solution to make up to 100 mL, and then filter and sterilize in a sterile environment using a 0.22 μm filter membrane to obtain cryoprotection solutions F1-F3 for mesenchymal stem cells to be used.

[0086] The only difference between cell cryoprotectant solutions F1-F3 and D8, E15, and E16 is that 0.9g of glucose is added.

[0087] Cryopreservation: After harvesting mesenchymal stem cells, wash the cells with sodium chloride injection containing 0.2% HSA, centrifuge at 1200 rpm for 5 min, and discard the supernatant; add cell cryoprotectant solutions D5, E15, and E16 respectively, resuspend and mix the cells, and freeze at a density of 1.50 × 10⁶ cells / year. 6 Total cells / mL; place cryovials in a programmed cooling box and store at -80°C for 24 hours, then transfer to liquid nitrogen and freeze for 5 days.

[0088] Resuscitation of mesenchymal stem cells: Remove mesenchymal stem cells from the liquid nitrogen tank and quickly place them in a 37°C constant temperature water bath to thaw. Once only a small amount of ice remains, transfer them to a biosafety cabinet, mix well, count the cells, and calculate the cell viability.

[0089] The cryopreservation results are shown in Table 7 below:

[0090] Table 7

[0091] Table 5 shows that adding 0.9g of glucose to cell cryoprotectant D8, which contains only PEG400, hydroxyethyl starch 200 / 0.5, 20% HSA, trehalose, and sodium chloride injection, actually reduces cell viability after cryopreservation and thawing. Adding 0.9g of glucose to cell cryoprotectant E15, which contains PEG400, hydroxyethyl starch 200 / 0.5, 20% HSA, trehalose, dextran 40, and sodium chloride injection, further improves cell viability after cryopreservation; however, adding 0.9g of glucose to cell cryoprotectant E16, which has a high dextran 40 content, has no effect on improving cell viability.

[0092] Example 6

[0093] This embodiment uses the preferred cell cryoprotectant formulations D8, E15, E16, and F1-F3 from Example 5 as an example to study the effect of cryopreservation density on the survival rate of mesenchymal stem cells after cryopreservation and thawing. The specific operation is as follows:

[0094] Cryopreservation: After harvesting mesenchymal stem cells, wash the cells with sodium chloride injection containing 0.2% HSA, centrifuge at 1200 rpm for 5 min, and discard the supernatant; add cell cryoprotectant solutions D5, E15, E16, and F1-F3 respectively, resuspend and mix the cells, and freeze at cell densities of 1.50 × 10⁻⁶ cells / mL. 6 Total cells / mL, 3.0 × 10 6 Total cells / mL, 5.0 × 10 6 Total cells / mL; place cryovials in a programmed cooling box and store at -80°C for 24 hours, then transfer to liquid nitrogen and freeze for 5 days.

[0095] Resuscitation of mesenchymal stem cells: Remove mesenchymal stem cells from the liquid nitrogen tank and quickly place them in a 37°C constant temperature water bath to thaw. Once only a small amount of ice remains, transfer them to a biosafety cabinet, mix well, count the cells, and calculate the cell viability.

[0096] The cryopreservation results are shown in Table 8 below:

[0097] Table 8

[0098] Table 8 shows that the survival rate of mesenchymal stem cells after cryopreservation and thawing decreases with increasing cryopreservation density. The survival rate is lowest when the cryopreservation density is 1.50 × 10⁻⁶. 6 The optimal cryopreservation effect was achieved when the total number of cells / mL was 10, with the survival rate decreasing by less than 10% after thawing.

[0099] Example 7

[0100] This embodiment uses the preferred cell cryoprotectant formulations D8, E15, E16, and F1-F3 from Example 5 as an example to study the effect of the type of injection solution on the survival rate of mesenchymal stem cells after cryopreservation and thawing. The specific operation is as follows:

[0101] By replacing the sodium chloride injection in cell cryoprotectant formulations D8, E15, E16, and F1-F3 with an equal volume of compound electrolyte injection, cell cryoprotectant solutions G1-G6 were obtained.

[0102] Cryopreservation: After harvesting mesenchymal stem cells, wash the cells with sodium chloride injection containing 0.2% HSA, centrifuge at 1200 rpm for 5 min, and discard the supernatant. Add cell cryoprotectant solutions D8, E15, E16, and F1-F3 respectively; and wash the cells with compound electrolyte injection containing 0.2% HSA, centrifuge at 1200 rpm for 5 min, and discard the supernatant. Add cell cryoprotectant solutions G1-G6 respectively, resuspend and mix the cells, and the cell cryopreservation density is 1.50 × 10⁶ cells / year. 6 Total cells / mL; place cryovials in a programmed cooling box and store at -80°C for 24 hours, then transfer to liquid nitrogen and freeze for 5 days.

[0103] Resuscitation of mesenchymal stem cells: Remove mesenchymal stem cells from the liquid nitrogen tank and quickly place them in a 37°C constant temperature water bath to thaw. Once only a small amount of ice remains, transfer them to a biosafety cabinet, mix well, count the cells, and calculate the cell viability.

[0104] The cryopreservation results are shown in Table 9 below:

[0105] Table 9

[0106] As shown in Table 9, the cell cryoprotectant solution prepared with compound electrolyte injection can further improve the survival rate of mesenchymal stem cells after cryopreservation and thawing.

[0107] Comparative Example 1

[0108] Commercially available DMSO-free and glycerol-free cryopreservatives H1 (Nanjing 3SBio Co., Ltd., catalog number: YB050050) and H2 (FUJIFILM Irvine Scientific, catalog number: 91140) were used for cryopreservation of mesenchymal stem cells.

[0109] Cryopreservation: After harvesting mesenchymal stem cells, wash the cells with sodium chloride injection containing 0.2% HSA, centrifuge at 1200 rpm for 5 min, discard the supernatant, and add cell cryoprotectant solutions H1 and H2 respectively; and wash the cells with compound electrolyte injection containing 0.2% HSA, centrifuge at 1200 rpm for 5 min, discard the supernatant, and add cell cryoprotectant solutions H1 and H2 respectively.

[0110] Resuspend and mix the cells; the cell density for cryopreservation is 1.50 × 10⁻⁶. 6 Total cells / mL; place cryovials in a programmed cooling box and store at -80°C for 24 hours, then transfer to liquid nitrogen and freeze for 5 days.

[0111] Resuscitation of mesenchymal stem cells: Remove mesenchymal stem cells from the liquid nitrogen tank and quickly place them in a 37°C constant temperature water bath to thaw. Once only a small amount of ice remains, transfer them to a biosafety cabinet, mix well, count the cells, and calculate the cell viability.

[0112] The cryopreservation results are shown in Table 10 below:

[0113] Table 10

[0114] As shown in Table 10, after washing the harvested mesenchymal stem cells with sodium chloride injection containing 0.2% HSA and then cryopreserving them with commercially available H1 and H2 solutions, the cell viability after cryopreservation and thawing decreased by more than 14%. Under the same conditions, the cell viability after thawing mesenchymal stem cells cryopreserved with the cell cryoprotectants D8, E15, E16, and F1-F3 provided by this invention decreased by less than 10%. Furthermore, after washing the harvested mesenchymal stem cells with compound electrolyte injection containing 0.2% HSA and then cryopreserving them with commercially available H1 and H2 solutions, the cell viability after cryopreservation and thawing was lower than that with the cell cryoprotectants G1-G6 prepared by this invention. In particular, the cell viability after cryopreservation and thawing using G5 solutions decreased by only 0.51%, which is far superior to the effects of cell cryoprotectants H1 and H2.

[0115] Comparative Example 2

[0116] This comparative example uses the preferred cell cryoprotectant formulations D8, E15, E16, and F1-F3 from Example 5 to study the effectiveness of the above cell cryoprotectant for cryopreserving 293T cells. The specific operation is as follows:

[0117] Cryopreservation: After harvesting 293T cells, wash the cells with sodium chloride injection containing 0.2% HSA, centrifuge at 1200 rpm for 5 min, and discard the supernatant. Add cell cryoprotectant solutions D8, E15, E16, and F1-F3 respectively, resuspend and mix the cells, and freeze at a density of 1.50 × 10⁻⁶ cells / year. 6 Total cells / mL; place cryovials in a programmed cooling box and store at -80°C for 24 hours, then transfer to liquid nitrogen and freeze for 5 days.

[0118] 293T cell resuscitation: 293T cells were removed from the liquid nitrogen tank and quickly placed in a 37°C constant temperature water bath to thaw until only a small amount of ice remained. The cells were then transferred to a biosafety cabinet, mixed, counted, and the cell viability was calculated.

[0119] The cryopreservation results are shown in Table 11 below:

[0120] Table 11

[0121] As shown in Table 11, when 293T cells were cryopreserved using cell cryoprotectants D8, E15, E16, and F1-F3, the cell viability decreased by more than 40% after thawing. In contrast, the cell viability of cryopreserved mesenchymal stem cells decreased by no more than 10%, and could be as low as 3.27%. Therefore, the cell cryoprotectant provided by this invention is specific for the cryopreservation and protection of mesenchymal stem cells.

[0122] The embodiments described above are merely preferred examples to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A cell cryopreservation solution, characterized in that, The cell cryopreservation solution contains at least the following components: polyethylene glycol, hydroxyethyl starch, human serum albumin, and trehalose; the molecular weight of the polyethylene glycol is 200-600.

2. The cell cryopreservation solution according to claim 1, characterized in that, Per 100 mL of cell cryopreservation solution: polyethylene glycol content is 0.5-2 g, hydroxyethyl starch content is 6-12 g, human serum albumin content is 0.4-1.2 g, and trehalose content is 0.1-2 g.

3. The cell cryopreservation solution according to claim 1 or 2, characterized in that, The polyethylene glycol is PEG400; And / or, the hydroxyethyl starch is hydroxyethyl starch 200 / 0.

5.

4. The cell cryopreservation solution according to claim 1, characterized in that, The cell cryopreservation solution also includes sodium chloride injection and / or compound electrolyte injection. Preferably, each 100 mL of cell cryopreservation solution contains: 1 g of PEG400, 12 g of hydroxyethyl starch 200 / 0.5, 4 mL of human serum albumin 20%, 0.85 g of trehalose, and the remainder is sodium chloride injection and / or compound electrolyte injection.

5. The cell cryopreservation solution according to claim 1, characterized in that, The cell cryopreservation solution also contains dextran; Preferably, the content of dextran in each 100 mL of cell cryopreservation solution is 1-10 g.

6. The cell cryopreservation solution according to claim 5, characterized in that, The dextran is dextran 40; And / or, the mass ratio of the hydroxyethyl starch to dextran and trehalose is 1:(0.17-0.41):(0.07-0.14).

7. The cell cryopreservation solution according to claim 6, characterized in that, Each 100 mL of cell cryopreservation solution contains: 1 g of PEG400, 9-12 g of hydroxyethyl starch 200 / 0.5, 4 mL of human serum albumin 20%, 0.85-1.7 g of trehalose, 2-5 g of dextran 40, and the remainder is sodium chloride injection and / or compound electrolyte injection.

8. The cell cryopreservation solution according to claim 1 or 7, characterized in that, The cell cryopreservation solution also contains glucose; Preferably, the glucose content in each 100 mL of cell cryopreservation solution is 0.5-1.5 g.

9. The cell cryopreservation solution according to claim 8, characterized in that, Each 100 mL of cell cryopreservation solution contains: 1 g of PEG400, 12 g of hydroxyethyl starch 200 / 0.5, 4 mL of human serum albumin 20%, 1.7 g of trehalose, 2 g of dextran 40, 0.9 g of glucose, and the remainder is sodium chloride injection and / or compound electrolyte injection.

10. The use of the cell cryopreservation solution according to any one of claims 1-9 in the cryopreservation of mesenchymal stem cells.

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