A mesenchymal stem cell cryopreservation solution

Abstract

The present application provides a clinical level DMSO-free, serum-free and any protein component-free mesenchymal stem cell cryopreservation solution, which can be used for long-term preservation of mesenchymal stem cells in scientific research and clinical practice, such as establishment of mesenchymal stem cell bank, etc. Since the cryopreservation solution does not contain DMSO, serum and any protein and other animal-derived components, it is safer and more suitable for clinical needs. In particular, there is no need for programmed cooling when freezing cells, which simplifies the freezing process and unexpectedly improves the recovery rate and viability of the frozen cells.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry, and specifically to a mesenchymal stem cell cryopreservation solution that is clinically applicable and free of DMSO, serum, and any protein components. Background Technology

[0002] Cell cryopreservation is a technique that places cells in a low-temperature environment to reduce cellular metabolism for long-term storage. It is one of the main methods of cell preservation. The basic principle of cell cryopreservation is slow freezing; experiments have shown that this maximizes cell viability by reducing the formation of intracellular ice crystals, thereby minimizing cell damage caused by ice crystal formation. To reduce ice crystal formation and provide nutrients to the cells, existing cell cryopreservation solutions typically contain components such as dimethyl sulfoxide (DMSO) and serum.

[0003] DMSO has certain cytotoxic properties and is also toxic to organs such as blood vessels, liver, and kidneys. Clinically, when cells frozen in DMSO are reinfused into the human body to treat diseases, it may cause side effects such as nausea, vomiting, and skin rashes in patients.

[0004] Serum or serum analogues are generally derived from animals, and their specific components are complex and unclear. Due to batch-to-batch differences, the quality of cryopreserved solutions can be unstable. Moreover, foreign substances can pose safety risks to humans, such as animal-derived viral infections and allergic reactions.

[0005] Mesenchymal stem cells (MSCs) are pluripotent stem cells, possessing all the common characteristics of stem cells, namely self-renewal and multi-lineage differentiation capabilities. Under specific conditions, MSCs can be induced to differentiate into various types of tissue cells, such as skeletal muscle cells, chondrocytes, adipocytes, osteoblasts, or cardiomyocytes, thus having wide applications in the treatment of various diseases and tissue engineering research. Due to their wide range of applications, effectively improving the viability and recovery rate of cryopreserved MSCs after thawing is an urgent problem to be solved.

[0006] Therefore, it is necessary to provide a new cryopreservation solution for mesenchymal stem cells to overcome the above-mentioned defects and achieve efficient, safe and stable cell cryopreservation. Summary of the Invention

[0007] In view of the above-mentioned prior art, the present invention provides a clinical-grade mesenchymal stem cell cryopreservation solution free of DMSO, serum, and any protein components. This solution can be used for the long-term preservation of mesenchymal stem cells in research and clinical settings, such as for establishing mesenchymal stem cell banks. Because the cryopreservation solution does not contain DMSO, serum, any proteins, or other animal-derived components, it is safer and better meets clinical needs. In particular, it eliminates the need for programmed cooling during cell cryopreservation, simplifying the cryopreservation process and unexpectedly improving the recovery rate and viability of cryopreserved cells.

[0008] This invention is achieved through the following technical solution:

[0009] According to one aspect of the present invention, a mesenchymal stem cell cryopreservation solution is provided, comprising the following components:

[0010] Sodium chloride 9000 mg / L;

[0011] Disodium hydrogen phosphate·7H2O 726mg / L;

[0012] Potassium dihydrogen phosphate 210 mg / L;

[0013] Glycine 5000–20000 mg / L;

[0014] L-cysteine ​​hydrochloride 1000~10000 mg / L;

[0015] L-cystine hydrochloride 5000~10000 mg / L;

[0016] L-glutamine 1000~10000 mg / L;

[0017] L-Lysine hydrochloride 5000~20000 mg / L;

[0018] L-proline 10000~40000 mg / L;

[0019] p-Hydroxybenzoic acid 0.5–1.5 mg / L;

[0020] Glucose 1000–5000 mg / L;

[0021] Ascorbic acid 50-200 mg / L;

[0022] Vitamin E 20-100 mg / L;

[0023] Glutathione 50–200 mg / L;

[0024] Mannitol 20000~70000 mg / L;

[0025] Propylene glycol 20,000–80,000 mg / L;

[0026] Glycerin 20,000–70,000 mg / L;

[0027] Hydroxyethyl starch 10000~80000 mg / L;

[0028] Dextran-40 20000~100000 mg / L;

[0029] Poloxam-188, by volume percentage: 0.02–0.2%;

[0030] water.

[0031] Preferably, the cryopreservation solution is composed of the above-mentioned components.

[0032] Preferably, the water is ultrapure water.

[0033] Preferably, the pH of the above cryopreservation solution is adjusted to 7.1-7.4 using 4M HCl and / or NaOH aqueous solution when necessary.

[0034] Preferably, the mesenchymal stem cell cryopreservation solution of the present invention contains:

[0035] Sodium chloride 9000 mg / L;

[0036] Disodium hydrogen phosphate·7H2O 726mg / L;

[0037] Potassium dihydrogen phosphate 210 mg / L;

[0038] Glycine 12500 mg / L;

[0039] L-cysteine ​​hydrochloride 5000 mg / L;

[0040] L-cystine hydrochloride 7500 mg / L;

[0041] L-glutamine 5000 mg / L;

[0042] L-Lysine hydrochloride 12500 mg / L;

[0043] L-proline 20000 mg / L;

[0044] p-Hydroxybenzoic acid 1.0 mg / L;

[0045] Glucose 3500 mg / L;

[0046] Ascorbic acid 125 mg / L;

[0047] Vitamin E 60mg / L;

[0048] Glutathione 125 mg / L;

[0049] Mannitol 45000 mg / L;

[0050] Propylene glycol 50000 mg / L;

[0051] Glycerin 45000 mg / L;

[0052] Hydroxyethyl starch 45000 mg / L;

[0053] Dextran-40 60000 mg / L;

[0054] Polosham-188 is 0.1% by volume;

[0055] The remainder is water, and the pH is adjusted to 7.2 using a 4M aqueous solution of HCl and / or NaOH.

[0056] Preferably, the water is ultrapure water. Those skilled in the art will understand that the mesenchymal cell cryopreservation solution of the present invention is suitable for cryopreserving various mesenchymal stem cells, such as umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, dental pulp stem cells, bone marrow stem cells, placental stem cells, etc.

[0057] According to another aspect of the present invention, the present invention provides a method for preparing the above-mentioned mesenchymal stem cell cryopreservation solution, comprising the following steps: dissolving all components except water in water, measuring the pH, adjusting the pH to 7.1 to 7.4 with 4M HCl and / or NaOH aqueous solution if necessary, and filtering with a 0.22μm filter membrane for sterilization, thereby obtaining the solution.

[0058] According to another aspect of the present invention, the present invention provides the application of the above-described mesenchymal stem cell cryopreservation solution in the cryopreservation of mesenchymal stem cells.

[0059] Preferably, the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp stem cells, bone marrow stem cells, placental stem cells, etc.

[0060] The above-described applications of the present invention are not intended for the diagnosis and treatment of diseases.

[0061] According to another aspect of the present invention, the present invention provides a method for cryopreserving mesenchymal stem cells, wherein the mesenchymal stem cells are cryopreserved using the above-mentioned mesenchymal stem cell cryopreservation solution.

[0062] Preferably, the mesenchymal stem cells are suspended in the above-mentioned mesenchymal stem cell cryopreservation solution, stored at -80°C for 12 hours, and then stored in liquid nitrogen.

[0063] Preferably, the cell density is 5 × 10⁻⁶. 5 ~2×10 7 Cells / mL.

[0064] Preferably, the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp stem cells, bone marrow stem cells, placental stem cells, etc.

[0065] The methods described above in this invention are not intended for the diagnosis and treatment of diseases.

[0066] The cryopreservation solution of this invention is formulated based on the characteristics and cryopreservation principles of mesenchymal stem cells, making it particularly suitable for the cryopreservation of mesenchymal stem cells. This cell cryopreservation solution improves the recovery rate of cryopreserved mesenchymal stem cells, has a wider range of cryopreservation adaptability, and achieves an average recovery rate of over 85%. Furthermore, this product can be stored at 2–8°C, and no programmed cooling is required during cell cryopreservation; cells can be directly placed at -80°C for freezing and then directly stored in liquid nitrogen after 12 hours, greatly simplifying the cryopreservation process. The product is also more stable, eliminating concerns about the effects of repeated freeze-thaw cycles.

[0067] This cryopreservation solution is formulated by rationally adjusting the concentrations of various substances based on their ability to cross cell membranes. This balances the permeable and non-permeable components within the cryopreservation solution, allowing the intracellular and extracellular protective solutions to reach protective concentrations quickly, thus mitigating damage caused by osmotic pressure. The extracellular protectants hydroxyethyl starch and dextran-40 work synergistically to bind extracellular water molecules and stabilize the cell membrane. The intracellular protectants mannitol, glycerol, and propylene glycol also work synergistically, resulting in higher fold expansion and viability after thawing. Simultaneously, other water-binding substances and cell-stabilizing factors are added to reduce cell membrane damage caused by water of crystallization. By addressing these two main aspects, this cryopreservation solution has been developed, achieving performance levels approaching or even exceeding those of traditional cryopreservation solutions containing DMSO.

[0068] This cryopreservation solution can be used for cryopreserved human infusion and related stem cell therapies. After cell thawing, there is no need for cell washing, reducing cell loss and the probability of cell contamination, truly achieving immediate use and greatly facilitating clinical stem cell therapy. This product does not contain any toxic components, and all excipients used have been approved by the drug regulatory authority. It can be administered intravenously to humans without significant side effects.

[0069] Unlike most cryopreservation solutions on the market, this product contains no complex components of culture media, no animal-derived components such as serum, and no protein components, reducing the risk of biotoxicity and sensitization. It eliminates the risk of animal viral infection, contains no DMSO, and is completely free of the side effects associated with DMSO in humans. This significantly simplifies the approval process for clinical applications and greatly increases the safety of clinical use. Furthermore, its performance is superior compared to other cryopreservation solutions of the same market segment. Attached Figure Description

[0070] Figure 1 Traditional cryopreservation solution for 72 hours to thaw umbilical cord mesenchymal stem cells;

[0071] Figure 2 Example 2: Resuscitating umbilical cord mesenchymal stem cells with cryopreservation solution for 72 hours;

[0072] Figure 3 Umbilical cord mesenchymal stem cells were revived for 72 hours using P cryopreservation solution from the United States.

[0073] Figure 4 Umbilical cord mesenchymal stem cells were revived for 72 hours using Japanese Q cryopreservation solution.

[0074] Figure 5 .CN110024775A Umbilical cord mesenchymal stem cells were revived in cryopreservation solution for 72 hours;

[0075] Figure 6 Traditional cryopreservation solution for reviving adipose-derived mesenchymal stem cells for 72 hours;

[0076] Figure 7 Example 2: Resuscitating adipose-derived mesenchymal stem cells in cryopreservation solution for 72 hours;

[0077] Figure 8 American P cryopreservation solution resuscitates adipose-derived mesenchymal stem cells for 72 hours;

[0078] Figure 9 Japanese Q cryopreservation solution revives adipose-derived mesenchymal stem cells for 72 hours;

[0079] Figure 10 .CN110024775A Frozen solution was used to revive adipose-derived mesenchymal stem cells for 72 hours;

[0080] Figure 11 Cryopreservation solution A revives umbilical cord mesenchymal stem cells for 72 hours;

[0081] Figure 12 Cryopreservation solution B was used to revive umbilical cord mesenchymal stem cells for 72 hours.

[0082] Figure 13 Umbilical cord mesenchymal stem cells were revived in cryopreservation solution C for 72 hours;

[0083] Figure 14 1. Resuscitate umbilical cord mesenchymal stem cells with cryopreservation solution D for 72 hours;

[0084] Figure 15 Umbilical cord mesenchymal stem cells were revived in cryopreservation solution E for 72 hours;

[0085] Figure 16 Umbilical cord mesenchymal stem cells were revived in cryopreservation solution F for 72 hours. Detailed Implementation

[0086] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0087] Example 1

[0088] The mesenchymal stem cell cryopreservation solution in this embodiment consists of the following components:

[0089] Sodium chloride 9000 mg / L;

[0090] Disodium hydrogen phosphate·7H2O 726mg / L;

[0091] Potassium dihydrogen phosphate 210 mg / L;

[0092] Glycine 5000 mg / L;

[0093] L-cysteine ​​hydrochloride 1000 mg / L;

[0094] L-cystine hydrochloride 5000 mg / L;

[0095] L-glutamine 1000 mg / L;

[0096] L-Lysine hydrochloride 5000 mg / L;

[0097] L-proline 10000 mg / L;

[0098] p-Hydroxybenzoic acid 0.5 mg / L;

[0099] Glucose 1000 mg / L;

[0100] Ascorbic acid 50 mg / L;

[0101] Vitamin E 20 mg / L;

[0102] Glutathione 50 mg / L;

[0103] Mannitol 20000 mg / L;

[0104] Propylene glycol 20000 mg / L;

[0105] Glycerin 20000 mg / L;

[0106] Hydroxyethyl starch 10000 mg / L;

[0107] Dextran-40 20000 mg / L;

[0108] Polosham-188 contains 0.02% by volume;

[0109] The remainder is ultrapure water, and the pH is adjusted to 7.2 using 4M HCl and / or NaOH aqueous solution.

[0110] The preparation method is as follows: dissolve all components except ultrapure water in ultrapure water, adjust the pH value to 7.2 with 4M HCl and / or NaOH aqueous solution, filter and sterilize with a 0.22μm filter membrane, and then dispense and store at -4℃.

[0111] Example 2

[0112] The mesenchymal stem cell cryopreservation solution in this embodiment consists of the following components:

[0113] Sodium chloride 9000 mg / L;

[0114] Disodium hydrogen phosphate·7H2O 726mg / L;

[0115] Potassium dihydrogen phosphate 210 mg / L;

[0116] Glycine 12500 mg / L;

[0117] L-cysteine ​​hydrochloride 5000 mg / L;

[0118] L-cystine hydrochloride 7500 mg / L;

[0119] L-glutamine 5000 mg / L;

[0120] L-Lysine hydrochloride 12500 mg / L;

[0121] L-proline 20000 mg / L;

[0122] p-Hydroxybenzoic acid 1.0 mg / L;

[0123] Glucose 3500 mg / L;

[0124] Ascorbic acid 125 mg / L;

[0125] Vitamin E 60mg / L;

[0126] Glutathione 125 mg / L;

[0127] Mannitol 45000 mg / L;

[0128] Propylene glycol 50000 mg / L;

[0129] Glycerin 45000 mg / L;

[0130] Hydroxyethyl starch 45000 mg / L;

[0131] Dextran-40 60000 mg / L;

[0132] Polosham-188 is 0.1% by volume;

[0133] The remainder is ultrapure water, and the pH is adjusted to 7.2 using 4M HCl and / or NaOH aqueous solution.

[0134] The preparation method is as follows: dissolve all components except ultrapure water in ultrapure water, adjust the pH value to 7.2 with 4M HCl and / or NaOH aqueous solution, filter and sterilize with a 0.22μm filter membrane, and then dispense and store at -4℃.

[0135] Example 3

[0136] The mesenchymal stem cell cryopreservation solution in this embodiment consists of the following components:

[0137] Sodium chloride 9000 mg / L;

[0138] Disodium hydrogen phosphate·7H2O 726mg / L;

[0139] Potassium dihydrogen phosphate 210 mg / L;

[0140] Glycine 20000 mg / L;

[0141] L-cysteine ​​hydrochloride 10000 mg / L;

[0142] L-cystine hydrochloride 10000 mg / L;

[0143] L-glutamine 10000 mg / L;

[0144] L-Lysine hydrochloride 20000 mg / L;

[0145] L-proline 40000 mg / L;

[0146] p-Hydroxybenzoic acid 1.5 mg / L;

[0147] Glucose 5000 mg / L;

[0148] Ascorbic acid 200 mg / L;

[0149] Vitamin E 100mg / L;

[0150] Glutathione 200 mg / L;

[0151] Mannitol 70000 mg / L;

[0152] Propylene glycol 80000 mg / L;

[0153] Glycerin 70000 mg / L;

[0154] Hydroxyethyl starch 80000 mg / L;

[0155] Dextran-40 100,000 mg / L;

[0156] Polosham-188 is 0.2% by volume.

[0157] The remainder is ultrapure water, and the pH is adjusted to 7.2 using 4M HCl and / or NaOH aqueous solution.

[0158] The preparation method is as follows: dissolve all components except ultrapure water in ultrapure water, adjust the pH value to 7.2 with 4M HCl and / or NaOH aqueous solution, filter and sterilize with a 0.22μm filter membrane, and then dispense and store at -4℃.

[0159] Example 4

[0160] Two common types of mesenchymal stem cells (human umbilical cord mesenchymal stem cells and human adipose-derived mesenchymal stem cells) were cryopreserved using the cryopreservation solution of this invention for testing. The cryopreservation method was as follows: the cells to be cryopreserved were digested using conventional trypsin digestion, counted, centrifuged to remove the supernatant, resuspended in the cryopreservation solution, and the density was adjusted. The cryopreservation density of umbilical cord mesenchymal stem cells was 1.47 × 10⁻⁶. 6 / mL; the cryopreserved density of adipose-derived mesenchymal stem cells was 2.55×10⁻⁶. 6 / mL, cryopreserved in 1.5mL cryovials, 1mL per tube, directly placed at -80℃, and then directly placed in liquid nitrogen for storage after 12 hours. For cell thawing, thaw in a 37℃ water bath for 1–2 minutes, add fresh culture medium (for umbilical cord mesenchymal stem cells, use serum-free mesenchymal stem cell culture medium (catalog number NC0103) + serum-free additive (catalog number NC0105.S) from Youkang Biotechnology (Beijing) Co., Ltd.; for adipose-derived mesenchymal stem cells, use serum-free mesenchymal stem cell culture medium (catalog number NC0103) + serum-free additive (NC0104.S) from Youkang Biotechnology (Beijing) Co., Ltd., the same below), centrifuge to remove the cryoprotectant, resuspend the cells in fresh culture medium, and seed. The comparative cryopreservation solutions were: the commercially available cryopreservation solution containing DMSO and albumin from Youkang Biotechnology (Beijing) Co., Ltd. (catalog number NC1001.1, hereinafter referred to as conventional cryopreservation solution); the US PRIME cryopreservation solution (catalog number 91140, hereinafter referred to as US P cryopreservation solution); the Japanese ZENOAQ cryopreservation solution (catalog number 11890, hereinafter referred to as Japanese Q cryopreservation solution); and the cryopreservation solution prepared in Example 4 of CN110024775A (hereinafter referred to as CN110024775A cryopreservation solution). The cryopreservation operation of each comparative cryopreservation solution was carried out in accordance with its product instructions or patent application specifications.

[0161] Three parallel experimental groups were conducted. Cells were harvested 72 hours after inoculation, and the cell recovery rate and viability were calculated and averaged as follows. The results are shown in Tables 1 and 2. Cells that did not stain with trypan blue were considered viable cells.

[0162] Recovery rate = (Number of cells with cellular structure after recovery / Number of cells with cellular structure before cryopreservation) * 100%;

[0163] Viability = Number of viable cells after resuscitation / Number of cells with cellular structure after resuscitation * 100%.

[0164] Table 1. Recovery rate and viability of umbilical cord mesenchymal stem cells

[0165]

[0166] Table 2. Recovery rate and viability of adipose-derived mesenchymal stem cells

[0167]

[0168]

[0169] As shown in Tables 1 and 2, umbilical cord mesenchymal stem cells cryopreserved using the cell cryopreservation solution of this invention do not require gradient cooling, and without the use of DMSO and serum, the average cell recovery rate reaches 86.9%, significantly better than traditional cryopreservation solutions, US P, Japanese Q, and CN110024775A. The average viability reaches 97.5%, also significantly better than the other four cryopreservation solutions. The cell cryopreservation solution of this invention also has a similar cryopreservation effect on adipose-derived mesenchymal stem cells as on umbilical cord mesenchymal stem cells, with a recovery rate of 87.5% and a viability of 97.3%. After 72 hours of culture, the cells exhibit a short spindle-shaped stem cell morphology, arranged in a regular whorl pattern, indicating good cell condition. This cryopreservation solution has a good cryopreservation effect on mesenchymal stem cells. In this example, the microscopic cell images of umbilical cord mesenchymal stem cells thawed by various cryopreservation solutions after 72 hours are as follows. Figures 1-5 72 hours after resuscitation, adipose-derived mesenchymal stem cells were observed under a microscope. Figures 6-10 .

[0170] Example 5

[0171] Cryopreservation solutions A, B, C, D, E, and F were prepared, all of which, compared to the cryopreservation solution of Example 2, only lacked hydroxyethyl starch. Cryopreservation solutions B, C, D, E, and F were also prepared, each lacking only hydroxyethyl starch, dextran-40, glycerol, and propylene glycol. Human umbilical cord mesenchymal stem cells to be cryopreserved were digested using conventional trypsin digestion. Cells were counted, centrifuged to remove the supernatant, and resuspended in the cryopreservation solution of Example 2, as well as the aforementioned cryopreservation solutions A, B, C, D, E, and F, respectively, adjusting the density to 1.47 × 10⁻⁶ cells / mL. 6 / mL, cryopreserved in 1.5mL cryovials, 1mL per tube, directly placed at -80℃, and then directly transferred to liquid nitrogen for storage after 12 hours. For cell resuscitation, cells were incubated in a 37℃ water bath for 1–2 minutes, fresh culture medium was added, centrifuged to remove the cryoprotectant, and the cells were resuspended in fresh culture medium and seeded into T25 cell culture flasks, with 200,000 cells per flask. Cells were harvested after 72 hours, and the fold increase and viability were calculated. The fold increase was calculated as: harvested cells / seeded cells.

[0172] The comparison results of cryopreservation solutions A and B and Example 2 are shown in Table 3 below. Microscopic examination of umbilical cord mesenchymal stem cells thawed in cryopreservation solutions A and B after 72 hours shows... Figure 11 and 12 The cells were sparse, irregular in shape, and in poor condition.

[0173] Table 3. Expansion fold and viability of umbilical cord mesenchymal stem cells

[0174] Group Cell number Expansion fold Viability Cryopreservation solution A 880000 4.4 92.30 Cryopreservation solution B 766000 3.83 85.10％ Example 2 cryopreservation solution 1552000 7.76 97.10％

[0175] The comparison results of cryopreservation solutions C, D, E, F and Example 2 are shown in Table 4 below. Microscopic observations of umbilical cord mesenchymal stem cells thawed in cryopreservation solutions C, D, E, and F after 72 hours show... Figures 13-16 The cells were sparse and irregular in shape, not the typical spindle-shaped morphology of mesenchymal stem cells, and the cell arrangement was not the typical whorl arrangement of mesenchymal stem cells, indicating that the cells were in poor condition.

[0176] Table 4. Expansion fold and viability of umbilical cord mesenchymal stem cells

[0177] Cell number Expansion fold Viability Cryopreservation solution C 328000 1.64 77.60％ Cryopreservation solution D 336000 1.68 73.80％ Cryopreservation solution E 400000 2.00 70.4％ Cryopreservation solution F 653000 3.27 89.7％ Example 2 cryopreservation solution 1552000 7.76 97.10％

[0178] The above results indicate that the extracellular protectants hydroxyethyl starch and dextran-40 contained in the cryopreservation solution of this invention have a synergistic effect; the intracellular protectants mannitol, propylene glycol and glycerol also have a synergistic effect, which is reflected in the higher amplification fold and higher viability of the first passage (72h) after thawing.

[0179] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cryopreservation solution for mesenchymal stem cells, characterized in that, It consists of the following components: Sodium chloride 9000 mg / L; Disodium hydrogen phosphate·7H2O 726mg / L; Potassium dihydrogen phosphate 210 mg / L; Glycine 5000–20000 mg / L; L-cysteine ​​hydrochloride 1000~10000 mg / L; L-cystine hydrochloride 5000~10000 mg / L; L-glutamine 1000~10000 mg / L; L-Lysine hydrochloride 5000~20000 mg / L; L-proline 10000~40000 mg / L; p-Hydroxybenzoic acid 0.5–1.5 mg / L; Glucose 1000–5000 mg / L; Ascorbic acid 50-200 mg / L; Vitamin E 20-100 mg / L; Glutathione 50–200 mg / L; Mannitol 20000~70000 mg / L; Propylene glycol 20,000–80,000 mg / L; Glycerin 20,000–70,000 mg / L; Hydroxyethyl starch 10000~80000 mg / L; Dextran-40 20000~100000 mg / L; Poloxam-188, by volume percentage: 0.02–0.2%; water.

2. The mesenchymal stem cell cryopreservation solution as described in claim 1, characterized in that, The water is ultrapure water.

3. The mesenchymal stem cell cryopreservation solution as described in claim 1, characterized in that, If necessary, adjust the pH of the cryopreservation solution to 7.1-7.4 using a 4M aqueous solution of HCl and / or NaOH.

4. The mesenchymal stem cell cryopreservation solution as described in claim 1, characterized in that, In the cryopreservation solution: Sodium chloride 9000 mg / L; Disodium hydrogen phosphate·7H2O 726mg / L; Potassium dihydrogen phosphate 210 mg / L; Glycine 12500 mg / L; L-cysteine ​​hydrochloride 5000 mg / L; L-cystine hydrochloride 7500 mg / L; L-glutamine 5000 mg / L; L-Lysine hydrochloride 12500 mg / L; L-proline 20000 mg / L; p-Hydroxybenzoic acid 1.0 mg / L; Glucose 3500 mg / L; Ascorbic acid 125 mg / L; Vitamin E 60mg / L; Glutathione 125 mg / L; Mannitol 45000 mg / L; Propylene glycol 50000 mg / L; Glycerin 45000 mg / L; Hydroxyethyl starch 45000 mg / L; Dextran-40 60000 mg / L; Polosham-188 is 0.1% by volume; The remainder is water, and the pH is adjusted to 7.2 using a 4M aqueous solution of HCl and / or NaOH.

5. The mesenchymal stem cell cryopreservation solution as described in claim 4, characterized in that, The water is ultrapure water.

6. The method for preparing the mesenchymal stem cell cryopreservation solution according to any one of claims 1-5, characterized in that, The process includes the following steps: dissolve all components except water in water, measure the pH, and adjust the pH to 7.1–7.4 with 4M HCl and / or NaOH aqueous solution if necessary. Filter the solution through a 0.22μm filter membrane for sterilization.

7. The use of the mesenchymal stem cell cryopreservation solution as described in any one of claims 1-5 in the cryopreservation of mesenchymal stem cells.

8. A method for cryopreserving mesenchymal stem cells, characterized in that, Mesenchymal stem cells were cryopreserved using the cryopreservation solution for mesenchymal stem cells as described in any one of claims 1-5.

9. The cryopreservation method as described in claim 8, characterized in that, Mesenchymal stem cells were suspended in the cryopreservation solution of any one of claims 1-5, and first stored at -80°C for 12 hours, and then stored in liquid nitrogen.

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