Serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation liquid

Abstract

The invention relates to the technical field of cell culture, and discloses a serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution. The invention discloses a serum-free and DMSO (dimethyl sulfoxide)-free human umbilical cord mesenchymal stem cell cryopreservation solution. The serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution is prepared from the following components: ethylene glycol with the final concentration of 1000 to 1500 mmol / L, trehalose with the final concentration of 50 to 150 mmol / L, proline with the final concentration of 50 to 150 mmol / L and 5 to 15 percent of human serum albumin injection. The invention further discloses application of the cell cryopreservation liquid in cryopreservation of cells. The serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation liquid disclosed by the invention does not contain a dimethyl sulfoxide (DMSO) component and a basic culture medium component, the components are clear, the batch is stable, and the serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation liquid meets the industrial transformation requirements. Besides, the activity and dryness of the cells can be stably maintained for a long time when the cells are cryopreserved, the recovered cells can be directly used for clinical application, and the safety requirement and the effectiveness requirement of the clinical application are met.

Description

Technical Field

[0001] This invention relates to the field of cryopreservation solution preparation technology, specifically to a method for preparing a serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution. Background Technology

[0002] Human umbilical cord mesenchymal stem cells (UC-MSCs) are pluripotent stem cells found in the umbilical cord tissue of newborns. They possess a strong self-renewal capacity and can differentiate into many types of tissue cells. UC-MSCs have the following advantages: 1. Self-renewal, rapid proliferation, stable passage, and rapid cell growth after passage; 2. Low immunogenicity: UC-MSCs do not express histocompatibility antigens such as HLA-DR, HLA-DP, HLA-DA, and surface co-stimulatory molecules CD80, CD86, and CD40, but express immunosuppressive factors such as HLA-G, indoleamine 2,3-peroxidase (IDO), PGE2, IL-10, IL-6, vascular endothelial growth factor (VEGF), and TGF-β. These factors give UC-MSCs low immunogenicity and the ability to induce immune tolerance; 3. Potential for induced differentiation: UC-MSCs can be induced into adipocytes, chondrocytes, osteocytes, neurons, cardiomyocytes, etc., in appropriate induction media.

[0003] In traditional medicine, umbilical cords are considered waste and often disposed of carelessly. However, the ISO / TC276 International Biotechnology Committee has clearly defined their new value: through collection and specific processing, umbilical cords can be transformed into valuable biological resources, figuratively referred to as "seeds of life" in life banks. Globally, umbilical cord mesenchymal stem cells have been widely used in various clinical studies, demonstrating significant potential, particularly in the treatment of common and intractable diseases. These diseases include, but are not limited to, arthritis, stroke, liver disease, diabetes, and cardiovascular disease. Clinical research results indicate that umbilical cord mesenchymal stem cells have significant therapeutic effects in promoting tissue repair, regulating immune responses, and improving disease symptoms.

[0004] Fresh cell products have a short shelf life. To extend the shelf life or preserve cell products long-term, cell cryopreservation is typically performed, stored in liquid nitrogen at -80°C or -196°C. The cryopreservation process requires cryoprotective solutions to minimize cell damage. Cryoprotectants are generally classified into two main types based on whether they penetrate the cell membrane: permeable cryoprotectants and non-permeable cryoprotectants. Permeable cryoprotectants are mostly small-molecule neutral substances, including glycerol, DMSO, propylene glycol, ethylene glycol, acetamide, and methanol. Non-permeable cryoprotectants are mostly large-molecule substances, including trehalose, polyethylene glycol, hydroxyethyl starch, polyvinylpyrrolidone (PVP), dextran, and albumin.

[0005] DMSO is the most commonly used permeable cryoprotectant, widely used for cell cryopreservation. At the cellular level, in addition to being a DNA teratogen, DMSO can penetrate into cells, and the S=O bonds in its molecule may react chemically with intracellular proteins, causing protein denaturation. Clinically, the use of cells cryopreserved with DMSO can cause widespread physical discomfort, the most common being diarrhea (approximately 50%), and in severe cases, it can cause damage to the nervous and respiratory systems or even death (0.2%).

[0006] Therefore, there is an urgent need to find a new type of serum-free, DMSO-free, no-wash, no-procedure cooling clinical-grade cell cryopreservation solution. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a serum-free, DMSO-free, wash-free, and temperature-controlled clinical-grade cell cryopreservation solution. To this end, this invention proposes a serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution, its preparation method, and its application. This stem cell cryopreservation solution avoids the toxic side effects of DMSO in traditional stem cell cryopreservation solutions and avoids the risk of exogenous viral factors introduced from serum / animal-derived protein components in traditional stem cell cryopreservation solutions. Furthermore, it reduces the need for washing cryopreservation solutions in a clean laboratory before clinical use of traditional cryopreserved cells, preventing potential microbial contamination risks during washing, reducing safety hazards, and simplifying the clinical use process of umbilical cord mesenchymal stem cell products.

[0008] This invention also proposes a method for preparing a serum-free, DMSO-free cryopreservation solution for human umbilical cord mesenchymal stem cells.

[0009] This invention also proposes the application of a serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution in the cryopreservation of umbilical cord mesenchymal stem cells.

[0010] This invention also proposes a method for cryopreservation of umbilical cord mesenchymal stem cells.

[0011] A first aspect of the present invention provides a serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution, comprising the following components: Ethylene glycol 1000~1500 mmol / L Trehalose 50~150 mmol / L L-proline 50~150 mmol / L Human serum albumin 5%~15% Remaining amount of solvent for cell injection The solvent for cell injection is selected from at least one of compound electrolyte injection, sodium chloride injection, Normosol-R injection, dextran 40 glucose injection, compound dextran 40 injection, and pulex A injection.

[0012] The clinical-grade umbilical cord mesenchymal stem cell cryopreservation solution according to embodiments of the present invention has at least the following beneficial effects: (1) The serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution of the present invention does not contain DMSO, which can reduce the toxic side effects of DMSO in traditional stem cell cryopreservation solutions on the human body, such as gastrointestinal adverse reactions, cardiovascular and respiratory adverse reactions, skin disease reactions, nervous system reactions, urinary and reproductive system reactions, etc.

[0013] (2) The serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution of the present invention does not contain basic culture medium components, which can reduce the need for traditional cryopreserved cells to be washed in a clean laboratory before clinical use, prevent the risk of microbial contamination that may be caused by the washing process, reduce safety hazards, and simplify the clinical use process of stem cell products.

[0014] (3) The serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution of the present invention contains ethylene glycol and trehalose. Ethylene glycol, as a biomimetic ice-controlling material, can combine with water molecules to form hydrates during the freezing process. Trehalose, as a vitrification freezing component, has high solubility and low freezing point. The two work together to significantly reduce the activity of water and inhibit the growth rate of ice crystals, which is crucial for maintaining cell viability.

[0015] In some embodiments of the present invention, the solvent for cell injection is selected from at least one of compound electrolyte injection, sodium chloride injection, Normosol-R injection, dextran 40 glucose injection, compound dextran 40 injection, and pulex A injection.

[0016] In some embodiments of the present invention, the clinical-grade umbilical cord mesenchymal stem cell cryopreservation solution further comprises a pH adjuster. The pH adjuster is used to adjust the pH value of the stem cell cryopreservation solution to a suitable storage pH.

[0017] In some embodiments of the present invention, the pH adjuster includes, but is not limited to, sodium chloride and sodium hydroxide.

[0018] In some embodiments of the present invention, the pH value of the serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution is 7.2 to 7.6.

[0019] In a second aspect, the present invention provides a method for preparing a serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution, wherein the components of the serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution described in any one of the first aspects are mixed and then filtered for sterilization.

[0020] The preparation method according to embodiments of the present invention has at least the following beneficial effects: The present invention provides a simple and low-cost process for preparing a serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution, which is suitable for mass production.

[0021] In some embodiments of the present invention, the method for preparing a serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution includes the following steps: S1. Dissolve trehalose and L-proline in an appropriate amount of cell injection solvent to obtain trehalose solution and L-proline solution, respectively. S2. Mix the trehalose solution, L-proline solution, ethylene glycol, human serum albumin, and the remaining cell injection solvent separately, and then filter them under sterile conditions to obtain the final product.

[0022] In some embodiments of the present invention, the pH is adjusted to 7.2-7.6 before the sterilization filtration.

[0023] A serum-free, DMSO-free cryopreservation solution for human umbilical cord mesenchymal stem cells according to an embodiment of the present invention has at least the following beneficial effects: The present invention provides a serum-free, DMSO-free cryopreservation solution for human umbilical cord mesenchymal stem cells that can be cryopreserved at low temperatures and can be directly used for clinical injection after thawing.

[0024] A fourth aspect of the present invention provides the application of the serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution described in the first aspect in the cryopreservation of stem cells.

[0025] A fifth aspect of the present invention provides a method for cryopreservation of stem cells, comprising the following steps: The serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution described in any of the first aspects is mixed with umbilical cord mesenchymal stem cells at a ratio of 1 mL: 5 × 10⁻⁶. 6 ~12×10 6 The mixtures were mixed in proportion to a certain ratio, then placed at -75 to -80°C for 6 to 12 hours, and then transferred to liquid nitrogen for storage.

[0026] The method for cryopreservation of human umbilical cord mesenchymal stem cells according to embodiments of the present invention has at least the following beneficial effects: The human umbilical cord mesenchymal stem cell cryopreservation method of the present invention does not require programmed cooling during cell cryopreservation and does not require washing after cell thawing. It can not only avoid the introduction of exogenous unknown components into stem cell products, reduce safety risks, and ensure product safety, but also greatly simplify the cryopreservation cooling process.

[0027] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The viability of human umbilical cord mesenchymal stem cells before and after 3, 6, and 12 months of treatment with the cryopreservation solution of Examples 1-3 and Comparative Example 1 of the present invention was measured.

[0029] Figure 2 The apoptosis rate of human umbilical cord mesenchymal stem cells after treatment with cryopreservation solution of Examples 1-3 and Comparative Example 1 for 3 months, 6 months and 12 months was determined.

[0030] Figure 3 The proliferation capacity of human umbilical cord mesenchymal stem cells after 12 months of treatment with cryopreservation solution of Examples 1-3 and Comparative Example 1 was measured.

[0031] Figure 4 The morphological changes of human umbilical cord mesenchymal stem cells after 12 months of treatment with cryopreservation solution of human umbilical cord mesenchymal stem cells in Examples 1-3 and Comparative Example 1 of this invention were investigated. Detailed Implementation

[0032] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0033] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0034] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0035] The sources of the active ingredients in the products involved in the following examples and comparative examples are as follows (only the active ingredients are shown; other necessary excipients contained in commercially available raw materials are not described): Ethylene glycol was purchased from Jinan Gerunda Pharmaceutical Technology Co., Ltd.; dextran 40 glucose injection (containing 6% dextran 40 and 5% glucose) was purchased from Shandong Lukang Chenxin Pharmaceutical Co., Ltd.; trehalose was purchased from Xi'an Jinxiang Pharmaceutical Excipients Co., Ltd.; L-proline was purchased from Chengdu Anminotai Pharmaceutical Technology Co., Ltd.; human serum albumin (20% 50mL*10g) was purchased from Instituto Grifols, SA; Normosol-R injection was purchased from Nanjing Yixun Biotechnology Co., Ltd.; fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; DMSO was purchased from Sigma-Aldrich; and SUPERCULTURE MSC-specific basal culture medium (type B) was purchased from Shenzhen Dakwei Biotechnology Co., Ltd. Example 1

[0036] This embodiment provides a serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution, which contains the components shown in Table 1.

[0037] Table 1: Components of cell cryopreservation solution Components Final concentration Ethylene glycol 1250 mmol / L Trehalose 100 mmol / L L-proline 100 mmol / L Human serum albumin 10% Dextran 40 glucose injection 10% Normosol-R injection Balance (constant volume) This embodiment also provides a method for preparing serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution, including the following steps: S1. Dissolve trehalose and L-proline in an appropriate amount of Normosol-R injection solution to obtain trehalose solution and L-proline solution with a final concentration of 100 mmol / L, respectively. S2. According to the final concentration in Example 1, a certain amount of trehalose solution, L-proline solution, ethylene glycol, human serum albumin solution and remaining cell injection solvent are mixed evenly.

[0038] S3. Adjust the pH of the mixed solution obtained in step S2 to 7.2-7.6 with 1 mol / L NaOH solution, and then filter to remove bacteria to obtain the final product.

[0039] Example 2 This embodiment provides a method for preparing a serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution, which contains the components shown in Table 2.

[0040] Table 2: Components of cell cryopreservation solution Components Final concentration Ethylene glycol 1500 mmol / L Trehalose 150 mmol / L L-proline 150 mmol / L Human serum albumin 10% Dextran 40 glucose injection 10% Normosol-R injection Balance (constant volume) This embodiment also provides a method for preparing serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution. The specific preparation method is as described in Example 1, and the content of each component is shown in Table 2.

[0041] Example 3 This embodiment provides a method for preparing a serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution, which contains the components shown in Table 3.

[0042] Table 3: Components of Cell Cryopreservation Solution Components Final concentration Ethylene glycol 1000 mmol / L Trehalose 50 mmol / L L-proline 50 mmol / L Human serum albumin 10% Dextran 40 glucose injection 10% Normosol-R injection Balance (constant volume) This embodiment also provides a method for preparing serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution. The specific preparation method is as described in Example 1, and the content of each component is shown in Table 3.

[0043] Comparative Example 1 This comparative example provides a conventional cell cryopreservation solution containing serum and DMSO, which contains the components shown in Table 4 by volume percentage.

[0044] Table 4: Components of Cell Cryopreservation Solution Components Final concentration Superculture MSC-specific basal medium (Type B) 70% fetal bovine serum 20 DMSO 10 This comparative example describes a method for preparing a conventional cell cryopreservation solution containing serum and DMSO, comprising the following steps: S1. Take 7 portions of SUPERCULTURE MSC-specific basal culture medium (type B), 2 portions of fetal bovine serum and 1 portion of DMSO and mix them evenly; S2. After filtering the mixture obtained in step S1 through a 0.22μm needle filter, a cell cryopreservation solution containing 20% ​​animal serum and 10% DMSO is prepared. S3. Store the sterile cell cryopreservation solution obtained in step S2 at 4°C for later use.

[0045] Detection 1: Cell viability and apoptosis rate detection Human umbilical cord mesenchymal stem cells were used as test cells. Serum-free and DMSO-free cryopreservation solutions from Examples 1-3 were used as test cryopreservation solutions, while a conventional cell cryopreservation solution containing serum and DMSO from Comparative Example 1 was used as a control cryopreservation solution. Human umbilical cord mesenchymal stem cells frozen for 3 months, 6 months, and 12 months were thawed according to the cryopreservation and thawing methods described in this invention. Cell viability was measured, and the preservation of cell activity by different cryopreservation solutions was compared. The specific method includes the following steps: (1) Using SUPERCULTURE MSC-specific basal medium (type B) and UltraGRO-Advanced cell nutrient additive (addition amount 5%), human umbilical cord mesenchymal stem cells of generation P4 were expanded and cultured at 37℃ and 5% CO2, and then the cells were collected. (2) The cell cryopreservation solutions of Examples 1-3 and Comparative Example 1 were respectively mixed with the collected human umbilical cord mesenchymal stem cells at a ratio of 1 mL: 5 × 10⁻⁶ mcg / mL. 6 ~12×10 6 The mixtures were mixed in proportion to a certain ratio, then individually packaged and frozen, with each frozen container measuring approximately 10 x 10. 6 One vial / 1mL / vial; (3) The above-mentioned pre-packaged human umbilical cord mesenchymal stem cells to be cryopreserved were transferred to a cryopreservation box, and then placed directly at -80℃ overnight using non-programmed cooling before being transferred to liquid nitrogen for cryopreservation. (4) Cells were revived after 3, 6, and 12 months of cryopreservation. Four 50 mL centrifuge tubes were labeled as T1, T2, T3, and C1, respectively. During revival, 20 mL of 0.9% sodium chloride injection solution was first transferred to a 50 mL centrifuge tube, and the cryopreserved cells were placed in a 37°C water bath. After the cells thawed, they were added to the corresponding centrifuge tubes for resuspending. After balancing, the mixture was centrifuged at 400 g for 5 min, the supernatant was discarded, and 20 mL of complete culture medium was added to the precipitate and mixed well. 20 μL of cell suspension and 20 μL of trypan blue solution (0.2%) were mixed and then analyzed by a Countstar cell fluorescence analyzer to obtain cell viability and cell concentration.

[0046] The cell viability test results are shown in Table 5.

[0047] The test results showed that when human umbilical cord mesenchymal stem cells were preserved using the serum-free and DMSO-free cryopreservation solution of the present invention, they still had a viability of over 90% after 12 months of preservation. Among them, when the serum-free and DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution of Example 1 was used for preservation, the cell viability after 12 months of cryopreservation was close to that of Comparative Example 1.

[0048] The results of the apoptosis rate detection are shown in Table 6.

[0049] The test results showed that when human umbilical cord mesenchymal stem cells were preserved using the serum-free and DMSO-free cryopreservation solution of the present invention, the cell apoptosis rate was between 13.76% and 18.35% after 3 months, 6 months and 12 months of preservation. The cryopreservation effect of Examples 1 and 2 was better than that of Example 3, but slightly worse than that of Comparative Example 1.

[0050] Test 2: Cell morphology and proliferation capacity detection This test example examines the cell morphology and proliferation capacity of human umbilical cord mesenchymal stem cells preserved using the cell cryopreservation solutions of Examples 1-3 and Comparative Example 1. The specific methods include the following steps: S1. Remove the cryopreservation tube containing human umbilical cord mesenchymal stem cells from the liquid nitrogen tank and thaw it quickly in a water bath at 37~38 ℃. S2. Transfer the cryopreservation solution containing human umbilical cord mesenchymal stem cells into a 50 mL centrifuge tube, add 0.9% sodium chloride injection, and centrifuge for 5 min. S3. Discard the supernatant, add an appropriate amount of complete culture medium, and distribute it evenly into several T175 culture flasks at an appropriate passage ratio. Perform cell counting after 3 days.

[0051] The cell count results are shown in Table 8.

[0052] Group Example 1 Example 2 Example 3 Comparative Example 1 <![CDATA[Quantity (×10 7 )]]> 1.29±0.07 1.21±0.13 1.07±0.06 1.04±0.03 After 3 days of culture, the cell count in Comparative Example 1 was (1.29 ± 0.07) × 10⁻⁶. 7 The number of samples was significantly higher than that of control sample 1 (1.04 ± 0.03) × 10⁻⁶. 7 indivual( P <0.05), and the cell proliferation fold was 4.21. Simultaneously, microscopic examination revealed that the human umbilical cord mesenchymal stem cells preserved for 12 months using the cell cryopreservation solutions of Examples 1-3 and Comparative Example 1 exhibited normal adhesion, proliferation, and morphology, with no significant cell aging or morphological abnormalities observed.

[0053] Detection 3: Cell Flow Cytometry Analysis This test example performs flow cytometry analysis on umbilical cord mesenchymal stem cells preserved using the cell cryopreservation solutions of Examples 1-3 and Comparative Example 1, respectively. The specific method includes the following steps: S1. Using serum-free umbilical cord mesenchymal stem cell culture medium, P4 generation human umbilical cord mesenchymal stem cells were expanded and cultured at 37℃ and 5% CO2, and then the cells were collected. S2. The cell cryopreservation solutions of Examples 1-3 and Comparative Example 1 were respectively mixed with the collected human umbilical cord mesenchymal stem cells at a ratio of approximately 1 mL: 10 × 10⁻⁶. 6 The mixtures were mixed in proportion to a certain ratio, then individually packaged and frozen, with each frozen container measuring approximately 10 x 10. 6 One vial / 1mL / vial; S3. Transfer the pre-packaged human umbilical cord mesenchymal stem cells to a cryopreservation box, use non-programmed cooling, place them directly at -80℃ overnight, and then transfer them to liquid nitrogen for cryopreservation. S4. After cryopreservation for 3, 6, and 12 months, cells were thawed, and the expression of surface markers of umbilical cord mesenchymal stem cells after cryopreservation and thawing was analyzed by flow cytometry. The expression of cell surface marker CD105 was also statistically analyzed. + CD73 + CD90 + CD34 + and CD45 + Expression level.

[0054] The statistical results of cell flow cytometry analysis are shown in Table 7.

[0055] Indicator Groups <![CDATA[CD105 + ]]> <![CDATA[CD73 + ]]> <![CDATA[CD90 + ]]> <![CDATA[CD34 + ]]> <![CDATA[CD45 + ]]> Example 1 99.87% 99.82% 99.78% 0.01% 0.02% Example 2 99.91% 99.84% 99.89% 0.01% 0.01% Example 3 99.78% 99.81% 99.91% 0.02% 0.03% Comparative Example 1 99.82% 99.86% 99.84% 0.01% 0.02% The above flow cytometry analysis results showed that after cryopreservation of human umbilical cord mesenchymal stem cells in Examples 1-3 and Comparative Example 1, all positive markers (CD105) were reduced. + CD73 + CD90 + ) and negative markers (CD34) + CD45 + All samples met the identification criteria for umbilical cord mesenchymal stem cells established by the International Cell Therapy Association. Overall, compared to the comparative example, there was no significant difference in the expression rates of positive and negative cell surface markers in human umbilical cord mesenchymal stem cells. Cell surface markers are related to specific cell functions, further demonstrating that the serum-free, DMSO-free cryopreservation solution for human umbilical cord mesenchymal stem cells of this invention does not affect the specific functions of human umbilical cord mesenchymal stem cells.

Claims

1. A serum-free, DMSO-free cryopreservation solution for human umbilical cord mesenchymal stem cells, characterized in that, The serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution is composed of the following components: Ethylene glycol 1000~1250 mmol / L Trehalose 50~100 mmol / L Proline 50~100 mmol / L Human serum albumin 5%~10% Remaining amount of solvent for cell injection.

2. The serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution according to claim 1, characterized in that, The solvent for cell injection is selected from at least one of compound electrolyte injection, sodium chloride injection, Normosol-R injection, dextran 40 glucose injection, compound dextran 40 injection, and pulex A injection.

3. The serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution according to claim 2, characterized in that, The pH value of the serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution is 7.2-7.

6.

4. A method for preparing a serum-free, DMSO-free cryopreservation solution for human umbilical cord mesenchymal stem cells, characterized in that, The components of the serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution according to any one of claims 1 to 3 are mixed and then filtered for sterilization to obtain the solution.

5. A serum-free, DMSO-free cryopreservation solution for human umbilical cord mesenchymal stem cells, characterized in that, It comprises a serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution and human umbilical cord mesenchymal stem cells as described in any one of claims 1 to 4.

6. A method for cryopreservation of stem cells, characterized in that, Includes the following steps: The serum-free, DMSO-free human umbilical cord mesenchymal stem cell cryopreservation solution according to any one of claims 1 to 5 is mixed with human umbilical cord mesenchymal stem cells at a ratio of 1 mL: 5 × 10⁻⁶. 6 ~12×10 6 The mixtures were mixed in a specific ratio, then placed at -75 to -80°C for 6 to 12 hours, and then transferred to liquid nitrogen for storage.

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