Mesenchymal stem cell cryopreservation solution and method of preparation thereof

Abstract

This invention provides a cryopreservation solution for mesenchymal stem cells and its preparation method. The cryopreservation solution comprises the following components at the following concentrations: 60-70 g / L trehalose, 50-70 g / L hydroxyethyl starch, 40-60 mg / L L-ascorbic acid, 40-60 g / L albumin, 20-25 mg / L human insulin, 15-20 μg / L basic fibroblast growth factor, 5-20 μg / L epidermal growth factor, 10-50 μg / L platelet-derived growth factor, and basal culture medium. This invention optimizes the composition of the cryopreservation solution by removing DMSO and FBS, using natural components with good biocompatibility and low toxicity, and supplementing them with growth factors that aid in cell recovery and functional maintenance. This significantly improves the cell survival rate, functional recovery, and biocompatibility after cryopreservation and thawing.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to a mesenchymal stem cell cryopreservation solution and its preparation method. Background Technology

[0002] Mesenchymal stem cell cryopreservation solutions are liquid solutions specifically designed for cryopreserving cells, primarily to protect their viability at low temperatures. Because mesenchymal stem cells (MSCs) have poor tolerance to low temperatures and are easily damaged by ice crystals during freezing, cryopreservation solutions help MSCs survive this process through various components and maintain their activity and function after thawing. Current MSC cryopreservation solutions include dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS). DMSO is often used in conjunction with FBS as a cryopreservation agent to protect cell membranes and improve cell viability after thawing. However, DMSO is toxic, and FBS may contain bovine spongiform encephalopathy (BSE) virus, posing potential risks in clinical applications. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a mesenchymal stem cell cryopreservation solution and its preparation method, which significantly improves the survival rate, functional recovery, and biocompatibility of mesenchymal stem cells after cryopreservation and thawing.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] First, the present invention provides a mesenchymal stem cell cryopreservation solution comprising the following components at the following concentrations: 60-70 g / L trehalose, 50-70 g / L hydroxyethyl starch, 40-60 mg / L L-ascorbic acid, 40-60 g / L albumin, 20-25 mg / L human insulin, 15-20 μg / L basic fibroblast growth factor, 5-20 μg / L epidermal growth factor, 10-50 μg / L platelet-derived growth factor, and basal culture medium.

[0006] Preferably, the concentration of trehalose is 65 g / L, the concentration of hydroxyethyl starch is 60 g / L, the concentration of L-ascorbic acid is 50 mg / L, the concentration of albumin is 50 g / L, the concentration of human insulin is 25 mg / L, the concentration of basic fibroblast growth factor is 20 μg / L, the concentration of epidermal growth factor is 15 μg / L, and the concentration of platelet-derived growth factor is 50 μg / L.

[0007] Preferably, the basal culture medium is DMEM / F12 medium.

[0008] Secondly, the present invention provides a method for preparing the mesenchymal stem cell cryopreservation solution, comprising the following steps:

[0009] Trehalose, hydroxyethyl starch, L-ascorbic acid, albumin, human insulin, basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor were mixed into the culture medium to obtain a mixture.

[0010] Adjust the pH of the mixture to 7.4-7.6, filter, and obtain the mesenchymal stem cell cryopreservation solution.

[0011] Preferably, after adjusting the pH value of the mixture, it is filtered and sterilized to obtain a mesenchymal stem cell cryopreservation solution.

[0012] Preferably, the filter is a 0.22μm filter.

[0013] Finally, the present invention provides a method for using a mesenchymal stem cell cryopreservation solution, wherein the mesenchymal stem cell cryopreservation solution is mixed with mesenchymal stem cells and then frozen for preservation.

[0014] Preferably, the freezing temperature is -196°C.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] (1) By optimizing the composition of the cryopreservation solution, the present invention removes DMSO and FBS and uses natural components with good biocompatibility and low toxicity. At the same time, it supplements growth factors that help cell recovery and function maintenance, thereby significantly improving the survival rate, functional recovery and biocompatibility of mesenchymal stem cells after cryopreservation and thawing.

[0017] (2) The growth factors of the present invention include epidermal growth factor and platelet-derived growth factor. Epidermal growth factor helps cell proliferation and repair and can promote the recovery process of mesenchymal stem cells after cryopreservation. Platelet-derived growth factor can promote tissue repair, cell migration and proliferation and can support the survival and regeneration of mesenchymal stem cells. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0019] Example 1

[0020] A mesenchymal stem cell cryopreservation solution is provided, comprising the following components at the following concentrations: 60 g / L trehalose, 50 g / L hydroxyethyl starch, 40 mg / L L-ascorbic acid, 40 g / L albumin, 20 mg / L human insulin, 15 μg / L basic fibroblast growth factor, 5 μg / L epidermal growth factor, 10 μg / L platelet-derived growth factor, and basal culture medium.

[0021] The method for preparing mesenchymal stem cell cryopreservation solution is as follows: 60 g / L trehalose, 50 g / L hydroxyethyl starch, 40 mg / L L-ascorbic acid, 40 g / L albumin, 20 mg / L human insulin, 15 μg / L basic fibroblast growth factor, 5 μg / L epidermal growth factor, and 10 μg / L platelet-derived growth factor are mixed into the culture medium to obtain a mixture; the pH of the mixture is adjusted to 7.4-7.6, and then filtered through a 0.22 μm filter for sterilization to obtain the mesenchymal stem cell cryopreservation solution, which is then stored at 20°C in the dark.

[0022] Example 2

[0023] A mesenchymal stem cell cryopreservation solution is provided, comprising the following components at the following concentrations: 65 g / L trehalose, 60 g / L hydroxyethyl starch, 50 mg / L L-ascorbic acid, 50 g / L albumin, 23 mg / L human insulin, 18 μg / L basic fibroblast growth factor, 15 μg / L epidermal growth factor, 35 μg / L platelet-derived growth factor, and basal culture medium.

[0024] The preparation method of the mesenchymal stem cell cryopreservation solution is the same as in Example 1.

[0025] Example 3

[0026] A mesenchymal stem cell cryopreservation solution is provided, comprising the following components at the following concentrations: 70 g / L trehalose, 70 g / L hydroxyethyl starch, 60 mg / L L-ascorbic acid, 60 g / L albumin, 25 mg / L human insulin, 20 μg / L basic fibroblast growth factor, 20 μg / L epidermal growth factor, 50 μg / L platelet-derived growth factor, and basal culture medium.

[0027] The preparation method of the mesenchymal stem cell cryopreservation solution is the same as in Example 1.

[0028] Example 4

[0029] A mesenchymal stem cell cryopreservation solution is provided, comprising the following components at the following concentrations: 65 g / L trehalose, 60 g / L hydroxyethyl starch, 50 mg / L L-ascorbic acid, 50 g / L albumin, 25 mg / L human insulin, 20 μg / L basic fibroblast growth factor, 15 μg / L epidermal growth factor, 50 μg / L platelet-derived growth factor, and basal culture medium.

[0030] The preparation method of the mesenchymal stem cell cryopreservation solution is the same as in Example 1.

[0031] Comparative Example 1

[0032] A mesenchymal stem cell cryopreservation solution is provided, comprising fetal bovine serum (FBS) + 10% dimethyl sulfoxide (DMSO).

[0033] Comparative Example 2

[0034] A mesenchymal stem cell cryopreservation solution is provided, with the same formulation and preparation method as in Example 4, except that epidermal growth factor is not added.

[0035] Comparative Example 3

[0036] A mesenchymal stem cell cryopreservation solution is provided, with the same formulation and preparation method as in Example 4, except that platelet-derived growth factor is not added.

[0037] Comparative Example 4

[0038] A mesenchymal stem cell cryopreservation solution is provided, with the same formulation and preparation method as in Example 4, except that epidermal growth factor and platelet-derived growth factor are not added.

[0039] Performance Tests and Results

[0040] The mesenchymal stem cell cryopreservation solutions prepared in Example 4 and Comparative Examples 1-4 were used to freeze the same amount of tissue at -196°C for 6 months. After thawing, cell viability and survival rate were measured for each cell sample. Cell viability was analyzed using a creatine kinase kit (Sino Biologics Inc.), and the results are expressed as ΔOD values. The results are shown in Table 1. Cell survival rate was detected using trypan blue staining. The results are shown in Table 1.

[0041] Table 1

[0042]

[0043] As shown in Table 1, compared to Comparative Example 1, the mesenchymal stem cell cryopreservation solution prepared in Example 4 exhibited higher cell viability and survival rate during cell preservation. A comparison of Example 4 with Comparative Examples 2-4 shows that the addition of epidermal growth factor and platelet-derived growth factor can improve cell survival rate and viability.

[0044] All other raw materials or structures not specifically described in this invention already exist in the prior art and can be purchased directly from the market.

[0045] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A cryopreservation solution for mesenchymal stem cells, characterized in that, Including the following concentrations of ingredients: 60-70 g / L trehalose, 50-70 g / L hydroxyethyl starch, 40-60 mg / L L-ascorbic acid, 40-60 g / L albumin, 20-25 mg / L human insulin, 15-20 μg / L basic fibroblast growth factor, 5-20 μg / L epidermal growth factor, 10-50 μg / L platelet-derived growth factor, and basal culture medium.

2. The mesenchymal stem cell cryopreservation solution according to claim 1, characterized in that, The concentrations of the trehalose, hydroxyethyl starch, L-ascorbic acid, albumin, human insulin, basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor are all specified. The concentrations of the trehalose, hydroxyethyl starch, L-ascorbic acid, albumin, human insulin, basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor are all specified. The concentration of the trehalose is 65 g / L, the concentration of the hydroxyethyl starch is 60 g / L, the concentration of the hydroxyethyl starch is 15 μg / L, and the concentration of the hydroxyethyl starch is 50 μg / L.

3. The mesenchymal stem cell cryopreservation solution according to claim 1, characterized in that, The basal culture medium is DMEM / F12 medium.

4. The method for preparing mesenchymal stem cell cryopreservation solution according to any one of claims 1-3, characterized in that, Includes the following steps: Trehalose, hydroxyethyl starch, L-ascorbic acid, albumin, human insulin, basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor were mixed into the culture medium to obtain a mixture. Adjust the pH of the mixture to 7.4-7.6, filter, and obtain the mesenchymal stem cell cryopreservation solution.

5. The method according to claim 4, characterized in that, After adjusting the pH of the mixture, it is filtered through a filter to remove bacteria, thus obtaining a mesenchymal stem cell cryopreservation solution.

6. The method according to claim 5, characterized in that, The filter is a 0.22μm filter.

7. The method of using the mesenchymal stem cell cryopreservation solution according to any one of claims 1-3, characterized in that, The mesenchymal stem cell cryopreservation solution was mixed with the mesenchymal stem cells and then cryopreserved.

8. The method of use according to claim 7, characterized in that, The freezing temperature is -196℃.

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