Mesenchymal stem cell cryopreservation liquid and preparation method thereof

Abstract

The invention provides a mesenchymal stem cell cryopreservation solution and a preparation method thereof. The mesenchymal stem cell cryopreservation solution comprises the following components in concentration: 60-70 g / L of trehalose, 50-70 g / L of hydroxyethyl starch, 40-60 mg / L of L-ascorbic acid, 40-60 g / L of albumin, 20-25 mg / L of human insulin, 15-20 [mu] g / L of basic fibroblast growth factors, 5-20 [mu] g / L of epidermal growth factors, 10-50 [mu] g / L of platelet-derived growth factors and a basal culture medium. The components of the cryopreservation liquid are optimized, DMSO and FBS are removed, natural components with good biocompatibility and low toxicity are adopted, and growth factors beneficial to cell recovery and function maintenance are supplemented at the same time, so that the survival rate, function recovery and biocompatibility of cells after cryopreservation and unfreezing are remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of cell biology, and particularly relates to a mesenchymal stem cell freezing solution and a preparation method thereof. Background Art

[0002] Mesenchymal stem cell cryopreservation fluid is a liquid solution specifically used for cryopreservation of cells. Its main function is to protect the viability of cells at low temperatures. Since mesenchymal stem cells have poor tolerance to low temperatures and are easily damaged by ice crystals during the freezing process, the role of the cryopreservation fluid is to help mesenchymal stem cells survive this process through various ingredients and maintain their activity and function after thawing. Current mesenchymal stem cell cryopreservation fluids include dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS). DMSO is often used together with FBS as a cryopreservation agent to protect cell membranes and increase cell activity after recovery. However, DMSO is toxic and FBS may contain mad cow disease virus, which poses potential risks in clinical applications. Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention provides a mesenchymal stem cell cryopreservation solution and a preparation method thereof, which significantly improves the survival rate, functional recovery and biocompatibility of mesenchymal stem cells after cryopreservation and thawing.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: First, the present invention provides a mesenchymal stem cell freezing solution, comprising the following components in 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 a basal culture medium.

[0005] Preferably, the trehalose concentration is 65 g / L, the hydroxyethyl starch concentration is 60 g / L, the L-ascorbic acid concentration is 50 mg / L, the albumin concentration is 50 g / L, the human insulin concentration is 25 mg / L, the basic fibroblast growth factor concentration is 20 μg / L, the epidermal growth factor concentration is 15 μg / L, and the platelet-derived growth factor concentration is 50 μg / L.

[0006] Preferably, the basal culture medium is DMEM / F12 culture medium.

[0007] Secondly, the present invention provides a method for preparing the mesenchymal stem cell cryopreservation solution, comprising the following steps: mixing trehalose, hydroxyethyl starch, L-ascorbic acid, albumin, human insulin, basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor into the culture medium to obtain a mixed solution; The pH value of the mixed solution was adjusted to 7.4-7.6, and the mixture was filtered to obtain a mesenchymal stem cell cryopreservation solution.

[0008] Preferably, after adjusting the pH value of the mixed solution, the mixed solution is filtered and sterilized through a filter to obtain a mesenchymal stem cell cryopreservation solution.

[0009] Preferably, the filter is a 0.22 μm filter.

[0010] 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 cryopreserved.

[0011] Preferably, the cryopreservation temperature is -196°C.

[0012] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention optimizes the composition of the cryopreservation solution, removes DMSO and FBS, adopts natural ingredients with good biocompatibility and low toxicity, and 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 freezing and thawing.

[0013] (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 DESCRIPTION

[0014] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0015] Example 1 Provided is a mesenchymal stem cell freezing solution, comprising the following components in 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 a basal culture medium.

[0016] The method for preparing a mesenchymal stem cell freezing 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 mixed solution; the pH value of the mixed solution is adjusted to 7.4-7.6, and then filtered and sterilized with a 0.22 um filter to obtain a mesenchymal stem cell freezing solution, and the solution is stored at 20° C. in the dark.

[0017] Example 2 Provided is a mesenchymal stem cell freezing solution, comprising the following components in 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 a basal culture medium.

[0018] The preparation method of mesenchymal stem cell cryopreservation solution is the same as that in Example 1.

[0019] Example 3 Provided is a mesenchymal stem cell freezing solution, comprising the following components in 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 a basal culture medium.

[0020] The preparation method of mesenchymal stem cell cryopreservation solution is the same as that in Example 1.

[0021] Example 4 Provided is a mesenchymal stem cell freezing solution, comprising the following components in 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 a basal culture medium.

[0022] The preparation method of mesenchymal stem cell cryopreservation solution is the same as that in Example 1.

[0023] Comparative Example 1 Provided is a mesenchymal stem cell freezing solution comprising fetal bovine serum (FBS) + dimethyl sulfoxide (DMSO) with a mass fraction of 10%.

[0024] Comparative Example 2 A mesenchymal stem cell cryopreservation solution is provided, the formula and preparation method of which are the same as those of Example 4, except that epidermal growth factor is not added.

[0025] Comparative Example 3 A mesenchymal stem cell cryopreservation solution is provided, the formula and preparation method of which are the same as those of Example 4, except that platelet-derived growth factor is not added.

[0026] Comparative Example 4 A mesenchymal stem cell cryopreservation solution is provided, the formula and preparation method of which are the same as those of Example 4, except that epidermal growth factor and platelet-derived growth factor are not added.

[0027] Performance testing and results The mesenchymal stem cell cryopreservation solutions prepared in Example 4 and Comparative Examples 1-4 were frozen at -196°C for 6 months and then thawed to test the cell viability and survival rate of each cell. The cell viability was analyzed by a creatine kinase kit (Zhongsheng Beikong Biological Company), and the cell viability results were expressed as △OD values, and the results are shown in Table 1. The cell survival rate was detected by trypan blue staining, and the results are shown in Table 1.

[0028] Table 1

[0029] It can be seen from Table 1 that the mesenchymal stem cell cryopreservation solution prepared in Example 4 has higher cell viability and cell survival rate when preserving cells compared to Comparative Example 1. Comparing Example 4 with Comparative Examples 2-4, it can be seen that the addition of epidermal growth factor and platelet-derived growth factor can improve cell survival rate and viability.

[0030] Other raw materials or structures not specifically described in the present invention already exist in the prior art and can be directly purchased from the market.

[0031] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A mesenchymal stem cell cryopreservation solution, characterized in that: Includes the following ingredients in 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.

2. The mesenchymal stem cell cryopreservation solution according to claim 1, characterized in that: The trehalose concentration is 65 g / L, the hydroxyethyl starch concentration is 60 g / L, the L-ascorbic acid concentration is 50 mg / L, the albumin concentration is 50 g / L, the human insulin concentration is 25 mg / L, the basic fibroblast growth factor concentration is 20 μg / L, the epidermal growth factor concentration is 15 μg / L, and the platelet-derived growth factor concentration is 50 μg / L.

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

4. The method for preparing a mesenchymal stem cell cryopreservation solution according to any one of claims 1 to 3, characterized in that: The following steps are involved: mixing trehalose, hydroxyethyl starch, L-ascorbic acid, albumin, human insulin, basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor into the culture medium to obtain a mixed solution; The pH value of the mixed solution was adjusted to 7.4-7.6, and the mixture was filtered to obtain a mesenchymal stem cell cryopreservation solution.

5. The method according to claim 4, characterized in that After adjusting the pH value of the mixed solution, the mixed solution is filtered and sterilized through a filter to obtain a mesenchymal stem cell freezing solution.

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

7. The method for using the mesenchymal stem cell cryopreservation solution according to any one of claims 1 to 3, characterized in that: The mesenchymal stem cell freezing solution is 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°C.

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