A MSC culture medium additive and its application

Abstract

The present invention relates to a culture medium additive for MSC and application thereof, relating to the field of cell culture technology. The culture medium additive comprises the following components: 5-13 μg / L of composite growth factor, 1-3 g / L of plant component microemulsion, 10-20 mg / L of amino acids, 0.3-1.5 g / L of plant polysaccharides, 0.1-0.2 g / L of hyaluronic acid, 100-150 mg / L of betaine, 40-80 mg / L of vitamin C, 0.6-1.2 mg / L of trace elements, 10-30 mg / L of natural plant extracts, 10-30 mg / L of hydroxypropyl beta-cyclodextrin, and 2-4 mmol / L of acetylglutamine. The culture medium additive can effectively promote MSC proliferation, maintain cell activity and differentiation potential.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, in particular to a MSC culture medium additive and application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) show promising applications in regenerative medicine, cell therapy, and cosmetic procedures due to their multipotential differentiation, immunomodulatory properties, and ease of harvest. However, during in vitro expansion and culture, MSCs face challenges such as slow proliferation, difficulty maintaining cell viability, and loss of differentiation potential, which significantly limit their large-scale culture and clinical application.

[0003] MSC culture medium is the foundation for MSC culture, providing a suitable living environment for MSC cells. Currently, the culture medium used for MSC expansion and culture in vitro usually relies on animal-derived serum (such as FBS) or synthetic growth factors (such as bFGF). Animal-derived serum may introduce pathogens or trigger an immune response, leading to MSC growth stagnation or rapid aging. Traditional growth factor combinations are prone to protein denaturation, resulting in fluctuations in cell proliferation efficiency; single-factor regulation makes it difficult to simultaneously maintain stem cell stemness and differentiation potential; and signal pathway crosstalk caused by non-specific binding of growth factors often leads to increased cell heterogeneity. Existing MSCs also have the option of not adding animal-derived serum and synthetic growth factors during the culture process, but the culture effect is not ideal, with technical defects such as slow cell proliferation, low expression, and a limited number of proliferated cells.

[0004] To address these issues, a common practice is to incorporate MSC culture medium additives into MSC culture media. While commercially available MSC culture medium additives can promote MSC growth to a certain extent, they still suffer from suboptimal results, complex ingredients, and high costs. For example, Chinese invention patent CN112941022B discloses a culture medium specifically for culturing bone marrow mesenchymal stem cells and its applications. This culture medium also includes matrix metalloproteinase 2 (MMP2). Experimental studies have demonstrated that BMSCs treated with MMP2-containing medium exhibit no phenotype change but significantly increase their growth rate. Therefore, this MMP2-containing culture medium can be used to culture bone marrow-derived MSCs, enabling them to be more effective in clinical treatment and cosmetic applications. However, the lack of synergy between its components limits its differentiation potential, and its compatibility, safety, and stability in serum-free culture still require further improvement.

[0005] It can be seen that developing a MSC culture medium additive that can effectively promote MSC proliferation, maintain cell activity and differentiation potential has important practical significance. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a MSC culture medium additive that can effectively promote MSC proliferation, maintain cell activity and differentiation potential.

[0007] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is a MSC culture medium additive, comprising the following components: 5-13 μg / L of complex growth factor, 1-3 g / L of plant component microemulsion, 10-20 mg / L of amino acid, 0.3-1.5 g / L of plant polysaccharide, 0.1-0.2 g / L of hyaluronic acid, 100-150 mg / L of betaine, 40-80 mg / L of vitamin C, 0.6-1.2 mg / L of trace elements, 10-30 mg / L of natural plant extract, 10-30 mg / L of hydroxypropyl β-cyclodextrin, and 2-4 mmol / L of acetylglutamine.

[0008] Preferably, the composite growth factor is a mixture of BMP-2, TGF-β1, EGF, and IGF-1 in a mass ratio of (0.5-1.2):(0.3-0.8):(2-4):(3-8).

[0009] Preferably, the method for preparing the plant ingredient microemulsion comprises the following steps: dispersing curcumin, silybin, and epigallocatechin gallate in an organic solvent, adding an emulsifier, and mixing uniformly to obtain a mixed solution; mixing the mixed solution with deionized water, and treating the mixed solution with a high-pressure homogenizer at a pressure of 100-150 MPa for 5-8 times to obtain the plant ingredient microemulsion.

[0010] Preferably, the mass ratio of curcumin, silybin, epigallocatechin gallate, organic solvent and emulsifier is (6-8):(4-6):(3-5):(40-60):(0.65-1.5).

[0011] Preferably, the organic solvent is at least one of anhydrous ethanol and propylene glycol.

[0012] Preferably, the emulsifier is at least one of Poloxamer 188 and Tween 80.

[0013] Preferably, the volume ratio of the mixed solution to deionized water is 1:3.

[0014] Preferably, the amino acid is at least one of proline, glycine, glutamic acid and arginine.

[0015] Preferably, the plant polysaccharide is at least one of seaweed polysaccharide and glucan.

[0016] Preferably, the weight average molecular weight of the hyaluronic acid is 100-500 kDa.

[0017] Preferably, the trace elements are Se and Zn mixed in a mass ratio of (0.1-0.3):(0.5-1).

[0018] Preferably, the natural plant extract is a mixture of green tea polyphenols and rosmarinic acid in a mass ratio of 3:1.

[0019] Another object of the present invention is to provide a method for preparing the MSC culture medium additive, comprising the following steps: mixing the ingredients, adding them to deionized water, stirring them evenly at 4-10°C in the dark, then placing them into a freeze-drying bottle, pre-freezing them at -60°C for 2 hours, and then drying them in a freeze dryer for 25-30 hours to obtain the MSC culture medium additive.

[0020] Preferably, the drying vacuum degree is 5-10 Pa and the temperature is -40°C.

[0021] Another object of the present invention is to provide a use of the MSC culture medium additive in MSC cell culture, wherein the MSC culture medium additive is added to a basal cell culture medium for MSC cell culture; the addition amount of the MSC culture medium additive is 0.1-0.5 g / L.

[0022] Preferably, the basal cell culture medium is DMEM / F12 complete medium.

[0023] Due to the application of the above technical solution, the present invention has the following beneficial effects:

[0024] (1) The MSC culture medium additive disclosed in the present invention has a simple and easy preparation method, convenient operation and control, high preparation efficiency and finished product qualification rate, low dependence on equipment, and is easy to be produced on a large scale industrially. It is suitable for in vitro expansion and culture of MSCs from various sources and has high promotion and application value.

[0025] (2) The MSC culture medium additive disclosed in the present invention comprises the following components: 5-13 μg / L of composite growth factor, 1-3 g / L of plant component microemulsion, 10-20 mg / L of amino acid, 0.3-1.5 g / L of plant polysaccharide, 0.1-0.2 g / L of hyaluronic acid, 100-150 mg / L of betaine, 40-80 mg / L of vitamin C, 0.6-1.2 mg / L of trace elements, 10-30 mg / L of natural plant extract, 10-30 mg / L of hydroxypropyl β-cyclodextrin, and 2-4 mmol / L of acetylglutamine. Through the synergistic interaction of its ingredients, the resulting product effectively promotes MSC proliferation, maintaining cell viability, and differentiation potential. The botanical microemulsion contains curcumin, silybin, and epigallocatechin gallate. The antioxidant signaling pathway activated by curcumin works in concert with the autophagy process triggered by silybin to reduce intracellular oxidative stress products and waste products, providing a healthy and efficient intracellular environment for epigallocatechin gallate-promoted cell cycle progression. The physical protective film formed by plant polysaccharides and hyaluronic acid, combined with the enhanced cellular stress resistance and metabolic regulation capabilities of amino acids and trace elements, further reinforces the botanical ingredient complex's role in promoting MSC proliferation and maintaining stemness. The composite growth factor is a mixture of BMP-2, TGF-β1, EGF, and IGF-1 in a mass ratio of (0.5-1.2):(0.3-0.8):(2-4):(3-8); the synergistic effect of multiple growth factors can activate the proliferation signal pathway of MSC and promote the cell cycle process; the natural plant extract is a mixture of green tea polyphenols and rosmarinic acid in a mass ratio of 3:1, forming a composite defense system for oxidative stress, in which green tea polyphenols provide immediate antioxidant protection, and rosmarinic acid achieves long-term defense through pathway regulation, forming a "scavenging-inhibiting" dual antioxidant mechanism. In addition, they maintain the stemness of MSC through the synergistic effect of epigenetic and transcriptional regulation, and improve the anti-interference ability of the culture system; and are combined with hydroxypropyl β-cyclodextrin to enhance the chemical stability of the culture medium.

[0026] (3) The MSC culture medium additive disclosed in the present invention constructs a complex regulatory network that simulates the in vivo microenvironment through the coordinated design of multiple components such as composite growth factors, plant-based microemulsions, amino acids, and plant polysaccharides. This multi-target, multi-faceted intervention strategy breaks through the single action mode of existing technologies and represents a technological leap from "single factor regulation" to "systemic microenvironment simulation," enabling MSCs to maintain their complete differentiation capacity while rapidly proliferating. DETAILED DESCRIPTION

[0027] The following description is intended to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are merely examples, and those skilled in the art may conceive of other obvious variations.

[0028] Example 1

[0029] A MSC culture medium additive comprises the following components: 5 μg / L of composite growth factor, 1 g / L of plant component microemulsion, 10 mg / L of amino acids, 0.3 g / L of plant polysaccharides, 0.1 g / L of hyaluronic acid, 100 mg / L of betaine, 40 mg / L of vitamin C, 0.6 mg / L of trace elements, 10 mg / L of natural plant extracts, 10 mg / L of hydroxypropyl β-cyclodextrin, and 2 mmol / L of acetylglutamine; the composite growth factor is a mixture of BMP-2, TGF-β1, EGF, and IGF-1 in a mass ratio of 0.5:0.3:2:3.

[0030] The method for preparing a plant component microemulsion comprises the following steps: dispersing curcumin, silybin, and epigallocatechin gallate in an organic solvent, adding an emulsifier, and mixing uniformly to obtain a mixed solution; mixing the mixed solution with deionized water, and treating the mixed solution five times with a high-pressure homogenizer at a pressure of 100 MPa to obtain a plant component microemulsion; the mass ratio of the curcumin, silybin, epigallocatechin gallate, organic solvent, and emulsifier is 6:4:3:40:0.65; the organic solvent is anhydrous ethanol; the emulsifier is poloxamer 188; and the volume ratio of the mixed solution to deionized water is 1:3.

[0031] The amino acid is proline; the plant polysaccharide is seaweed polysaccharide; the weight-average molecular weight of the hyaluronic acid is 100 kDa; the trace elements are Se and Zn mixed in a mass ratio of 0.1:0.5; and the natural plant extract is green tea polyphenols and rosmarinic acid mixed in a mass ratio of 3:1.

[0032] A method for preparing the MSC culture medium additive comprises the following steps: mixing the components, adding them to deionized water, stirring them uniformly at 4°C in the dark, then placing them into a freeze-drying bottle, pre-freezing them at -60°C for 2 hours, and then drying them in a freeze dryer for 25 hours to obtain the MSC culture medium additive; the drying process is carried out at a vacuum degree of 5 Pa and a temperature of -40°C.

[0033] A use of the MSC culture medium additive in MSC cell culture, wherein the MSC culture medium additive is added to a basal cell culture medium for MSC cell culture; the addition amount of the MSC culture medium additive is 0.3 g / L; and the basal cell culture medium is a DMEM / F12 complete culture medium.

[0034] Example 2

[0035] A MSC culture medium additive comprises the following components: 7 μg / L of composite growth factor, 1.5 g / L of plant component microemulsion, 13 mg / L of amino acids, 0.5 g / L of plant polysaccharides, 0.13 g / L of hyaluronic acid, 110 mg / L of betaine, 50 mg / L of vitamin C, 0.8 mg / L of trace elements, 15 mg / L of natural plant extracts, 15 mg / L of hydroxypropyl β-cyclodextrin, and 2.5 mmol / L of acetylglutamine; the composite growth factor is a mixture of BMP-2, TGF-β1, EGF, and IGF-1 in a mass ratio of 0.7:0.5:2.5:4.

[0036] The method for preparing a plant component microemulsion comprises the following steps: dispersing curcumin, silybin, and epigallocatechin gallate in an organic solvent, adding an emulsifier, and mixing uniformly to obtain a mixed solution; mixing the mixed solution with deionized water, and treating the mixed solution six times with a high-pressure homogenizer at a pressure of 110 MPa to obtain a plant component microemulsion; the mass ratio of the curcumin, silybin, epigallocatechin gallate, organic solvent, and emulsifier is 6.5:4.5:3.5:45:0.8; the organic solvent is propylene glycol; the emulsifier is Tween 80; and the volume ratio of the mixed solution to deionized water is 1:3.

[0037] The amino acid is glycine; the plant polysaccharide is dextran; the weight-average molecular weight of the hyaluronic acid is 200 kDa; the trace elements are Se and Zn mixed in a mass ratio of 0.15:0.7; and the natural plant extract is green tea polyphenols and rosmarinic acid mixed in a mass ratio of 3:1.

[0038] A method for preparing the MSC culture medium additive comprises the following steps: mixing the components, adding them to deionized water, stirring them uniformly at 6°C in the dark, then placing them into a freeze-drying bottle, pre-freezing them at -60°C for 2 hours, and then drying them in a freeze dryer for 27 hours to obtain the MSC culture medium additive; the drying process is carried out at a vacuum degree of 6 Pa and a temperature of -40°C.

[0039] A use of the MSC culture medium additive in MSC cell culture, wherein the MSC culture medium additive is added to a basal cell culture medium for MSC cell culture; the addition amount of the MSC culture medium additive is 0.3 g / L; and the basal cell culture medium is a DMEM / F12 complete culture medium.

[0040] Example 3

[0041] A MSC culture medium additive comprises the following components: 9 μg / L of composite growth factor, 2 g / L of plant component microemulsion, 15 mg / L of amino acids, 1 g / L of plant polysaccharides, 0.15 g / L of hyaluronic acid, 130 mg / L of betaine, 60 mg / L of vitamin C, 0.9 mg / L of trace elements, 20 mg / L of natural plant extracts, 20 mg / L of hydroxypropyl β-cyclodextrin, and 3 mmol / L of acetylglutamine; the composite growth factor is a mixture of BMP-2, TGF-β1, EGF, and IGF-1 in a mass ratio of 0.9:0.6:3:5.

[0042] The method for preparing a plant component microemulsion comprises the following steps: dispersing curcumin, silybin, and epigallocatechin gallate in an organic solvent, adding an emulsifier, and mixing uniformly to obtain a mixed solution; mixing the mixed solution with deionized water, and treating the mixed solution seven times with a high-pressure homogenizer at a pressure of 130 MPa to obtain the plant component microemulsion; the mass ratio of the curcumin, silybin, epigallocatechin gallate, organic solvent, and emulsifier is 7:5:4:50:1.1; the organic solvent is anhydrous ethanol; the emulsifier is Tween 80; and the volume ratio of the mixed solution to deionized water is 1:3.

[0043] The amino acid is glutamic acid; the plant polysaccharide is seaweed polysaccharide; the weight-average molecular weight of the hyaluronic acid is 300 kDa; the trace elements are Se and Zn mixed in a mass ratio of 0.2:0.75; and the natural plant extract is green tea polyphenols and rosmarinic acid mixed in a mass ratio of 3:1.

[0044] A method for preparing the MSC culture medium additive comprises the following steps: mixing the components, adding them to deionized water, stirring them uniformly at 7°C in the dark, then placing them into a freeze-drying bottle, pre-freezing them at -60°C for 2 hours, and then drying them in a freeze dryer for 28 hours to obtain the MSC culture medium additive; the drying process is carried out at a vacuum degree of 7.5 Pa and a temperature of -40°C.

[0045] A use of the MSC culture medium additive in MSC cell culture, wherein the MSC culture medium additive is added to a basal cell culture medium for MSC cell culture; the addition amount of the MSC culture medium additive is 0.3 g / L; and the basal cell culture medium is a DMEM / F12 complete culture medium.

[0046] Example 4

[0047] A MSC culture medium additive comprises the following components: 12 μg / L of composite growth factors, 2.5 g / L of plant component microemulsion, 18 mg / L of amino acids, 1.3 g / L of plant polysaccharides, 0.18 g / L of hyaluronic acid, 140 mg / L of betaine, 75 mg / L of vitamin C, 1.1 mg / L of trace elements, 25 mg / L of natural plant extracts, 25 mg / L of hydroxypropyl β-cyclodextrin, and 3.5 mmol / L of acetylglutamine; the composite growth factors are prepared by mixing BMP-2, TGF-β1, EGF, and IGF-1 in a mass ratio of 1.1:0.7:3.5:7.

[0048] The method for preparing a plant component microemulsion comprises the following steps: dispersing curcumin, silybin, and epigallocatechin gallate in an organic solvent, adding an emulsifier, and uniformly mixing to obtain a mixed solution; mixing the mixed solution with deionized water, and treating the mixed solution seven times with a high-pressure homogenizer at a pressure of 140 MPa to obtain the plant component microemulsion; the mass ratio of the curcumin, silybin, epigallocatechin gallate, organic solvent, and emulsifier is 7.5:5.5:4.5:55:1.3; the organic solvent is a mixture of anhydrous ethanol and propylene glycol in a mass ratio of 1:2; the emulsifier is a mixture of poloxamer 188 and Tween 80 in a mass ratio of 3:5; and the volume ratio of the mixed solution to deionized water is 1:3.

[0049] The amino acids are proline, glycine, glutamic acid, and arginine mixed in a mass ratio of 1:1:2:3; the plant polysaccharide is seaweed polysaccharide and dextran mixed in a mass ratio of 1:2; the weight-average molecular weight of the hyaluronic acid is 400 kDa; the trace elements are Se and Zn mixed in a mass ratio of 0.25:0.9; and the natural plant extract is green tea polyphenols and rosmarinic acid mixed in a mass ratio of 3:1.

[0050] A method for preparing the MSC culture medium additive comprises the following steps: mixing the components, adding them to deionized water, stirring them uniformly at 9°C in the dark, then placing them into a freeze-drying bottle, pre-freezing them at -60°C for 2 hours, and then drying them in a freeze dryer for 29 hours to obtain the MSC culture medium additive; the drying process is carried out at a vacuum degree of 9 Pa and a temperature of -40°C.

[0051] A use of the MSC culture medium additive in MSC cell culture, wherein the MSC culture medium additive is added to a basal cell culture medium for MSC cell culture; the addition amount of the MSC culture medium additive is 0.3 g / L; and the basal cell culture medium is a DMEM / F12 complete culture medium.

[0052] Example 5

[0053] A MSC culture medium additive comprises the following components: 13 μg / L of composite growth factors, 3 g / L of plant component microemulsion, 20 mg / L of amino acids, 1.5 g / L of plant polysaccharides, 0.2 g / L of hyaluronic acid, 150 mg / L of betaine, 80 mg / L of vitamin C, 1.2 mg / L of trace elements, 30 mg / L of natural plant extracts, 30 mg / L of hydroxypropyl β-cyclodextrin, and 4 mmol / L of acetylglutamine; the composite growth factors are prepared by mixing BMP-2, TGF-β1, EGF, and IGF-1 in a mass ratio of 1.2:0.8:4:8.

[0054] The method for preparing a plant component microemulsion comprises the following steps: dispersing curcumin, silybin, and epigallocatechin gallate in an organic solvent, adding an emulsifier, and mixing uniformly to obtain a mixed solution; mixing the mixed solution with deionized water, and treating the mixed solution eight times with a high-pressure homogenizer at a pressure of 150 MPa to obtain a plant component microemulsion; the mass ratio of the curcumin, silybin, epigallocatechin gallate, organic solvent, and emulsifier is 8:6:5:60:1.5; the organic solvent is anhydrous ethanol; the emulsifier is poloxamer 188; and the volume ratio of the mixed solution to deionized water is 1:3.

[0055] The amino acid is arginine; the plant polysaccharide is seaweed polysaccharide; the weight-average molecular weight of the hyaluronic acid is 500 kDa; the trace elements are Se and Zn mixed in a mass ratio of 0.3:1; and the natural plant extract is green tea polyphenols and rosmarinic acid mixed in a mass ratio of 3:1.

[0056] A method for preparing the MSC culture medium additive comprises the following steps: mixing the components, adding them to deionized water, stirring them uniformly at 10°C in the dark, then placing them into a freeze-drying bottle, pre-freezing them at -60°C for 2 hours, and then drying them in a freeze dryer for 30 hours to obtain the MSC culture medium additive; the drying process is carried out at a vacuum degree of 10 Pa and a temperature of -40°C.

[0057] A use of the MSC culture medium additive in MSC cell culture, wherein the MSC culture medium additive is added to a basal cell culture medium for MSC cell culture; the addition amount of the MSC culture medium additive is 0.3 g / L; and the basal cell culture medium is a DMEM / F12 complete culture medium.

[0058] Comparative Example 1

[0059] A MSC culture medium additive and application thereof are basically the same as those in Example 1, except that no plant component microemulsion is added.

[0060] Comparative Example 2

[0061] A MSC culture medium additive and its application are basically the same as those in Example 1, except that hyaluronic acid and betaine are not added.

[0062] To further illustrate the beneficial technical effects of the MSC culture medium additives involved in each embodiment of the present invention, relevant performance tests were conducted on the MSC culture medium additives involved in Examples 1-5 and Comparative Examples 1-2. The test results are shown in Table 1. The testing method is as follows: According to the application method of each example, cells were cultured in an incubator at 37°C and 5% CO2. Cell growth was observed daily, and the medium was changed every 3 days. After 7 days of culture, cell survival rate was calculated using trypan blue staining. Cell proliferation was also detected using the CCK-8 method, and the MSC proliferation rate was calculated after 72 hours. The MSCs used in the tests were umbilical cord tissue-derived MSCs.

[0063] Table 1 Performance test results of MSC culture medium additives

[0064] project Example 1 Example 2 Example 3 Example 4 Example 5 Comparative Example 1 Comparative Example 2 Cell survival rate (%) 98.5 99.0 99.3 99.5 99.9 96.2 97.0 Cell proliferation rate (%) 355.6 358.0 360.0 361.5 363.3 337.8 343.9

[0065] As can be seen from Table 1, the MSC culture medium additives involved in the embodiments of the present invention can significantly improve the survival rate and proliferation rate of MSCs, and the effect is significant compared with the comparative example; the combined use of plant ingredient microemulsion, hyaluronic acid and betaine is beneficial to improving the above properties.

[0066] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A culture medium additive for MSCs, characterized in that It includes the following ingredients: complex growth factor 5-13μg / L, plant ingredient microemulsion 1-3g / L, amino acid 10-20mg / L, plant polysaccharide 0.3-1.5g / L, hyaluronic acid 0.1-0.2g / L, betaine 100-150mg / L, vitamin C 40-80mg / L, trace elements 0.6-1.2mg / L, natural plant extract 10-30mg / L, hydroxypropyl β-cyclodextrin 10-30mg / L, acetylglutamine 2-4mmol / L; The composite growth factor is a mixture of BMP-2, TGF-β1, EGF, and IGF-1 in a mass ratio of (0.5-1.2): (0.3-0.8): (2-4): (3-8); the preparation method of the plant component microemulsion comprises the following steps: dispersing curcumin, silybin, and epigallocatechin gallate in an organic solvent, adding an emulsifier, and mixing uniformly to obtain a mixed solution; mixing the mixed solution with deionized water, and treating the mixed solution with a high-pressure homogenizer at a pressure of 100-150 MPa for 5-8 times to obtain the plant component microemulsion; the curcumin, silybin, epigallocatechin gallate, and the emulsifier are mixed uniformly to obtain a mixed solution; The mass ratio of epigallocatechin gallate, organic solvent and emulsifier is (6-8):(4-6):(3-5):(40-60):(0.65-1.5); the amino acid is at least one of proline, glycine, glutamic acid and arginine; the plant polysaccharide is at least one of seaweed polysaccharide and glucan; the trace elements are Se and Zn mixed in a mass ratio of (0.1-0.3):(0.5-1); the natural plant extract is green tea polyphenols and rosmarinic acid mixed in a mass ratio of 3:1; the organic solvent is at least one of anhydrous ethanol and propylene glycol.

2. The MSC culture medium additive according to claim 1, characterized in that The emulsifier is at least one of poloxamer 188 and Tween 80; the volume ratio of the mixed solution to deionized water is 1:

3.

3. The MSC culture medium additive according to claim 1, characterized in that The weight average molecular weight of the hyaluronic acid is 100-500 kDa.

4. A method for preparing the MSC culture medium additive according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: mixing the components, adding the components into deionized water, stirring evenly at 4-10°C in the dark, then placing the components into a freeze-drying bottle, pre-freezing the components at -60°C for 2 hours, and then drying the components in a freeze dryer for 25-30 hours to obtain a culture medium additive for MSCs; the drying process is carried out at a vacuum degree of 5-10 Pa and a temperature of -40°C.

5. Use of the MSC culture medium additive according to any one of claims 1 to 3 in MSC cell culture, characterized in that: The MSC culture medium additive is added to a basal cell culture medium for MSC cell culture; the addition amount of the MSC culture medium additive is 0.1-0.5 g / L.

6. Use of the MSC culture medium additive according to claim 5 in MSC cell culture, characterized in that: The basal cell culture medium is DMEM / F12 complete culture medium.