Supplementary agent of cell culture medium, serum-free mesenchymal stem cell culture medium and application of serum-free mesenchymal stem cell culture medium

Abstract

The invention relates to the technical field of biology, in particular to a supplement of a cell culture medium, a serum-free mesenchymal stem cell culture medium and application of the serum-free mesenchymal stem cell culture medium. The invention provides a supplement for a cell culture medium. The supplement comprises the following components: nicotinamide, modified polygonatum odoratum polysaccharide, vitamin C and an aloe extract. The supplement can improve the multiplication capacity and the survival rate of the mesenchymal stem cells, and can delay cell aging under the condition of maintaining the differentiation potential and the self-renewal capacity of the mesenchymal stem cells. The invention further provides a serum-free mesenchymal stem cell culture medium, serum in a conventional culture medium is replaced with a serum substitute, and the problem of microbial contamination caused by introduction of serum in the culture process of mesenchymal stem cells is solved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a cell culture medium supplement, a serum-free mesenchymal stem cell culture medium and applications thereof. Background Art

[0002] Mesenchymal stem cells are a type of multipotent stem cells that have all the common characteristics of stem cells, namely, self-renewal and multidirectional differentiation capabilities. Under specific induction conditions in vivo or in vitro, they can differentiate into a variety of tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, endothelium, etc., and are ideal seed cells for regenerative medicine. Mesenchymal stem cells exist in a variety of tissues such as bone marrow, umbilical cord blood and umbilical cord tissue, placental tissue, and adipose tissue. Because they can differentiate into a wide range of tissue types, their clinical application value is very high.

[0003] The traditional culture medium currently used for the culture of mesenchymal stem cells is a mixture of basal culture medium and serum. Fetal bovine serum is rich in growth factors required for in vitro cell culture, but the specific composition of the serum is uncertain, which leads to large batch differences between different batches of cells, making it unsuitable for repeatable experiments; and the serum culture medium contains animal-derived serum components, which can easily cause exogenous contamination and cell mutation.

[0004] Based on this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a cell culture medium supplement and a serum-free mesenchymal stem cell culture medium which can significantly improve cell proliferation multiples and cell viability.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a cell culture medium supplement, comprising the following components in parts by weight: 1 to 10 parts of nicotinamide, 1 to 5 parts of modified polygonatum odoratum polysaccharide, 3 to 7 parts of vitamin C and 0.01 to 0.05 parts of aloe extract.

[0008] Preferably, the preparation method of the modified Polygonatum odoratum polysaccharide comprises the following steps:

[0009] (1) mixing polygonatum odoratum polysaccharide and acetic anhydride to obtain a mixed solution;

[0010] (2) dialyzing the mixed solution for 24 to 36 hours and drying to obtain modified Polygonatum odoratum polysaccharide;

[0011] The mass ratio of the polygonatum odoratum polysaccharide and acetic anhydride is 1:1-3.

[0012] Preferably, in step (1), the method of mixing the polygonatum odoratum polysaccharide and acetic anhydride is a pH driven method;

[0013] In step (2), the drying is vacuum freeze drying; the vacuum degree of the drying is 10 to 20 Pa; the drying temperature is -40 to -30°C; and the drying time is 2 to 4 hours.

[0014] Preferably, the preparation method of the aloe extract comprises the following steps:

[0015] 1) mixing aloe vera and an extraction solvent, performing ultrasonic extraction, filtering, and collecting a filtrate to obtain a crude extract;

[0016] 2) Mixing the crude extract and anhydrous ethanol for 2 to 5 hours, centrifuging, and taking the supernatant to obtain the aloe extract.

[0017] Preferably, in step 1), the extraction solvent is urea and choline chloride;

[0018] The molar ratio of urea to choline chloride is 1 to 3:1;

[0019] The mass ratio of the aloe vera to the extraction solvent is 1:4-6.

[0020] Preferably, in step 1), the ultrasonic extraction time is 30 to 50 minutes; the ultrasonic extraction temperature is 45 to 55° C.; the ultrasonic extraction power is 100 to 300 W;

[0021] In step 2), the centrifugal speed is 3000-5000 rpm, and the centrifugal time is 5-15 min.

[0022] The present invention provides a serum-free mesenchymal stem cell culture medium, which uses DMEM / F12 culture medium as a basic culture medium and further comprises the following components at final concentrations: 1-10 μg / L of recombinant human basic fibroblast growth factor, 3-8 mg / L of L-glutamine, 10-20 μg / L of selenomethionine, 4-8 mg / L of non-essential amino acids, 20-30 mg / L of recombinant human insulin, 3-6 μg / L of recombinant human epidermal growth factor, 1-10 mg / L of ferric citrate, 12-20 mg / L of transferrin, 15-25 μg / L of hydrocortisone, 0.5-2.5 mg / L of gentamicin, 2-6 mg / L of recombinant human serum protein, 0.5-1.5 mg / L of albumin, and 5-23 mg / L of supplements;

[0023] The supplement is the described supplement.

[0024] Preferably, the non-essential amino acids include one or more of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid.

[0025] The present invention also provides a method for preparing the serum-free mesenchymal stem cell culture medium, comprising the following steps: mixing DMEM / F12 culture medium and basic fibroblast growth factor, L-glutamine, selenomethionine, non-essential amino acids, recombinant human insulin, recombinant human epidermal growth factor, ferric citrate, transferrin, hydrocortisone, gentamicin, recombinant human serum protein, albumin, and a supplement to obtain a serum-free mesenchymal stem cell culture medium.

[0026] The present invention also provides the use of the supplement and the serum-free mesenchymal stem cell culture medium in culturing mesenchymal stem cells.

[0027] Beneficial effects of the present invention:

[0028] The present invention prepares a cell culture medium supplement by combining nicotinamide, modified polygonatum polysaccharide, vitamin C and aloe extract, which can improve the proliferation ability and survival rate of mesenchymal stem cells, and can also delay cell aging while maintaining the differentiation potential and self-renewal ability of mesenchymal stem cells. Among them, nicotinamide, vitamin C and aloe extract all have high antioxidant activity and can protect cells from oxidative stress damage; vitamin C can also promote cell growth and development, and vitamin C and aloe extract can repair cells during proliferation and maintain cell activity; modified polygonatum polysaccharide can not only enhance the adhesion and proliferation ability of cells, but also reduce cell damage and improve cell survival rate when combined with nicotinamide and aloe extract.

[0029] The present invention also provides a serum-free mesenchymal stem cell culture medium, which uses a serum substitute to replace the serum in a conventional culture medium, thereby avoiding the problem of microbial contamination caused by the introduction of serum into the mesenchymal stem cells during the culture process; the serum-free mesenchymal stem cell culture medium of the present invention has clear components and addition amounts, and the mesenchymal stem cells obtained by culture are of good quality and high purity, and the culture results are stable, and are suitable for repeatable experiments. DETAILED DESCRIPTION

[0030] The present invention provides a cell culture medium supplement, comprising the following components in parts by weight:

[0031] 1 to 10 parts of niacinamide, preferably 4 to 7 parts, more preferably 5.5 parts;

[0032] 1 to 5 parts of modified Polygonatum odoratum polysaccharide, preferably 2 to 4 parts, more preferably 3 parts;

[0033] 3 to 7 parts of vitamin C, preferably 4 to 6 parts, more preferably 5 parts;

[0034] The amount of the aloe extract is 0.01 to 0.05 parts, preferably 0.02 to 0.04 parts, and more preferably 0.03 parts.

[0035] In the present invention, the preparation method of the modified Polygonatum odoratum polysaccharide comprises the following steps:

[0036] (1) mixing polygonatum odoratum polysaccharide and acetic anhydride to obtain a mixed solution;

[0037] (2) dialyzing the mixed solution for 24 to 36 hours and drying to obtain modified Polygonatum odoratum polysaccharide;

[0038] The mass ratio of the polysaccharide and acetic anhydride is 1:1-3, preferably 1:2.

[0039] In the present invention, in step (1), the method for mixing the polysaccharide and acetic anhydride is a pH-driven method, which specifically comprises the following steps: firstly preparing a saturated solution of the polysaccharide with water, and adjusting the pH of the saturated solution to 9.0 using a 20 vt% sodium hydroxide solution; adding acetic anhydride at 60° C. to obtain a mixed solution, and adjusting the pH of the mixed solution to 7.0 using a 10 vt% hydrochloric acid solution to obtain a mixed solution;

[0040] In step (2), the dialysis time is preferably 28 to 32 hours, preferably 30 hours; the molecular weight cutoff of the dialysis is 8 to 14 kDa, preferably 10 to 13 kDa, and more preferably 12 kDa;

[0041] The drying is vacuum freeze drying; the vacuum degree of the drying is 10 to 20 Pa, preferably 13 to 17 Pa, and more preferably 15 Pa; the drying temperature is -40 to -30°C, preferably -37 to -33°C, and more preferably -35°C; the drying time is 2 to 4 hours, preferably 2.5 to 3.5 hours, and more preferably 3 hours.

[0042] In the present invention, the preparation method of the aloe extract comprises the following steps:

[0043] 1) mixing aloe vera and an extraction solvent, performing ultrasonic extraction, filtering, and collecting a filtrate to obtain a crude extract;

[0044] 2) Mixing the crude extract and anhydrous ethanol for 2 to 5 hours, centrifuging, and taking the supernatant to obtain the aloe extract.

[0045] In the present invention, in step 1), the aloe vera and the extraction solvent need to be pre-treated before mixing, including the following steps: washing, peeling, and pulping the aloe vera raw material to obtain the aloe vera.

[0046] The extraction solvent is urea and choline chloride;

[0047] The molar ratio of urea to choline chloride is 1 to 3:1, preferably 2:1;

[0048] The mass ratio of the aloe vera to the extraction solvent is 1:4-6, preferably 1:5.

[0049] In step 1), the ultrasonic extraction time is 30 to 50 minutes, preferably 34 to 45 minutes, and more preferably 40 minutes; the ultrasonic extraction temperature is 45 to 55° C., preferably 48 to 52° C., and more preferably 50° C.; the ultrasonic extraction power is 100 to 300 W, preferably 150 to 250 W, and more preferably 200 W;

[0050] In the present invention, in step 2), the volume ratio of the crude extract and anhydrous ethanol is 1:2 to 4, preferably 1:3;

[0051] The mixing time of the crude extract and anhydrous ethanol is preferably 3 to 4 hours, more preferably 3.5 hours;

[0052] The centrifugal speed is 3000-5000 rpm, preferably 3500-4500 rpm, and more preferably 4000 rpm; the centrifugal time is 5-15 min, preferably 8-12 min, and more preferably 10 min.

[0053] The present invention provides a serum-free mesenchymal stem cell culture medium, which uses DMEM / F12 culture medium as a basic culture medium and further includes the following components at final concentrations:

[0054] Recombinant human basic fibroblast growth factor 1-10 μg / L, preferably 4-7 μg / L, more preferably 5.5 μg / L;

[0055] L-glutamine 3-8 mg / L, preferably 5-6 mg / L, more preferably 5.5 mg / L;

[0056] Selenomethionine 10-20 μg / L, preferably 13-17 μg / L, more preferably 15 μg / L;

[0057] Non-essential amino acids 4-8 mg / L, preferably 5-7 mg / L, more preferably 6 mg / L;

[0058] Recombinant human insulin 20-30 mg / L, preferably 23-27 mg / L, more preferably 25 mg / L;

[0059] Recombinant human epidermal growth factor 3-6 μg / L, preferably 4-5 μg / L, more preferably 4.5 μg / L;

[0060] Ferric citrate 1-10 mg / L, preferably 4-7 mg / L, more preferably 5.5 mg / L;

[0061] Transferrin 12-20 mg / L, preferably 14-18 mg / L, more preferably 16 mg / L;

[0062] Hydrocortisone 15-25 μg / L, preferably 18-22 μg / L, more preferably 20 μg / L;

[0063] Gentamycin 0.5-2.5 mg / L, preferably 1.0-2.0 mg / L, more preferably 1.5 mg / L;

[0064] Recombinant human serum albumin 2-6 mg / L, preferably 3-5 mg / L, more preferably 4 mg / L;

[0065] Albumin 0.5-1.5 mg / L, preferably 0.8-1.2 mg / L, more preferably 1.0 mg / L;

[0066] Supplement 5-23 mg / L, preferably 10-18 mg / L, more preferably 14 mg / L;

[0067] The supplement is the described supplement.

[0068] In the present invention, the non-essential amino acids include one or more of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid;

[0069] When the non-essential amino acids are glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid, the mass ratio of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid is 0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5, and more preferably 1:1:1:1:1:1:1:1:1:1:1.

[0070] The present invention also provides a method for preparing the serum-free mesenchymal stem cell culture medium, comprising the following steps: mixing DMEM / F12 culture medium and basic fibroblast growth factor, L-glutamine, selenomethionine, non-essential amino acids, recombinant human insulin, recombinant human epidermal growth factor, ferric citrate, transferrin, hydrocortisone, gentamicin, recombinant human serum protein, albumin, and a supplement to obtain a serum-free mesenchymal stem cell culture medium.

[0071] The present invention also provides the use of the supplement and the serum-free mesenchymal stem cell culture medium in culturing mesenchymal stem cells;

[0072] In the present invention, the mesenchymal stem cells include adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, and umbilical cord-derived mesenchymal stem cells.

[0073] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0074] Example 1 A cell culture medium supplement

[0075] Preparation of modified Polygonatum odoratum polysaccharide:

[0076] (1) First, 10 g of Polygonatum odoratum polysaccharide is prepared into a saturated solution with water, and the pH of the saturated solution is adjusted to 9.0 with a 20 vt% sodium hydroxide solution; 20 g of acetic anhydride is added at 60° C. to obtain a mixed solution, and the pH of the mixed solution is adjusted to 7.0 with a 10 vt% hydrochloric acid solution to obtain a mixed solution;

[0077] (2) The mixed solution was dialyzed using a sterile dialysis bag (molecular weight cutoff: 12 kDa) for 30 h, and dried at 15 Pa and -35°C for 3 h to obtain modified Polygonatum odoratum polysaccharide.

[0078] Preparation of Aloe Vera Extract:

[0079] 1) washing, peeling and beating the aloe raw material to obtain aloe; mixing 10 g of aloe with 50 g of extraction solvent (mixing urea and choline chloride in a molar ratio of 2:1), ultrasonically extracting at 200 W and 50° C. for 40 min, filtering, and taking the filtrate to obtain a crude extract;

[0080] 2) The crude extract and anhydrous ethanol were mixed at a volume ratio of 1:3 for 2.5 hours, centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected to obtain an aloe extract.

[0081] A cell culture medium supplement:

[0082] It is composed of 5g niacinamide, 3g modified polygonatum polysaccharide, 5g vitamin C and 0.03g aloe vera extract.

[0083] Example 2

[0084] The preparation of modified Polygonatum odoratum polysaccharide and Aloe vera extract is the same as in Example 1;

[0085] A cell culture medium supplement:

[0086] It is composed of 8g niacinamide, 2g modified polygonatum polysaccharide, 3g vitamin C and 0.05g aloe vera extract.

[0087] Example 3

[0088] The preparation of modified Polygonatum odoratum polysaccharide and Aloe vera extract is the same as in Example 1;

[0089] A cell culture medium supplement:

[0090] It is composed of 4g niacinamide, 5g modified polygonatum polysaccharide, 7g vitamin C and 0.02g aloe vera extract.

[0091] Example 4

[0092] A serum-free mesenchymal stem cell culture medium:

[0093] 6 μg of recombinant human basic fibroblast growth factor, 5 mg of L-glutamine, 15 μg of selenomethionine, 6 mg of non-essential amino acids (the mass ratio of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid, and glutamate is 1:1:1:1:1:1:1:1:1:1:1), 25 mg of recombinant human insulin, 4.5 μg of recombinant human epidermal growth factor, 5 mg of ferric citrate, 16 mg of transferrin, 20 μg of hydrocortisone, 1.5 mg of gentamicin, 4 mg of recombinant human serum albumin, 1 mg of albumin and 13.03 g of the supplement described in Example 1 were mixed, and the volume was fixed to 1 L using DMEM / F12 medium to obtain a serum-free mesenchymal stem cell culture medium.

[0094] Example 5

[0095] A serum-free mesenchymal stem cell culture medium:

[0096] 3 μg of recombinant human basic fibroblast growth factor, 6 mg of L-glutamine, 12 μg of selenomethionine, 8 mg of non-essential amino acids (the mass ratio of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid, and glutamate is 1:1:0.5:1:0.5:1.5:1:1:1.5:1), 22 mg of recombinant human insulin, 3 μg of recombinant human epidermal growth factor, 8 mg of ferric citrate, 12 mg of transferrin, 15 μg of hydrocortisone, 0.5 mg of gentamicin, 6 mg of recombinant human serum protein, 0.5 mg of albumin and 13.05 g of the supplement described in Example 2 were mixed, and the volume was fixed to 1 L using DMEM / F12 medium to obtain a serum-free mesenchymal stem cell culture medium.

[0097] Example 6

[0098] 10 μg of recombinant human basic fibroblast growth factor, 4 mg of L-glutamine, 20 μg of selenomethionine, 4 mg of non-essential amino acids (the mass ratio of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, and aspartic acid is 1:1:1:1:1:1:1:1:1:1), 29 mg of recombinant human insulin, 6 μg of recombinant human epidermal growth factor, 2 mg of ferric citrate, 17 mg of transferrin, 24 μg of hydrocortisone, 2.0 mg of gentamicin, 2 mg of recombinant human serum albumin, 1.5 mg of albumin and 16.02 g of the supplement described in Example 3 were mixed, and the volume was fixed to 1 L using DMEM / F12 medium to obtain a serum-free mesenchymal stem cell culture medium.

[0099] Comparative Example 1

[0100] The difference from Example 4 is that the supplement consists of 5g niacinamide, 3g Polygonatum odoratum polysaccharide, 5g vitamin C and 0.03g aloe extract; the other steps are the same as Example 4.

[0101] Comparative Example 2

[0102] The difference from Example 4 is that the supplement consists of 5g niacinamide, 3g modified Polygonatum odoratum polysaccharide and 5g vitamin C; the other steps are the same as Example 4.

[0103] Comparative Example 3

[0104] The difference from Example 4 is that the supplement consists of 5g niacinamide, 3g modified Polygonatum odoratum polysaccharide and 0.03g aloe extract; the other steps are the same as Example 4.

[0105] Comparative Example 4

[0106] The difference from Example 4 is that selenomethionine is replaced by sodium selenite; the other steps are the same as Example 4.

[0107] Comparative Example 5

[0108] The difference from Example 4 is that ferric citrate is not added; the other steps are the same as Example 4.

[0109] Comparative Example 6

[0110] The difference from Example 4 is that albumin is not added; the other steps are the same as Example 4.

[0111] Experimental Example 1

[0112] Resuscitated P4 human umbilical cord mesenchymal stem cells (UCMSC) (purchased from Qingdao Aoke Biotechnology Co., Ltd.) were inoculated (inoculation volume was 2×10 6) were cultured in the serum-free mesenchymal stem cell culture medium shown in Example 4 and Comparative Examples 1 to 6. After culturing for 3 days, the number of cells in the culture medium and the cell viability were counted, and the proliferation times were calculated. The results are shown in Table 1;

[0113] The calculation formula of the proliferation multiple is as follows:

[0114]

[0115] Table 1 Cultivation of UCMSCs in different culture media

[0116]

[0117]

[0118] As can be seen from Table 1, when UCMSCs were cultured using the culture medium described in Example 4, their proliferation multiples could be as high as 21.55, and the survival rate was 99.37%, which was significantly higher than that of Comparative Examples 1-6.

[0119] Experimental Example 2

[0120] Human adipose-derived mesenchymal stem cells (ADMSCs) (purchased from Shenzhen Haodi Huatuo Biotechnology Co., Ltd.) were cultured using the method described in Experimental Example 1. The culture conditions are shown in Table 2.

[0121] Table 1 Cultivation of ADMSCs in different culture media

[0122]

[0123] As can be seen from Table 2, when ADMSCs were cultured using the culture medium described in Example 4, their proliferation multiples could be as high as 20.95, and their survival rate was 98.95%, which was significantly higher than that of Comparative Examples 1-6.

[0124] It can be seen from the above embodiments that the present invention provides a cell culture medium supplement, a serum-free mesenchymal stem cell culture medium and applications thereof. The serum-free mesenchymal stem cell culture medium of the present invention can significantly improve the survival rate and proliferation multiple of mesenchymal stem cells.

[0125] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cell culture medium supplement, characterized in that The invention comprises the following components in parts by weight: 1 to 10 parts of nicotinamide, 1 to 5 parts of modified polygonatum odoratum polysaccharide, 3 to 7 parts of vitamin C and 0.01 to 0.05 parts of aloe extract.

2. The supplement according to claim 1, characterized in that The preparation method of the modified Polygonatum odoratum polysaccharide comprises the following steps: (1) mixing polygonatum odoratum polysaccharide and acetic anhydride to obtain a mixed solution; (2) dialyzing the mixed solution for 24 to 36 hours and drying to obtain modified Polygonatum odoratum polysaccharide; The mass ratio of the polygonatum odoratum polysaccharide and acetic anhydride is 1:1-3.

3. The supplement according to claim 2, characterized in that In step (1), the method of mixing the polygonatum odoratum polysaccharide and acetic anhydride is a pH driven method; In step (2), the drying is vacuum freeze drying; the vacuum degree of the drying is 10 to 20 Pa; the drying temperature is -40 to -30°C; and the drying time is 2 to 4 hours.

4. The supplement according to claim 3, characterized in that The preparation method of the aloe extract comprises the following steps: 1) mixing aloe vera and an extraction solvent, performing ultrasonic extraction, filtering, and collecting a filtrate to obtain a crude extract; 2) Mixing the crude extract and anhydrous ethanol for 2 to 5 hours, centrifuging, and taking the supernatant to obtain the aloe extract.

5. The supplement according to claim 4, characterized in that In step 1), the extraction solvent is urea and choline chloride; The molar ratio of urea to choline chloride is 1 to 3:1; The mass ratio of the aloe vera to the extraction solvent is 1:4-6.

6. The supplement according to claim 5, characterized in that In step 1), the ultrasonic extraction time is 30 to 50 minutes; the ultrasonic extraction temperature is 45 to 55° C.; the ultrasonic extraction power is 100 to 300 W; In step 2), the centrifugal speed is 3000-5000 rpm, and the centrifugal time is 5-15 min.

7. A serum-free mesenchymal stem cell culture medium, characterized in that: The serum-free mesenchymal stem cell culture medium is based on DMEM / F12 culture medium, and further comprises the following components at final concentrations: 1-10 μg / L recombinant human basic fibroblast growth factor, 3-8 mg / L L-glutamine, 10-20 μg / L selenomethionine, 4-8 mg / L non-essential amino acids, 20-30 mg / L recombinant human insulin, 3-6 μg / L recombinant human epidermal growth factor, 1-10 mg / L ferric citrate, 12-20 mg / L transferrin, 15-25 μg / L hydrocortisone, 0.5-2.5 mg / L gentamicin, 2-6 mg / L recombinant human serum albumin, 0.5-1.5 mg / L albumin, and 5-23 mg / L supplement; The supplement is the supplement according to any one of claims 1 to 6.

8. The serum-free mesenchymal stem cell culture medium according to claim 7, characterized in that The non-essential amino acids include one or more of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid.

9. The method for preparing the serum-free mesenchymal stem cell culture medium according to claim 7 or 8, characterized in that: The method comprises the following steps: mixing DMEM / F12 culture medium with basic fibroblast growth factor, L-glutamine, selenomethionine, non-essential amino acids, recombinant human insulin, recombinant human epidermal growth factor, ferric citrate, transferrin, hydrocortisone, gentamicin, recombinant human serum protein, albumin and supplements to obtain serum-free mesenchymal stem cell culture medium.

10. Use of the supplement according to any one of claims 1 to 6 or the serum-free mesenchymal stem cell culture medium according to claim 7 or 8 in culturing mesenchymal stem cells.

Images