Serum-free mesenchymal stem cell culture medium and preparation method thereof

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a serum-free mesenchymal stem cell culture medium and a preparation method thereof. The serum-free mesenchymal stem cell culture medium provided by the invention is prepared by taking a DMEM (Dulbecco Modified Eagle Medium) / F12 basal culture medium as a basis and adding supplementary factors, namely yeast hydrolysate, insulin, albumin, dexamethasone, taurine, salidroside, soy isoflavone, baicalein, human platelet lysate, momordica saponin and hyperoside. According to the serum-free mesenchymal stem cell culture medium provided by the invention, the normal form of cells can be maintained without supplementing serum, rapid proliferation is realized, and the biological characteristics of the mesenchymal stem cells are maintained.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and particularly relates to a serum-free mesenchymal stem cell culture medium and a preparation method thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are widely found in umbilical cord tissue, bone marrow, fat, skeletal muscle, lung, liver, pancreas and amniotic fluid. They are a type of pluripotent stem cells with high self-renewal ability and multidirectional differentiation potential. Under certain conditions, MSCs can differentiate into osteoblasts, epithelial cells, adipocytes, nerve cells, muscle cells and cardiomyocytes, etc., and have great clinical application prospects in tissue engineering repair, cell therapy and regenerative medicine.

[0003] Traditional mesenchymal stem cell culture medium requires the addition of a certain proportion of animal serum, such as fetal bovine serum (FBS), to the basal culture medium. Serum can provide a variety of nutrients necessary for cell growth, such as insulin, adrenocortical hormones, steroid hormones, albumin, vitamins, lipids, etc. However, since fetal bovine serum comes from animals and contains heterologous components, it is easy to cause immune rejection reactions in patients. There is also a safety hazard of carrying exogenous bacteria and viruses. In addition, different batches of serum differ in composition and content, which can lead to unstable cell culture effects and easily cause cell morphological variations, increasing the difficulty of production quality control and limiting the application of mesenchymal stem cells.

[0004] The culture effects of serum-free mesenchymal stem cell culture media currently developed on the market are mostly not as good as serum culture media, and the cell adhesion and proliferation performance are not ideal. For example, CN105420182A discloses a serum-free umbilical cord mesenchymal stem cell culture medium, including basal culture medium DMEM, epidermal growth factor, basic fibroblast growth factor, insulin, trypsin, 2-mercaptoethanol, sodium selenite and fibronectin, etc., and better adhesion performance is achieved by adding a higher concentration of fibronectin, but the cell proliferation rate is slow; CN102634482A discloses a serum-free complete culture medium for mesenchymal stem cells, which is composed of basal culture medium DMEM / F12 and a variety of added components, but the cultured stem cells are easy to differentiate and thus phenotypic changes occur. Therefore, it is of great significance to develop a serum-free culture medium that allows mesenchymal stem cells to proliferate stably and efficiently. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a serum-free mesenchymal stem cell culture medium, which selects specific supplementary factors to replace the function of animal serum according to the nutritional requirements of cells growing in vitro, thereby maintaining the normal morphology of cells and achieving rapid proliferation without the need for serum supplementation, and maintaining the biological characteristics of mesenchymal stem cells.

[0006] To achieve the above purpose, the specific technical solutions of the present invention are as follows: The first aspect of the present invention provides a serum-free mesenchymal stem cell culture medium, which is composed of DMEM / F12 basal culture medium, momordica charantia saponin, hyperoside, yeast hydrolyzate, insulin, albumin, dexamethasone, taurine, salidroside, soybean isoflavones, baicalein and human platelet lysate; in each 1L of serum-free mesenchymal stem cell culture medium, the mass of momordica charantia saponin is 126.98-761.88 mg, the mass of hyperoside is 0.232-2.32 mg, the mass of yeast hydrolyzate is 80.8-323.2 mg, and the mass of insulin is 1.5-2.0 mg. The mass of is 0.008~0.094mg, the mass of albumin is 0.016~0.094mg, the mass of dexamethasone is 0.0002~0.008mg, the mass of taurine is 0.0032~0.0128mg, the mass of salidroside is 0.001~0.005mg, the mass of soy isoflavones is 27.78~111.18mg, the mass of baicalein is 13.51~67.56mg, the volume fraction of human platelet lysate is 0.5%~5%, and the DMEM / F12 basal culture medium is supplemented. The human platelet lysate in the culture medium of the present invention is extracted from purified platelet-rich plasma, and contains many different cell growth factors. The growth factors derived from platelets promote the differentiation and proliferation of cells. There is no xenobiotic additive. The low-concentration human platelet lysate can partially replace fetal bovine serum and provide a variety of nutrients necessary for cell growth. Yeast hydrolysate contains nucleotides, nucleosides and bases, which increase the energy supply of cells. Insulin and dexamethasone can promote cells to utilize sugars, amino acids and the like in the culture medium for material synthesis and energy. Albumin can further improve the cell's wall adhesion performance and proliferation effect, and at the same time make the cell growth morphology complete and have good uniformity. ; Taurine is a sulfur-containing non-protein amino acid. Although it does not participate in protein synthesis, it is closely related to the metabolism of cystine and cysteine; Momordica charantia saponins, salidroside, hyperoside and baicalein can promote cell proliferation and improve cell activity; Soy isoflavones are flavonoid compounds with a similar structure to estrogen. The estrogenic effect of soy isoflavones affects hormone secretion, metabolic biological activity, protein synthesis, growth factor activity, and can reduce cell apoptosis; Adding Chinese medicinal ingredients such as momordica charantia saponins, salidroside, hyperoside, etc. to the culture medium can produce a good regulatory effect on the original cell culture medium, provide more nutrition and supplements for cells, and promote cell proliferation.

[0007] Furthermore, in each 1L of serum-free mesenchymal stem cell culture medium, the mass of momordica saponin is 253.96 mg, the mass of hyperoside is 0.928 mg, the mass of yeast hydrolysate is 161.6 mg, the mass of insulin is 0.032 mg, the mass of albumin is 0.032 mg, the mass of dexamethasone is 0.0004 mg, the mass of taurine is 0.0064 mg, the mass of salidroside is 0.003 mg, the mass of soy isoflavones is 55.56 mg, the mass of baicalein is 54.01 mg, and the volume fraction of human platelet lysate is 1%.

[0008] The second aspect of the present invention provides a use of the above-mentioned serum-free mesenchymal stem cell culture medium in culturing mesenchymal stem cells, wherein the culture medium is used to maintain the biological characteristics of mesenchymal stem cells.

[0009] Furthermore, the serum-free mesenchymal stem cell culture medium is used to maintain the expression of mesenchymal stem cell surface markers. Furthermore, the surface marker is any one or more of CD105, CD73 and CD90.

[0010] The third aspect of the present invention provides a use of the above-mentioned serum-free mesenchymal stem cell culture medium in promoting the proliferation of mesenchymal stem cells.

[0011] A fourth aspect of the present invention provides a method for preparing the above-mentioned serum-free mesenchymal stem cell culture medium, comprising the following steps: 126.98-761.88 mg of bitter melon saponin, 0.232-2.32 mg of hyperoside, 0.8-323.2 mg of yeast hydrolyzate, 0.008-0.094 mg of insulin, 0.016-0.094 mg of albumin, 0.0002-0.008 mg of dexamethasone, 0.0032-0.0128 mg of taurine, 0.001-0.005 mg of salidroside, 27.78-111.18 mg of soy isoflavones and 13.51-67.56 mg of baicalein are mixed, 10 mL of human platelet lysate is added, and then DMEM / F12 basal culture medium is used to make up to 1 L to obtain a mixed solution; the pH value and osmotic pressure of the mixed solution are adjusted, and the serum-free mesenchymal stem cell culture medium is obtained after sterilization.

[0012] Furthermore, the pH value of the serum-free mesenchymal stem cell culture medium is 7.2-7.4; the osmotic pressure of the serum-free mesenchymal stem cell culture medium is 285-300 mOsm / L.

[0013] A fifth aspect of the present invention provides a method for culturing mesenchymal stem cells, wherein the method comprises culturing mesenchymal stem cells using the serum-free mesenchymal stem cell culture medium described above.

[0014] Furthermore, the mesenchymal stem cells are bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, endometrial mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, synovial mesenchymal stem cells, lung tissue mesenchymal stem cells or menstrual blood mesenchymal stem cells.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The serum-free mesenchymal stem cell culture medium of the present invention is composed of DMEM / F12 basic culture medium, momordica charantia saponin, hyperoside, yeast hydrolyzate, insulin, albumin, dexamethasone, taurine, salidroside, soybean isoflavones, baicalein and human platelet lysate according to the nutritional requirements for cell growth.

[0016] The nutrients are balanced and the components cooperate with each other, effectively promoting the in vitro proliferation of mesenchymal stem cells. The culture medium does not contain heterologous animal-derived components and does not require the addition of fetal bovine serum to maintain the culture of mesenchymal stem cells. The present invention uses hyperoside and momordica charantia saponins as components of serum-free mesenchymal stem cell culture medium for the first time. Hyperoside, as a flavonoid compound, has anti-inflammatory and antioxidant effects. Momordica charantia saponins are mostly used in the fields of hypoglycemic and anti-cancer. Cell experiments have found that after adding hyperoside and momordica charantia saponins, the cell density, cell viability, cell growth rate and cell yield after passage of cultured mesenchymal stem cells can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is the morphology of MSCs cultured in culture medium; Figure 1 Figure A in the figure is the morphology of MSCs cultured in the culture medium of Comparative Example 1, Figure 1 Figure B is a morphological diagram of MSCs cultured in the culture medium of Example 1. Figure 1 Figure C is a morphological diagram of MSCs cultured in the culture medium of Comparative Example 2; magnification: ×40.

[0019] Figure 2 This is the growth curve of MSCs cultured in culture medium; Figure 3 The cell yield results of MSCs cultured in different culture media for three consecutive passages. P1 means cells were passaged once, P2 means cells were passaged twice, and P3 means cells were passaged three times. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0021] The DMEM / F12 basal culture medium in the present invention was purchased from Henan Punuoyi Biological Products Research Institute Co., Ltd., with the item number AD1016.

[0022] The concentration of the human platelet lysate in the present invention is 0.5% to 5%; the human platelet lysate was purchased from Henan Punuoyi Biological Products Research Institute Co., Ltd., with the item number DP1002.

[0023] Traditional mesenchymal stem cell culture medium requires the addition of a certain proportion of animal serum, such as fetal bovine serum, to the basal culture medium. However, since fetal bovine serum comes from animals and contains heterologous components, there is a safety hazard of carrying exogenous bacteria and viruses, and the cell culture effect is unstable. The culture effect of serum-free mesenchymal stem cell culture medium currently developed on the market is mostly inferior to that of serum culture medium, and the adhesion and proliferation performance are not ideal. Therefore, it is of great significance to develop a serum-free culture medium that can stably and efficiently proliferate mesenchymal stem cells.

[0024] The present invention provides a serum-free mesenchymal stem cell culture medium and a preparation method thereof. The serum-free mesenchymal stem cell culture medium provided by the present invention is prepared by adding supplementary factors: bitter melon saponin, hyperoside, yeast hydrolyzate, insulin, albumin, dexamethasone, taurine, salidroside, soybean isoflavones, baicalein and human platelet lysate based on DMEM / F12 basic culture medium; the preparation method is simple, the raw materials are easy to obtain, and experiments show that using the culture medium provided by the present invention to culture mesenchymal stem cells can achieve rapid proliferation of cells while maintaining the normal morphology and biological characteristics of the cells.

[0025] Example 1: A serum-free mesenchymal stem cell culture medium The formula of serum-free mesenchymal stem cell culture medium is shown in Table 1: Table 1 Formula of serum-free mesenchymal stem cell culture medium Preparation method of serum-free mesenchymal stem cell medium: Mix the culture medium components in Table 1 according to the corresponding proportions to obtain a mixed solution, adjust the pH value of the mixed solution to 7.3, then add sodium chloride to adjust the osmotic pressure of the solution to 290mOsm / kg, and then filter and sterilize through a 0.22μm filter membrane to obtain a serum-free mesenchymal stem cell medium.

[0026] Example 2: A serum-free mesenchymal stem cell culture medium The formula of serum-free mesenchymal stem cell culture medium is shown in Table 2: Table 2 Formula of serum-free mesenchymal stem cell culture medium Preparation method of serum-free mesenchymal stem cell medium: Mix the culture medium components in Table 2 in corresponding proportions to obtain a mixed solution, adjust the pH value of the mixed solution to 7.2, then add sodium chloride to adjust the osmotic pressure of the solution to 285mOsm / kg, and then filter and sterilize through a 0.22μm filter membrane to obtain a serum-free mesenchymal stem cell medium.

[0027] Example 3: A serum-free mesenchymal stem cell culture medium The formula of serum-free mesenchymal stem cell culture medium is shown in Table 3: Table 3 Formula of serum-free mesenchymal stem cell culture medium Preparation method of serum-free mesenchymal stem cell medium: Mix the culture medium components in Table 3 in corresponding proportions to obtain a mixed solution, adjust the pH value of the mixed solution to 7.4, then add sodium chloride to adjust the osmotic pressure of the solution to 300mOsm / kg, and then filter and sterilize through a 0.22μm filter membrane to obtain a serum-free mesenchymal stem cell medium.

[0028] Comparative Example 1: A serum-free mesenchymal stem cell culture medium The formula of serum-free mesenchymal stem cell culture medium is shown in Table 4: Table 4 Formula of serum-free mesenchymal stem cell culture medium Preparation method of serum-free mesenchymal stem cell medium: Mix the culture medium components in Table 4 in corresponding proportions to obtain a mixed solution, adjust the pH value of the mixed solution to 7.3, then add sodium chloride to adjust the osmotic pressure of the solution to 290mOsm / kg, and then filter and sterilize through a 0.22μm filter membrane to obtain a serum-free mesenchymal stem cell medium.

[0029] Comparative Example 2: A mesenchymal stem cell culture medium 10 v / v% FBS was added to the DMEM / F12 liquid basal medium and mixed to obtain a mesenchymal stem cell medium.

[0030] Examples 1 to 3 have similar effects. For the convenience of subsequent discussion and reference, the test results of Example 1 are taken as an example. The following is the test result of Example 1. The present invention further explores the ability of Example 1, Comparative Example 1 and Comparative Example 2 to culture mesenchymal stem cells through cell experiments of culturing umbilical cord blood mesenchymal stem cells.

[0031] Experimental Example 1: MSCs morphology observation, density and activity detection P3 generation MSCs were selected for the experiment and the cells were cultured at 4×10 4 The cells were inoculated at a density of / mL in a 12-well plate, with 3 replicate wells in each group. The plates were cultured in a 5% CO2 incubator at 37°C, and cell images were collected after 48 hours of culture.

[0032] The results are as follows Figure 1 As shown, Figure 1 B is a morphological diagram of MSCs cultured in Example 1, A is a morphological diagram of MSCs cultured in Comparative Example 1, and C is a morphological diagram of MSCs cultured in Comparative Example 2.

[0033] Depend on Figure 1 It can be seen that MSCs in each group grew in a single layer and adherent to the wall, and most of the cells were long spindle-shaped with irregular morphology.

[0034] After culturing for 72 h, MSCs from each group were collected by digestion with 0.25% trypsin solution, and the cell number and cell viability of each group were calculated using a Countstar cell counter. The results are shown in Table 5.

[0035] Table 5 MSCs density and activity test results As shown in Table 5, the density and activity of MSCs cultured in Example 1 and Comparative Example 1 are higher than those in Comparative Example 2, and the density and activity of MSCs cultured in Example 1 with added momordica charantia saponin and hyperoside are higher than those in Comparative Example 1.

[0036] Experimental Example 2: Detection of MSCs proliferation ability (1) P3 MSCs were selected for the experiment. The cells were seeded at a density of 2000 cells / well in a 96-well plate, with 3 replicates per group. The plates were cultured in a 5% CO2 incubator at 37°C. The CCK8 method was used to detect cell survival and proliferation every day.

[0037] (2) Before detection, replace the medium with 100 μL DMEM / F12 basal culture medium in each well.

[0038] (3) Add 10 μL of CCK8 working solution to each well and incubate in a constant temperature incubator for 3 h. Detect the OD value at 450 nm with an enzyme marker. Repeat the measurement 3 times for each group of samples and take the average value. Draw a growth curve based on the obtained OD value. The results are shown in the figure. Figure 2 shown.

[0039] from Figure 2 It can be seen from the cell growth curve that the culture medium of Comparative Example 2 can promote the growth of mesenchymal stem cells, the growth rate of the cells in the culture medium of Comparative Example 1 is similar to the growth rate of the cells in the culture medium of Comparative Example 2, and the growth rate of the cells cultured with the culture medium in Example 1 of the present invention is significantly higher than that of the Comparative Example 1 and Comparative Example 2 groups.

[0040] Experimental Example 3: Analysis of MSCs Subculture P3 generation MSCs were selected for the experiment and the cells were cultured at 4×10 4 / mL density was inoculated in a 12-well plate, and 3 replicate wells were set for each group. The plates were placed in a 5% CO2 incubator at 37°C, and the culture medium was replaced every 72 hours. When the cell confluence was about 85%, the cells were digested, the cell number was recorded, and the cells were continued to be cultured at 4×10 4 The cells were inoculated at a density of 100 μg / mL in 12-well plates for subculture, and the cells were subcultured three times.

[0041] Figure 3 The cell yields of MSCs cultured in different culture media for three consecutive passages are shown in Table 1. Figure 3 It can be seen that the cell yield of Example 1 is 1.65 times that of Comparative Example 1, and the cell yield of Comparative Example 1 is 1.21 times that of Comparative Example 2.

[0042] Experimental Example 4: Detection of MSCs surface markers P3 generation MSCs were selected for the experiment. The cells were cultured and passaged three times continuously. Cell immunostaining was performed using conventional methods, and marker analysis was performed by flow cytometry.

[0043] The test results are shown in Table 6. The mesenchymal stem cells cultured with the culture medium of Example 1 can well ensure that the biological characteristics of the mesenchymal stem cells remain unchanged. The expression rates of positive markers CD105, CD73 and CD90 are more than 95%, and CD34 and CD45 are negatively expressed, which is consistent with the biological characteristics of MSCs.

[0044] Table 6 Detection results of MSCs surface markers in each group It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0045] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A serum-free mesenchymal stem cell culture medium, characterized in that The serum-free mesenchymal stem cell culture medium consists of DMEM / F12 basal culture medium, momordica charantia saponin, hyperoside, yeast hydrolyzate, insulin, albumin, dexamethasone, taurine, salidroside, soybean isoflavones, baicalein and human platelet lysate; in each liter of the serum-free mesenchymal stem cell culture medium, the mass of momordica charantia saponin is 126.98-761.88 mg, the mass of hyperoside is 0.232-2.32 mg, the mass of yeast hydrolyzate is 80.8-323.2 mg, and the mass of insulin is 0.0 08~0.094mg, the mass of albumin is 0.016~0.094mg, the mass of dexamethasone is 0.0002~0.008mg, the mass of taurine is 0.0032~0.0128mg, the mass of salidroside is 0.001~0.005mg, the mass of soy isoflavones is 27.78~111.18mg, the mass of baicalein is 13.51~67.56mg, the volume fraction of human platelet lysate is 0.5%~5%, and the DMEM / F12 basal culture medium is supplemented.

2. The serum-free mesenchymal stem cell culture medium according to claim 1, characterized in that In each 1L serum-free mesenchymal stem cell culture medium, the mass of momordica charantia saponin is 253.96 mg, the mass of hyperoside is 0.928 mg, the mass of yeast hydrolysate is 161.6 mg, the mass of insulin is 0.032 mg, the mass of albumin is 0.032 mg, the mass of dexamethasone is 0.0004 mg, the mass of taurine is 0.0064 mg, the mass of salidroside is 0.003 mg, the mass of soy isoflavones is 55.56 mg, the mass of baicalein is 54.01 mg, and the volume fraction of human platelet lysate is 1%.

3. A method for preparing the serum-free mesenchymal stem cell culture medium according to claim 1, characterized in that: The following steps are involved: 126.98-761.88 mg of bitter melon saponin, 0.232-2.32 mg of hyperoside, 0.8-323.2 mg of yeast hydrolyzate, 0.008-0.094 mg of insulin, 0.016-0.094 mg of albumin, 0.0002-0.008 mg of dexamethasone, 0.0032-0.0128 mg of taurine, 0.001-0.005 mg of salidroside, 27.78-111.18 mg of soy isoflavones and 13.51-67.56 mg of baicalein are mixed, 10 mL of human platelet lysate is added, and then supplemented with DMEM / F12 basal culture medium to obtain a mixed solution; the pH value and osmotic pressure of the mixed solution are adjusted, and the serum-free mesenchymal stem cell culture medium is obtained after sterilization.

4. The preparation method according to claim 3, characterized in that: The pH value of the serum-free mesenchymal stem cell culture medium is 7.2-7.4; the osmotic pressure of the serum-free mesenchymal stem cell culture medium is 285-300 mOsm / L.

5. Use of the serum-free mesenchymal stem cell culture medium according to claim 1 in culturing mesenchymal stem cells, characterized in that: The serum-free mesenchymal stem cell culture medium is used to maintain the biological characteristics of mesenchymal stem cells.

6. Use of the serum-free mesenchymal stem cell culture medium according to claim 1 in promoting the proliferation of mesenchymal stem cells.

7. A method for culturing mesenchymal stem cells, characterized in that: Mesenchymal stem cells are cultured using the serum-free mesenchymal stem cell culture medium according to claim 1.

8. The culture method according to claim 7, characterized in that: The mesenchymal stem cells are bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, endometrial mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, synovial mesenchymal stem cells, lung tissue mesenchymal stem cells or menstrual blood mesenchymal stem cells.

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