Culture medium for stem cells as well as preparation method and application of culture medium

Abstract

The invention provides a culture medium for stem cells as well as a preparation method and application thereof, and the culture medium is prepared from a DMEM basic culture solution, serum, inorganic salt, a pH regulator, ascorbic acid, a growth factor, a growth factor substitute, antibiotics, a growth factor promoter and non-essential amino acid. Wherein the growth factor substitute is any one or a composition of more than one of oleanolic acid saponin, caffeine and 5-azacytidine, and by adding the growth factor substitute, the dosage of growth factors can be effectively reduced, the stem cell culture cost can be reduced, the cell culture period can be effectively shortened, and the cell culture efficiency can be improved. The stem cells are sequentially subjected to primary culture and subculture by using the culture medium provided by the invention, and then the subcultured stem cells are inoculated on a batch culture carrier, so that the batch culture efficiency of the stem cells is effectively improved, the yield of the stem cells can be improved by one order of magnitude, and the culture medium has the advantages of simplicity in operation, high cell culture efficiency and low cost. And popularization and implementation are facilitated.

Description

Technical Field

[0001] This application belongs to the technical field of cell culture, and specifically relates to a culture medium for stem cells, a preparation method thereof, and an application thereof. Background Art

[0002] In the prior art, human umbilical cord mesenchymal stem cells (hUC-MSCs) are a type of multipotent stem cells present in neonatal umbilical cord tissue, with the potential for self-renewal, high proliferation, and multi-directional differentiation. They can differentiate into many types of tissue cells, such as bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium, and myocardium, etc., and have broad application prospects in the fields of tissue engineering and regenerative medicine. They are widely used in spinal cord injury repair, Alzheimer's disease treatment, heart failure and coronary heart disease treatment, systemic lupus erythematosus treatment, and liver failure treatment.

[0003] In the prior art, during the culture process of human umbilical cord mesenchymal stem cells (hUC-MSCs), although they have broad application prospects, there are still some challenges related to culture efficiency. The existing methods for culturing human umbilical cord mesenchymal stem cells are usually the tissue block method and the enzyme digestion method. In the actual application process, on the one hand, the stem cells in the tissue block have a long growth cycle in the existing culture medium, and the consumption of growth factors is large, significantly increasing the culture cost of human umbilical cord mesenchymal stem cells and making it inconvenient for clinical promotion and use. On the other hand, the culture conditions of human umbilical cord mesenchymal stem cells are complex, the pollution risk is high, and the dosage of antibiotics is high, further reducing the culture efficiency and culture cost of human umbilical cord mesenchymal stem cells. Therefore, improvements are urgently needed now. Summary of the Invention

[0004] This application aims to solve the technical problems in the prior art that during the traditional culture process of human umbilical cord mesenchymal stem cells, affected by the existing culture medium, the culture cycle of human umbilical cord mesenchymal stem cells is long. After the primary cell culture is completed, batch culture for 5D - 7D is still required. During the whole culture process, to meet the proliferation requirements of cells, the consumption of growth factors is large, significantly increasing the culture cost of human umbilical cord mesenchymal stem cells, and the culture conditions of human umbilical cord mesenchymal stem cells are complex, the pollution risk is high, and the dosage of antibiotics is high, further reducing the culture efficiency of human umbilical cord mesenchymal stem cells. A culture medium for stem cells that can significantly shorten the culture cycle of human umbilical cord mesenchymal stem cells is proposed;

[0005] To solve the technical problems proposed in this application, this application also provides a preparation method of a culture medium for stem cells;

[0006] To solve the technical problems proposed in this application, this application also provides an application of a culture medium for stem cells.

[0007] The present application adopts the following solution. A culture medium for stem cells is composed of the following components: DMEM basal culture medium, 3.2 mg / L - 4.8 mg / L fetal bovine serum, inorganic salts, pH regulator, 50 mg / L - 100 mg / L ascorbic acid, growth factors, growth factor substitutes, antibiotics, growth factor promoters, and 5 mmol / L - 25 mmol / L non-essential amino acids;

[0008] The growth factor is selected from any one or a mixture of more than one of bFGF and transforming growth factor β;

[0009] The growth factor substitute is selected from any one or a combination of more than one of oleanolic acid saponin, caffeine, and 5-azacytidine;

[0010] The growth factor promoter is selected from any one or a combination of more than one of insulin, saturated transferrin, and adenine;

[0011] The non-essential amino acids are one or a mixture of two or more of glutamic acid, alanine, glycine, aspartic acid, cystine, proline, serine, or tyrosine.

[0012] In the actual implementation process, for the convenience of measurement, the concentration of each component represents the mass required for preparing 1 L of the culture medium for stem cell culture. Exemplarily: 3.2 mg / L fetal bovine serum means that 3.2 mg of fetal bovine serum is required for preparing 1 L of the culture medium for stem cell culture; 0.005 mol / L selenic acid means that 0.005 mol of selenic acid is required for preparing 1 L of the culture medium for stem cell culture.

[0013] In some feasible embodiments, the growth factor is selected as a mixture of 0.1 μg / L - 1.5 μg / L bFGF and 0.5 μg / L - 0.8 μg / L transforming growth factor β.

[0014] In the actual implementation process, both bFGF and transforming growth factor β are purchased from Gibco.

[0015] In some feasible embodiments, the growth factor substitute is selected as a mixture of 22 μg / L - 31 μg / L oleanolic acid saponin, 11 μg / L - 18 μg / L caffeine, and 2 μg / L - 4 μg / L 5-azacytidine.

[0016] In the actual implementation process, oleanolic acid saponin is purchased from Sigma-Aldrich.

[0017] In the actual implementation process, oleanolic acid saponins can regulate the proliferation and differentiation of NSCs by improving the microenvironment, transcription factors, and signaling pathways of NSCs. Oleanolic acid saponins can activate the phosphatidylinositol 3 (PI3K) / protein kinase B intracellular signaling pathway, thereby promoting the proliferation of stem cells.

[0018] In some feasible embodiments, the growth factor promoter is a mixture of 5 μg / L - 8 μg / L insulin, 7 μg / L - 10 μg / L saturated transferrin, and 10 μg / L - 15 μg / L adenine.

[0019] In the actual implementation process, saturated transferrin is purchased from Pepro-Tech.

[0020] In some feasible embodiments, the inorganic salt is any one or a combination of more than one of sodium chloride and sodium selenide;

[0021] The pH regulator is any one or a combination of more than one of PBS buffer, sodium bicarbonate, and selenic acid;

[0022] The antibiotic is any one or a combination of more than one of penicillin, erythromycin, tetracycline, kanamycin, streptomycin, and erythromycin.

[0023] In some feasible embodiments, the inorganic salt is a mixture of 1.05 g / L - 1.25 g / L sodium chloride solution and 10 μg / L - 15 μg / L sodium selenide solution;

[0024] The pH regulator is a mixture of 0.01 mol / L PBS buffer and 0.005 mol / L selenic acid;

[0025] The antibiotic is a mixture of 5 mg / L - 10 mg / L penicillin and 1 mg / L - 3.3 mg / L tetracycline.

[0026] To solve the technical problems proposed by this application, this application also provides a preparation method for a culture medium for stem cells, including the following steps: selecting the target components of the stem cell culture medium, and mixing all the target components in sequence according to the target dosages, then obtaining the culture medium for stem cells.

[0027] To solve the technical problems proposed by this application, this application also provides an application of a culture medium for stem cells, for batch culturing human umbilical cord mesenchymal stem cells;

[0028] Batch culturing human umbilical cord mesenchymal stem cells includes the following steps;

[0029] Primary culture: Tissue pieces are isolated from discarded umbilical cord tissue and adhered to the bottom of culture dish A. After adhesion, a culture medium for stem cells described in any one of claims 1-6 is added to culture dish A. After culturing for 3D-5D, primary human umbilical cord mesenchymal stem cells are obtained;

[0030] Subculture: Using a sterile pipette, trypsin and 0.01 mol / L PBS buffer are successively dropped into the primary human umbilical cord mesenchymal stem cells in culture dish A. After dropping, culture dish A is shaken. After shaking, an excessive amount of 3.2 mg / L fetal bovine serum is dropped into culture dish A to terminate the digestion of trypsin. After the digestion is terminated, an appropriate amount of sterile normal saline is dropped into culture dish A. Culture dish A is shaken and the cell suspension in culture dish A is collected. The cell density in the cell suspension is adjusted to 5×10 4 / mL. After the cell suspension is transferred to a centrifuge tube and centrifuged, the supernatant in the centrifuge tube is removed. Then, a culture medium for stem cells described in any one of claims 1-6 is added to the centrifuge tube. After the cells at the bottom of the centrifuge tube are mixed with the culture medium, cell culture solution X is obtained. Cell culture solution X is spread on culture dish B through a sterile pipette. After culturing for 2D-4D, N-generation human umbilical cord mesenchymal stem cells are obtained;

[0031] Batch culture: The batch culture carrier is placed in culture dish C. The N-generation human umbilical cord mesenchymal stem cells are activated and mixed with an appropriate amount of sterile normal saline. After mixing, cell culture solution Y is obtained. A culture medium for stem cells described in any one of claims 1-6 and cell culture solution Y are successively spread on the batch culture carrier, and then culture dish C is transferred to a cell culture incubator.

[0032] In the actual implementation process, during the stem cell culture process, by setting the batch culture carrier, the paracrine effect of stem cells can be enhanced, the concentration of stem cell autocrine growth factors can be significantly increased, thereby significantly shortening the stem cell culture cycle. With the cooperation of growth factor substitutes and low-concentration growth factors, the stem cells are activated, enabling the stem cells to enter the logarithmic growth phase 2D-4D earlier and significantly improving the stem cell culture efficiency.

[0033] In some feasible embodiments, the culture conditions for the primary culture, subculture, and batch culture are all 5% CO 2 , 37 °C.

[0034] In some feasible embodiments, the cell density for subculture is 5×10 4 / mL;

[0035] The initial seeding density in batch culture is 5×10 3 / mL, and the cell density for batch culture is 5×10 5-1×10 6 / mL.

[0036] Compared with the prior art, the present application has the following beneficial effects:

[0037] The present application provides a culture medium for stem cells, a preparation method thereof and an application. The culture medium is composed of DMEM basal culture solution, serum, inorganic salts, pH regulator, ascorbic acid, growth factors, growth factor substitutes, antibiotics, growth factor promoters and non-essential amino acids. The growth factor substitute is selected from any one or more combinations of oleanolic acid saponin, caffeine, 5-azacytidine, and the growth factor promoter is selected from any one or more combinations of insulin, saturated transferrin, adenine. Through the cooperation of the growth factor promoter and the growth factor substitute, not only can the dosage of the growth factor be effectively reduced, the cost of stem cell culture be reduced, but also the culture cycle of cells can be effectively shortened. During the batch culture process of stem cells, after the stem cells are successively subjected to primary culture and passage culture using the culture medium provided by the present application, and then the passaged stem cells are inoculated on the batch culture carrier, the batch culture efficiency of stem cells is effectively improved, and the yield of stem cells can be increased by an order of magnitude. It has the advantages of simple operation, high cell culture efficiency, significantly improving the commercial value of stem cells, and being convenient for popularization and implementation. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a schematic diagram during the batch culture process of stem cells of the present application;

[0039] Figure 2 is the stem cell growth curve during the batch culture process of stem cells in Examples 1-3 of the present application;

[0040] Figure 3 is the stem cell growth curve during the batch culture process of stem cells in Example 1 and Comparative Examples 1-3 of the present application;

[0041] Figure 4 is the stem cell growth curve during the batch culture process of stem cells in Example 1, Comparative Example 1 and Comparative Example 4 of the present application;

[0042] Figure 5 is the stem cell growth curve during the batch culture process of stem cells in Example 1, Comparative Example 1, Comparative Example 5 and Comparative Example 6 of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0043] Combined with Figures 1-5 , Examples 1-3 and Comparative Examples 1-6, the technical solution provided by the present application is further described.

[0044] Among them, for the convenience of measurement, the concentration of each component represents the mass required for preparing 1 L of the culture medium for stem cells. Exemplarily, 3.2 mg / L fetal bovine serum means that 3.2 mg of fetal bovine serum is required for preparing 1 L of the culture medium for stem cells; 0.005 mol / L selenic acid means that 0.005 mol of selenic acid is required for preparing 1 L of the culture medium for stem cells.

[0045] Among them, the discarded umbilical cord tissue is taken from the umbilical cords discarded by newborns in the obstetrics and gynecology department.

[0046] As Figure 1 shown, during the mass culture of stem cells, the mass culture carrier 1 is located at the center of the culture dish 2;

[0047] There is a spacing h1 between the mass culture carrier 1 and the culture dish 2, and the length of the mass culture carrier 1 is h2. h1 and h2 satisfy the following relationship: h2 / h1 = 3.5;

[0048] The height of the culture dish 2 is t1, and the height of the mass culture carrier 1 is t2. t1 and t2 satisfy the following relationship: t1 / t2 = 3.

[0049] The material of the mass culture carrier 1 is gelatin, and the porosity of the mass culture carrier 1 is 82%.

[0050] Example 1

[0051] (1) The preparation method of the culture medium includes the following steps:

[0052] After sequentially mixing the target components according to the component table shown in Table 1 according to the target dosages, the culture medium U is obtained.

[0053] (2) The mass culture of human umbilical cord mesenchymal stem cells includes the following steps:

[0054] Primary culture: Tissue blocks are isolated from the discarded umbilical cord tissue and adhered to the bottom of the culture dish A. After the adhesion is completed, the culture medium U is added to the culture dish A1. After culturing to the plateau phase under the conditions of 5% CO 2 , 37 °C, the primary human umbilical cord mesenchymal stem cells are obtained;

[0055] Subculture: Using a sterile pipette, trypsin and 0.01 mol / L PBS buffer are sequentially dropped into the primary human umbilical cord mesenchymal stem cells in the culture dish A1. After the dropping is completed, the culture dish A is shaken. After the shaking is completed, an excessive amount of 3.2 mg / L fetal bovine serum is dropped into the culture dish A1 to terminate the digestion of trypsin. After the digestion is terminated, an appropriate amount of sterile normal saline is dropped into the culture dish A, the culture dish A1 is shaken and the cell suspension in the culture dish A1 is collected, and the cell density in the cell suspension is adjusted to 5×10 4 / mL, transfer the cell suspension to a centrifuge tube. After centrifugation, remove the supernatant in the centrifuge tube, then add medium U to the centrifuge tube. After the cells at the bottom of the centrifuge tube are mixed with the medium, cell culture solution X is obtained. Spread cell culture solution X on culture dish B1 through a sterile pipette. Under the conditions of 5% CO 2 and cultured at 37 °C until the plateau phase is reached, then the P1 generation of human umbilical cord mesenchymal stem cells is obtained. Repeat the subculture until the P6 generation of human umbilical cord mesenchymal stem cells is obtained;

[0056] Batch culture: Place the batch culture carrier in culture dish C1, and the initial seeding density is 5×10 3 / mL. After activating the P6 generation of human umbilical cord mesenchymal stem cells and mixing them with an appropriate amount of sterile normal saline, after the mixing is completed, cell culture solution Y is obtained. After spreading medium U and cell culture solution Y on the batch culture carrier in sequence, transfer culture dish C1 to a cell incubator and culture it under the conditions of 5% CO 2 and 37 °C.

[0057] Example 2

[0058] (1) The preparation method of the medium includes the following steps:

[0059] Mix each target component in sequence according to the target dosage shown in Table 1, then medium V is obtained.

[0060] (2) The batch culture of human umbilical cord mesenchymal stem cells includes the following steps:

[0061] Primary culture: Isolate tissue blocks from discarded umbilical cord tissue and adhere the tissue blocks to the bottom of culture dish A. After the adhesion is completed, add medium V to culture dish A2. Under the conditions of 5% CO 2 and cultured at 37 °C until the plateau phase is reached, then the primary human umbilical cord mesenchymal stem cells are obtained;

[0062] Subculture: Use a sterile pipette to sequentially drop trypsin and 0.01 mol / L PBS buffer solution into the primary human umbilical cord mesenchymal stem cells in culture dish A2. After the dropping is completed, shake culture dish A. After the shaking is completed, drop an excessive amount of 3.2 mg / L fetal bovine serum into culture dish A2 to terminate the digestion of trypsin. After the digestion is terminated, drop an appropriate amount of sterile normal saline into culture dish A, shake culture dish A2 and collect the cell suspension in culture dish A2, and adjust the cell density in the cell suspension to 5×10 4 / mL. Transfer the cell suspension to a centrifuge tube. After centrifugation, remove the supernatant in the centrifuge tube, then add medium V to the centrifuge tube. After the cells at the bottom of the centrifuge tube are mixed with the medium, cell culture solution X is obtained. Spread cell culture solution X on culture dish B2 through a sterile pipette. Under the conditions of 5% CO 2Cultivate under the condition of 37°C until the plateau phase is reached, and the P1 generation of human umbilical cord mesenchymal stem cells is obtained. Repeat the subculture to obtain the P6 generation of human umbilical cord mesenchymal stem cells;

[0063] Batch cultivation: Place the batch cultivation carrier in Petri dish C2, with an initial inoculation density of 5×10 3 / mL. After activating the P6 generation of human umbilical cord mesenchymal stem cells and mixing them with an appropriate amount of sterile physiological saline, after the mixing is completed, cell culture medium Y is obtained. After sequentially spreading the culture medium V and cell culture medium Y on the batch cultivation carrier, transfer Petri dish C2 to the cell culture incubator and cultivate under the conditions of 5% CO 2 2 and 37°C.

[0064] Example 3

[0065] (1) The preparation method of the culture medium includes the following steps:

[0066] Mix each target component according to the target dosage in the component table shown in Table 1 in sequence, and then the culture medium W is obtained.

[0067] (2) The batch cultivation of human umbilical cord mesenchymal stem cells includes the following steps:

[0068] Primary culture: Isolate tissue blocks from discarded umbilical cord tissues, and adhere the tissue blocks to the bottom of Petri dish A. After the adhesion is completed, add the culture medium W to Petri dish A3, and cultivate under the conditions of 5% CO 2 2 and 37°C until the plateau phase is reached, and then the primary human umbilical cord mesenchymal stem cells are obtained;

[0069] Subculture: Use a sterile pipette to sequentially drip trypsin and 0.01 mol / L PBS buffer solution into the primary human umbilical cord mesenchymal stem cells in Petri dish A3. After the dripping is completed, shake Petri dish A. After the shaking is completed, drip an excessive amount of 3.2 mg / L fetal bovine serum into Petri dish A3 to terminate the digestion of trypsin. After the digestion is terminated, drip an appropriate amount of sterile physiological saline into Petri dish A, shake Petri dish A3 and collect the cell suspension in Petri dish A3. Adjust the cell density in the cell suspension to 5×10 4 / mL. After transferring the cell suspension to a centrifuge tube for centrifugation and removing the supernatant in the centrifuge tube, add the culture medium W to the centrifuge tube. After the cells at the bottom of the centrifuge tube are mixed with the culture medium, cell culture medium X is obtained. Spread cell culture medium X on Petri dish B3 through a sterile pipette, and cultivate under the conditions of 5% CO 2 2 and 37°C until the plateau phase is reached, and the P1 generation of human umbilical cord mesenchymal stem cells is obtained. Repeat the subculture to obtain the P6 generation of human umbilical cord mesenchymal stem cells;

[0070] Batch cultivation: Place the batch cultivation carrier in Petri dish C3, with an initial inoculation density of 5×10 3 / mL. After activating the passage 6 human umbilical cord mesenchymal stem cells and mixing them with an appropriate amount of sterile saline, after the mixing is completed, the cell culture medium Y is obtained. After successively spreading the culture medium W and the cell culture medium Y on the batch culture carrier, the culture dish C3 is transferred to a cell culture incubator and cultured under the conditions of 5% CO 2 and 37 °C.

[0071] Comparative Example 1-4 and Comparative Example 6

[0072] Prepare the culture medium according to the component table shown in Table 1, and use this culture medium to batch-culture human umbilical cord mesenchymal stem cells.

[0073] Comparative Example 5

[0074] Remove the batch culture carrier in the culture dish C1 during the batch culture process in Example 1, and directly culture the passage 6 human umbilical cord mesenchymal stem cells on the surface of the culture dish C1, with the remaining conditions remaining unchanged.

[0075] Table 1 Component table of Examples 1-3 and Comparative Examples 1-6

[0076]

[0077]

[0078] Continued Table 1

[0079]

[0080]

[0081] In Examples 1-3 and Comparative Examples 1-6, during the batch culture process, the culture medium was changed every day, and a cell counter was used for counting. After culturing for 10 days, with the number of culture days as the abscissa and the cell number as the ordinate, a growth curve was plotted, and the growth curve is as Figures 2-5 shown.

[0082] From Figure 2 it can be seen that the present application provides a culture medium for stem cells. By adding a growth factor substitute on the basis of a basal medium and performing amplification culture on a batch culture carrier, it can not only effectively shorten the batch culture cycle of stem cells, but also increase the single culture scale of stem cells to 10 6 levels, significantly improving the culture efficiency of stem cells and significantly enhancing the commercial value of stem cells;

[0083] From Table 1 and Figure 3It can be seen that in Comparative Example 1, the growth factor substitute was completely removed. During the stem cell culture process, the mechanism of action of bFGF is that bFGF binds to the tyrosine kinase receptor (FGFR) on the cell surface, activates the FGF / FGFR signaling pathway, promotes cell proliferation, and maintains the undifferentiated state of the cells; the mechanism of action of transforming growth factor β is that transforming growth factor β binds to the transforming growth factor β receptor on the cell surface to maintain the undifferentiated state of the cells. bFGF and transforming growth factor β cooperate to activate the intracellular proliferation signaling pathway simultaneously, and jointly promote the rapid proliferation of stem cells.

[0084] During the stem cell culture process, the dosages of bFGF and transforming growth factor β are usually 10 μg / L. As can be seen from Table 1, after completely removing the growth factor substitute in Comparative Example 1, the growth factor dosage is lower than the conventional dosage (only 0.1 μg / L), and the stem cell proliferation rate is significantly reduced. In Example 1, after the stem cells were cultured from 2D to 3D, the stem cells entered the logarithmic growth phase. After culturing for 4D - 5D, the stem cells entered the plateau phase, and the culture scale was 10 6 levels. In Comparative Example 1, after the stem cells were cultured for 5D - 7D, the stem cells entered the logarithmic growth phase. After culturing for 7D - 8D, the stem cells entered the plateau phase, and the culture scale was only 10 3 -10 4 levels, and the culture efficiency was significantly reduced, that is, the growth factor substitute can effectively replace the growth factor and reduce the dosage of the growth factor during the stem cell culture process.

[0085] As can be seen from Table 1 and Figure 3 it can be seen that in Comparative Example 2, oleanolic acid saponin was completely removed, and in Comparative Example 3, caffeine was completely removed. In Comparative Example 2, after the stem cells were cultured for 5D - 6D, the stem cells entered the logarithmic growth phase. After culturing for 7D - 8D, the stem cells entered the plateau phase, and the culture scale was 10 4 -10 5 levels. In Comparative Example 3, after the stem cells were cultured for 5D - 6D, the stem cells entered the logarithmic growth phase. After culturing for 6D - 7D, the stem cells entered the plateau phase, and the culture scale was 10 4 -10 5 levels, that is, both oleanolic acid saponin and caffeine can effectively replace the growth factor, and under the synergistic effect of oleanolic acid saponin and caffeine, the logarithmic growth phase of stem cells can be advanced by 2D - 4D, significantly improving the culture efficiency of stem cells.

[0086] During the process of stem cell culture, oleanolic acid saponins can promote the proliferation of stem cells by regulating the intracellular signaling pathways, inhibit the phosphorylation levels of signaling pathways such as ERK, p38, and JNK, reduce oxidative stress damage, slow down the aging rate of stem cells, and improve the cell proliferation efficiency; caffeine can promote the proliferation and migration of stem cells by regulating the intracellular cAMP level, activating the cAMP-dependent exchange protein, increasing the scale of cell proliferation, and Figure 3 it is known that there is a synergistic effect between oleanolic acid saponins and caffeine. Under the action of caffeine in regulating the metabolic efficiency of stem cells, the anti-inflammatory and antioxidant environment provided by oleanolic acid saponins can significantly improve the stem cell proliferation efficiency and increase the scale of stem cell culture under the condition of extremely low concentrations of growth factors.

[0087] As shown in Table 1 and Figure 4 it is known that after completely removing the growth factor substitute in the comparative example and reducing the growth factor to the conventional dosage, in Comparative Example 4, after culturing the stem cells for 5D - 6D, the stem cells enter the logarithmic growth phase, and after culturing for 7D - 8D, the stem cells enter the plateau phase, and the culture scale is 10 4 -10 54 level, that is, oleanolic acid saponins and caffeine can not only effectively replace growth factors, reduce the dosage of growth factors, but also produce a synergistic effect, significantly improve the stem cell proliferation efficiency, advance the logarithmic growth phase of stem cells by 2D - 4D, increase the scale of stem cell culture, and increase the stem cell culture scale from 10 4 to 10 6 .

[0088] As shown in Table 1 and Figure 5 it is known that in Comparative Example 5, after removing the batch culture carrier and directly inoculating the passaged stem cells on the culture dish, in Comparative Example 5, after culturing the stem cells for 2D - 3D, the stem cells enter the logarithmic growth phase, and after culturing for 4D - 5D, the stem cells enter the plateau phase, and the culture scale is 10 5 level, that is, on the premise of using a growth factor substitute, by designing the structure and material of the batch culture carrier, compared with directly inoculating cells on the surface of the culture dish, the cell proliferation scale can be increased from 10 5 to 10 6 while ensuring the cell proliferation efficiency.

[0089] During the process of stem cell culture, by using gelatin as the batch culture carrier and designing the porosity of gelatin, when P6-generation stem cells are inoculated on it, it can effectively promote the paracrine effect of stem cells, and under the action of the growth factor substitute, it can effectively improve the proliferation, attachment, and migration of stem cells, enabling the stem cells to rapidly proliferate on the batch carrier. The cell proliferation scale can be increased from 10 5 to 106 。

[0090] As can be seen from Table 1 and Figure 5 it can be known that in Comparative Example 6, the growth factor substitute was completely removed and an appropriate amount of antibiotics was added. After increasing the antibiotic dosage, the cell number increased slightly, and the proliferation efficiency did not increase significantly. That is, when the growth factor substitute was completely removed, low-concentration growth factors were not conducive to maintaining the anti-inflammatory, antioxidant, and antibacterial states of the culture dish. After appropriately supplementing antibiotics, it was beneficial to the recovery of cell number. That is, by adding growth factor analogs, the dosage of growth factors and antibiotics can be effectively reduced, the culture cost of stem cells can be effectively reduced, and the commercial value of stem cells can be increased.

[0091] The present application provides a culture medium for stem cells, its preparation method and application. The culture medium is composed of DMEM basal culture solution, serum, inorganic salts, pH regulator, ascorbic acid, growth factors, growth factor substitutes, antibiotics, growth factor promoters, and non-essential amino acids. The growth factor substitute is selected from any one or more combinations of oleanolic acid saponin, caffeine, and 5-azacytidine. The growth factor promoter is selected from any one or more combinations of insulin, saturated transferrin, and adenine. Through the cooperation of the growth factor promoter and the growth factor substitute, not only can the dosage of growth factors be effectively reduced, the culture cost of stem cells be reduced, but also the culture cycle of cells can be effectively shortened. During the batch culture process of stem cells, after the stem cells are successively subjected to primary culture and subculture using the culture medium provided by the present application, and then the subcultured stem cells are inoculated on the batch culture carrier, the batch culture efficiency of stem cells is effectively improved, and the yield of stem cells can be increased by an order of magnitude. It has the advantages of simple operation, high cell culture efficiency, significantly increasing the commercial value of stem cells, and being convenient for popularization and implementation.

[0092] The above are only the embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A culture medium for stem cells, characterized in that The invention is composed of the following components: DMEM basal culture medium, 3.2mg / L-4.8mg / L fetal bovine serum, inorganic salts, pH regulator, 50mg / L-100mg / L ascorbic acid, growth factors, growth factor substitutes, antibiotics, growth factor promoters and 5mmol / L-25mmol / L non-essential amino acids; The growth factor is selected from any one of bFGF and transforming growth factor β or a mixture of more than one of them; The growth factor substitute is selected from any one of oleanolic acid saponin, caffeine, 5-azacytidine or a combination of more than one of them; The growth factor promoter is selected from any one of insulin, saturated transferrin, and adenine, or a combination of more than one of them; The non-essential amino acid is one of glutamic acid, alanine, glycine, aspartic acid, cystine, proline, serine or tyrosine, or a mixture of two or more thereof.

2. A culture medium for stem cells according to claim 1, characterized in that: The growth factor is a mixture of 0.1 μg / L-1.5 μg / L bFGF and 0.5 μg / L-0.8 μg / L transforming growth factor β.

3. A culture medium for stem cells according to claim 1, characterized in that: The growth factor substitute is a mixture of 22 μg / L-31 μg / L oleanolic acid saponin, 11 μg / L-18 μg / L caffeine and 2 μg / L-4 μg / L 5-azacytidine.

4. A culture medium for stem cells according to claim 1, characterized in that: The growth factor promoter is a mixture of 5 μg / L-8 μg / L insulin, 7 μg / L-10 μg / L saturated transferrin and 10 μg / L-15 μg / L adenine.

5. A culture medium for stem cells according to claim 1, characterized in that: The inorganic salt is selected from any one of sodium chloride and sodium selenide or a combination of more than one of them; The pH regulator is selected from any one of PBS buffer, sodium bicarbonate, selenic acid, or a combination of more than one of them; The antibiotic is selected from any one of penicillin, erythromycin, tetracycline, kanamycin, streptomycin and erythromycin, or a combination of more than one of them.

6. A culture medium for stem cells according to claim 5, characterized in that: The inorganic salt is selected from a mixture of 1.05 g / L-1.25 g / L sodium chloride solution and 10 μg / L-15 μg / L sodium selenide solution; The pH regulator is a mixture of 0.01 mol / L PBS buffer and 0.005 mol / L selenic acid; The antibiotic is selected from a mixture of 5mg / L-10mg / L penicillin and 1mg / L-3.3mg / L tetracycline.

7. A method for preparing a culture medium for stem cells according to any one of claims 1 to 6, characterized in that: The following steps are involved: The target components of the stem cell culture medium are selected, and all the target components are mixed in sequence according to the target dosage to obtain the culture medium for stem cells.

8. Use of a culture medium for stem cells according to any one of claims 1 to 6, characterized in that: Used for mass culture of human umbilical cord mesenchymal stem cells; The batch culture of human umbilical cord mesenchymal stem cells includes the following steps: Primary culture: separating tissue blocks from discarded umbilical cord tissues and adhering the tissue blocks to the bottom of a culture dish A. After the adhesion is completed, adding a culture medium for stem cells according to any one of claims 1 to 6 to the culture dish A, and culturing for 3D to 5D to obtain primary human umbilical cord mesenchymal stem cells; Subculture: Use a sterile pipette to drip trypsin and 0.01 mol / L PBS buffer into the primary human umbilical cord mesenchymal stem cells in culture dish A in sequence. After the dripping is completed, shake the culture dish A. After the shaking is completed, drip an excess of 3.2 mg / L fetal bovine serum into the culture dish A to terminate the digestion of trypsin. After the digestion is terminated, drip an appropriate amount of sterile saline into the culture dish A, shake the culture dish A and collect the cell suspension in the culture dish A. Adjust the cell density in the cell suspension to 5×10 4 / mL, transferring the cell suspension to a centrifuge tube for centrifugation, removing the supernatant in the centrifuge tube, adding a culture medium for stem cells according to any one of claims 1 to 6 to the centrifuge tube, and mixing the cells at the bottom of the centrifuge tube with the culture medium to obtain a cell culture solution X, and spreading the cell culture solution X in a culture dish B through a sterile pipette, and culturing for 2D-4D to obtain N-generation human umbilical cord mesenchymal stem cells; Batch culture: a batch culture carrier is placed in a culture dish C, N-generation human umbilical cord mesenchymal stem cells are activated and mixed with an appropriate amount of sterile saline, and after mixing, a cell culture solution Y is obtained. A culture medium for stem cells according to any one of claims 1 to 6 and the cell culture solution Y are spread on the batch culture carrier in sequence, and the culture dish C is transferred to a cell culture incubator.

9. The use of a culture medium for stem cells according to claim 8, characterized in that: The culture conditions for the primary culture, subculture and batch culture are all 5% CO2 and 37°C.

10. The use of a culture medium for stem cells according to claim 8, characterized in that: The cell density of subculture was 5×10 4 (cells / ml); The cell density of batch culture was 5×10 5 -1×10 6 (cells / ml).

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