Umbilical cord mesenchymal stem cell adipogenesis induced differentiation culture medium and culture method

Abstract

The invention relates to the technical field of stem cell induced differentiation, and discloses an umbilical cord mesenchymal stem cell adipogenic induced differentiation culture medium and a culture method. The culture medium comprises the following substances: a DMEM (Dulbecco's Modified Eagle Medium) / F12 basal culture medium, fetal calf serum, insulin, penicillin, streptomycin, dexamethasone, 3-isobutyl-1-methylxanthine, indometacin, BMP-2 and FGF-1 (Fibroblast Growth Factor 1). The culture method comprises the following steps: (1) carrying out primary isolation culture on umbilical cord mesenchymal stem cells; (2) carrying out passage on the umbilical cord mesenchymal stem cells; and (3) inducing adipogenesis of the umbilical cord mesenchymal stem cells by using the adipogenesis induced differentiation culture medium. BMP-2 and FGF-1 are added on the basis of a traditional adipogenic induction culture medium to induce and promote the umbilical cord mesenchymal stem cells to be more efficiently differentiated into adipocytes under the combined action, so that the adipogenic differentiation time is shortened, and the adipogenic differentiation efficiency is improved; the umbilical cord mesenchymal stem cell adipogenic culture medium is stable in formula, simple and convenient in culture method and suitable for wide popularization.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell induction differentiation, and in particular to an umbilical cord mesenchymal stem cell adipogenic induction differentiation culture medium and a culture method. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Mesenchymal stem cells (MSCs) derived from the embryonic mesoderm are multipotent stem cells with high self-renewal ability and multidirectional differentiation potential. They are widely present in bone marrow, fat, amnion, placenta, umbilical cord blood and umbilical cord tissue, and can be induced to differentiate into adipocytes, osteoblasts, cardiomyocytes, islet-like cells, vascular endothelial cells and epidermal cells in vitro. In addition, human umbilical cord mesenchymal stem cells are widely used in experimental and clinical research in cell therapy and regenerative medicine because of their low immunogenicity, multidirectional differentiation ability, immunomodulatory and anti-inflammatory effects. In addition, their collection is simple and convenient, and their biological characteristics are still maintained after continuous subculture and cryopreservation. Transplanted human umbilical cord mesenchymal stem cells can secrete various effector factors, exert immunomodulatory effects, and have clear efficacy on various injuries, degenerative and inflammatory diseases, which is of great significance in stem cell therapy, tissue engineering therapy and regenerative medicine research and application.

[0004] The experiment of adipogenic differentiation of mesenchymal stem cells verified from a scientific perspective that MSCs have the biological characteristics of directional differentiation into adipocytes, providing a test basis for the evaluation of stem cell functions. However, there are many problems with the existing adipogenic differentiation technology: cells gradually fall off and die during the adipogenic differentiation process, the adipogenic differentiation induction time is long, the commercial induction differentiation medium is expensive, and only a small number of cells differentiate successfully during the induction differentiation process, the induction differentiation rate is low, and the operation is unstable. There is currently no simpler and more efficient method for adipogenic differentiation of mesenchymal stem cells.

[0005] In view of the shortcomings and deficiencies of existing adipogenic differentiation culture media, there is an urgent need for a method for inducing adipogenesis of mesenchymal stem cells with a high adipogenic differentiation rate, short differentiation time, convenient and stable operation. Summary of the invention

[0006] The purpose of the present invention is to provide an umbilical cord mesenchymal stem cell adipogenic differentiation medium and culture method to address the current problems of low differentiation efficiency, unsatisfactory adipogenic effect, and long differentiation time in stem cell induction into adipocytes, which can induce the differentiation of mesenchymal stem cells into adipocytes more efficiently and stably in a shorter time, and provide a faster detection method for the adipogenic function detection of mesenchymal stem cells.

[0007] The technical solution of the present invention is as follows:

[0008] An umbilical cord mesenchymal stem cell adipogenic differentiation medium, comprising the following components:

[0009] DMEM / F12 basal medium, fetal bovine serum, insulin, penicillin, streptomycin, dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, 8-15ng / mL BMP-2 and 8-15ng / mL FGF-1.

[0010] According to a preferred embodiment, the umbilical cord mesenchymal stem cell adipogenic differentiation medium comprises the following components:

[0011] DMEM / F12 basal medium, fetal bovine serum, insulin, penicillin, streptomycin, dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, 8-12 ng / mL BMP-2 and 8-12 ng / mL FGF-1.

[0012] According to a preferred embodiment, the umbilical cord mesenchymal stem cell adipogenic differentiation medium comprises the following components:

[0013] DMEM / F12 basal culture medium, volume concentration 8%-12% fetal bovine serum, 8-10μg / ml insulin, mass concentration 0.8%-1.2% penicillin, mass concentration 0.8%-1.2% streptomycin, 0.8-1.2μM dexamethasone, 0.01-0.05mM 3-isobutyl-1-methylxanthine, 200-250μM indomethacin, 8-15ng / mL BMP-2, 8-15ng / mL FGF-1.

[0014] According to a preferred embodiment, the mesenchymal stem cell adipogenic differentiation medium comprises the following components:

[0015] DMEM / F12 basal culture medium, volume concentration of 10% fetal bovine serum, 10 μg / ml insulin, mass concentration of 1% penicillin, mass concentration of 1% streptomycin, 1 μM dexamethasone, 0.05 mM 3-isobutyl-1-methylxanthine, 200 μM indomethacin, 10 ng / mL BMP-2, 10 ng / mL FGF-1.

[0016] Another aspect of the present invention provides an umbilical cord mesenchymal stem cell adipogenic differentiation medium as described above. The use of the adipogenic differentiation medium to induce adipogenic differentiation of mesenchymal stem cells has the characteristics of high differentiation induction rate, short induction time, high stability, and can meet the in vitro culture of MSCs and meet the MSCs quality standard detection. The mesenchymal stem cells are not limited to umbilical cord mesenchymal stem cells, but also include adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, amniotic mesenchymal stem cells or placental mesenchymal stem cells, etc.

[0017] Another aspect of the present invention provides a method for culturing adipogenic differentiation of umbilical cord mesenchymal stem cells, comprising the following steps:

[0018] Step (1): Isolation and culture of primary umbilical cord mesenchymal stem cells: Remove the veins and arteries from the umbilical cord tissue, clean the blood, and cut into 1 cm 3 Size, inoculated at T175cm 2 Add an appropriate amount of complete culture medium to the cell culture flask for culture. During the first 6 days of culture, replenish the medium every 2 days. Continue to culture for 7 to 12 days, replenishing or changing the medium every 2 days depending on the situation.

[0019] Step (2): Passaging of umbilical cord mesenchymal stem cells: After culturing for 10 to 12 days, when a large number of cells crawl out from the tissue block and the total amount reaches about 40 to 70%, the time for subculturing is ripe, and the cells are subcultured. The cells are seeded in a 6-well plate, and complete culture medium is used. The cells are placed in an incubator at 37° C. and 5% CO2 and cultured for 1 to 3 days;

[0020] Step (3): Inducing adipogenesis of umbilical cord mesenchymal stem cells: When the cell confluence reaches 70%-90%, aspirate the complete culture medium, add umbilical cord mesenchymal stem cell adipogenesis induction differentiation medium, and culture the cells in an incubator at 37°C, 5% CO2. Replace the umbilical cord mesenchymal stem cell adipogenesis induction differentiation medium with fresh one as described above every 2-3 days; continue the induction for more than one week, depending on the cell growth and adipogenesis.

[0021] The formula of the umbilical cord mesenchymal stem cell adipogenic differentiation medium is as follows:

[0022] DMEM / F12 basal culture medium, volume concentration 8%-12% fetal bovine serum, 8-10μg / L insulin, mass concentration 0.8%-1.2% penicillin, mass concentration 0.8%-1.2% streptomycin, 0.8-1.2μM dexamethasone, 0.01-0.05mM 3-isobutyl-1-methylxanthine, 200-250μM indomethacin, 10-15ng / mL BMP-2, 10-15ng / mL FGF-1.

[0023] Preferably, the complete culture medium in step (1) is DMEM / F12 basal culture medium with a volume concentration of 10% fetal bovine serum, 1.0% penicillin, and 1.0% streptomycin.

[0024] Preferably, the umbilical cord mesenchymal stem cells in step (3) are 2nd to 10th generation mesenchymal stem cells.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. An umbilical cord mesenchymal stem cell adipogenic differentiation medium of the present invention, wherein the induction medium used is compounded with two substances, BMP-2 and FGF-1, on the basis of fetal bovine serum, insulin, penicillin, streptomycin, dexamethasone, 3-isobutyl-1-methylxanthine, and indomethacin, which synergistically promotes the adipogenic differentiation of mesenchymal stem cells, shortens the induction time, and improves the success rate of adipogenic induction;

[0027] 2. The adipogenic differentiation medium of umbilical cord mesenchymal stem cells of the present invention is conducive to the stable growth of cells. Compared with the induction medium of existing products, cell apoptosis will not occur in the first 3 days, and the adipogenic differentiation ratio is high. The culture medium of the present invention simplifies the induction differentiation operation of umbilical cord mesenchymal stem cells into adipocytes, and it is found through Oil Red O staining that the adipogenic differentiation rate of umbilical cord mesenchymal stem cells of the present invention is very high under the same induction time, and the lipid droplets are also larger;

[0028] 3. The umbilical cord mesenchymal stem cell adipogenic differentiation medium of the present invention greatly shortens the induction time. Fat droplets can be observed under a microscope after about 3 days of induction, and a large number of fat droplets are formed after about 14 days of induction, which effectively shortens the cycle of adipogenic function detection of umbilical cord mesenchymal stem cells.

[0029] 4. The reagent components used in the culture medium of the present invention are clear, and only one culture medium is used in the induction method of the present invention, the formula is stable, and the operation is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a graph showing the results of flow cytometry detection of surface markers of human umbilical cord mesenchymal stem cells in Example 1;

[0031] Figure 2 The cell morphology diagram of the culture process of human umbilical cord mesenchymal stem cells in Example 1 in the adipogenic differentiation medium of the present invention and the purchased commercial adipogenic differentiation medium 1 and commercial adipogenic differentiation medium 2;

[0032] Figure 3 The graphs are the results of adipogenic differentiation culture of human umbilical cord mesenchymal stem cells in Example 1 on days 3, 7 and 14 in the adipogenic differentiation medium of the present invention;

[0033] Figure 4 The Oil Red O staining result diagram of human umbilical cord mesenchymal stem cells after culture in different adipogenic differentiation mediums of Example 1, wherein A corresponds to commercialized adipogenic differentiation medium 1 (culture day 21); B corresponds to purchased commercialized adipogenic differentiation medium 2 (culture day 21); C corresponds to the Oil Red O staining result diagram (400X) after culture in the adipogenic differentiation medium of the present invention (culture day 14);

[0034] Figure 5 This is a diagram showing the Oil Red O staining result of human umbilical cord mesenchymal stem cells induced to differentiate in the adipogenic differentiation medium of the present invention on day 14 in Example 2 (400X);

[0035] Figure 6 This is a diagram showing the Oil Red O staining result of human umbilical cord mesenchymal stem cells induced to differentiate in the adipogenic differentiation medium of the present invention on the 14th day of Example 3 (400X);

[0036] Figure 7 This is the Oil Red O staining result of Comparative Example 1 human umbilical cord mesenchymal stem cells induced to differentiate in the adipogenic differentiation medium of the present invention on the 14th day (400X);

[0037] Figure 8 This is the Oil Red O staining result of Comparative Example 2 of human umbilical cord mesenchymal stem cells induced to differentiate in the adipogenic differentiation medium of the present invention on the 14th day (400X). DETAILED DESCRIPTION

[0038] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail to highlight the purport of the present invention.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0040] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0041] Sources of materials in the embodiments and comparative examples of the present invention:

[0042] Umbilical cord mesenchymal stem cells, homemade;

[0043] DMEM / F12 medium, fetal bovine serum, and insulin were purchased from Gibco;

[0044] Penicillin and streptomycin were purchased from BI;

[0045] Insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and indomethacin were purchased from Sigma;

[0046] BMP-2 and FGF-1 were purchased from Peprotech;

[0047] BMP-2 stands for: bone morphogenetic protein 2;

[0048] FGF-1 is: acidic fibroblast growth factor;

[0049] Commercial adipogenic differentiation medium 1 was purchased from Stem Cell, catalog number: 05412;

[0050] Commercialized adipogenic differentiation medium 2 was purchased from BI, catalog number: 05-330-1B.

[0051] Example 1 A method for culturing adipogenic differentiation of umbilical cord mesenchymal stem cells

[0052] Step (1): Isolation and preparation of umbilical cord mesenchymal stem cells

[0053] Take a fresh piece of umbilical cord tissue and place it in a 100 mm dish. Use surgical instruments to remove the veins and arteries of the umbilical cord tissue, clean the blood, and cut it into 1 cm 3 Size. Inoculated in T75cm 2 Add an appropriate amount of complete culture medium to the cell culture flask for culture. During the first 6 days of culture, replenish the medium every 2 days. Continue to culture for 7 to 12 days, replenish or replace the medium every 2 days depending on the situation.

[0054] The umbilical cord mesenchymal stem cells prepared in Example 1 were phenotypically identified, and flow cytometry antibodies CD90-PC5, CD73-PE, CD44-APC-750, CD105-APC, CD45-KO525, CD34-PC7, CD11b-FITC, and HLA-DR-PB450 were added for surface labeling. The results of flow cytometry detection of surface markers of human umbilical cord mesenchymal stem cells are shown in the figure below. Figure 1 As shown, the presence of relevant markers was detected, which was consistent with the surface marker characteristics of umbilical cord mesenchymal stem cells, indicating that the umbilical cord mesenchymal stem cells were successfully prepared.

[0055] Step (2): Passaging of umbilical cord mesenchymal stem cells; after culturing for 10 to 12 days, when a large number of cells crawl out from the periphery of the tissue block and the total amount reaches about 40 to 70%, the time for passaging is ripe, and the cells are passaged and inoculated into a 6-well plate, using complete culture medium, and the cells are placed in an incubator at 37° C. and 5% CO2 for 1-3 days.

[0056] The digested mesenchymal stem cells were divided into 1×10 4 cells / cm 2 The cells were seeded at a density of 100 μg / mL in a six-well plate, 2 ml of DMEM complete medium was added to each well, and the cells were cultured in an incubator at 37°C and 5% CO2.

[0057] Step (3): Umbilical cord mesenchymal stem cells are induced to become adipogenic

[0058] Step (3.1): Prepare umbilical cord mesenchymal stem cell adipogenic differentiation medium: DMEM / F12 basal medium, volume concentration 10% fetal bovine serum, 10 μg / ml insulin, 1% penicillin, 1% streptomycin, 1 μmol / L dexamethasone, 0.05 mM 3-isobutyl-1-methylxanthine, 200 μM indomethacin, 10 ng / ml BMP-2, 10 ng / ml FGF-1.

[0059] Step (3.2): When the cell confluence reaches 70%-90%, aspirate the complete culture medium, add 2 ml of the mesenchymal stem cell adipogenic differentiation medium of the present invention, the purchased commercial adipogenic differentiation medium 1 and the purchased commercial adipogenic differentiation medium 2, respectively, and place the cells in an incubator at 37°C, 5% CO2 for continued culture.

[0060] Step (3.3): replacing the mesenchymal stem cell adipogenic differentiation medium of the present invention, the commercially purchased adipogenic differentiation medium 1, and the commercially purchased adipogenic differentiation medium 2 with fresh medium every 2 or 3 days;

[0061] Step (3.4): Continue the induction for more than 2 weeks, depending on the cell growth and adipogenesis conditions, and perform Oil Red O staining on the three groups.

[0062] The results are as follows Figure 2 As shown, the cells induced and cultured with the mesenchymal stem cell adipogenic differentiation medium of the present invention have a good growth state, while the cells induced and cultured with the purchased commercial adipogenic differentiation medium 1 and the purchased commercial adipogenic differentiation medium 2 all show varying degrees of apoptosis.

[0063] The results are as follows Figure 3 As shown, the mesenchymal stem cell adipogenic differentiation medium of the present invention can be observed under a microscope on the third day, while the purchased commercial adipogenic differentiation medium 1 and the purchased commercial adipogenic differentiation medium 2 need to be cultured for more than 14 days or even longer before fat droplets appear. As the number of days increases, the number of cell fat droplets induced by the adipogenic differentiation medium of the present application gradually increases, and the volume of fat droplets gradually increases.

[0064] Oil red O staining was performed on the mesenchymal stem cells induced by the adipogenic differentiation medium of the present invention, the commercial adipogenic differentiation medium 1 purchased, and the commercial adipogenic differentiation medium 2 purchased. The results are as follows: Figure 4 As shown, the number of stained lipid droplets in the mesenchymal stem cells induced by the mesenchymal stem cell adipogenic differentiation medium of the experimental group (the present invention) is significantly greater; and at this time, it is only the 14th day of induction, and its effect is better than the commercial reagent group 1 and the commercial reagent group 2, which are induced to the 21st day, indicating that the culture medium formula of the present invention can shorten the induction period.

[0065] Example 2 A method for culturing umbilical cord mesenchymal stem cells by adipogenic induction and differentiation

[0066] Step (1): Isolation and preparation of umbilical cord mesenchymal stem cells

[0067] Take a fresh piece of umbilical cord tissue and place it in a 100 mm dish. Use surgical instruments to remove the veins and arteries of the umbilical cord tissue, clean the blood, and cut it into 1 cm 3 Size. Inoculated in T75cm 2 Add an appropriate amount of complete culture medium to the cell culture flask for culture. During the first 6 days of culture, replenish the medium every 2 days. Continue to culture for 7 to 12 days, replenish or replace the medium every 2 days depending on the situation.

[0068] Step (2): Passaging of umbilical cord mesenchymal stem cells; after culturing for 10 to 12 days, when a large number of cells crawl out from the periphery of the tissue block and the total amount reaches about 40 to 70%, the time for passaging is ripe, and the cells are passaged and inoculated into a 6-well plate, using complete culture medium, and the cells are placed in an incubator at 37° C. and 5% CO2 for 1-3 days.

[0069] The digested mesenchymal stem cells were divided into 1×10 4 cells / cm 2 The cells were seeded at a density of 100 μg / mL in a six-well plate, 2 ml of DMEM complete medium was added to each well, and the cells were cultured in an incubator at 37°C and 5% CO2.

[0070] Step (3): Umbilical cord mesenchymal stem cells are induced to become adipogenic

[0071] Step (3.1): Prepare umbilical cord mesenchymal stem cell adipogenic differentiation medium: DMEM / F12 basal medium, volume concentration 10% fetal bovine serum, 10 μg / ml insulin, 1% penicillin, 1% streptomycin, 1 μmol / L dexamethasone, 0.05 mM 3-isobutyl-1-methylxanthine, 200 μM indomethacin, 8 ng / ml BMP-2, 8 ng / ml FGF-1.

[0072] Step (3.2): When the cell confluence reaches 70%-90%, the complete culture medium is removed, and 2 ml of the mesenchymal stem cell adipogenic differentiation medium of the present invention is added respectively, and the cells are placed in an incubator at 37° C. and 5% CO 2 for further culture.

[0073] Step (3.3): Replace the mesenchymal stem cell adipogenic differentiation medium described above with fresh medium every 2 or 3 days.

[0074] Step (3.4): Continue induction for more than 2 weeks, depending on the cell growth and adipogenesis conditions, and perform Oil Red O staining. The results of Oil Red O staining are shown in Figure 2. Figure 5 As shown, from Figure 5 It can be seen that more lipid droplets have been formed on the 14th day, and their number is much greater than that of commercial reagent group 1 and commercial reagent group 2 after culture until the 21st day.

[0075] Example 3 A method for culturing umbilical cord mesenchymal stem cells by adipogenic induction and differentiation

[0076] Step (1): Isolation and preparation of umbilical cord mesenchymal stem cells

[0077] Take a fresh piece of umbilical cord tissue and place it in a 100 mm dish. Use surgical instruments to remove the veins and arteries of the umbilical cord tissue, clean the blood, and cut it into 1 cm 3 Size. Inoculated in T75cm 2 Add an appropriate amount of complete culture medium to the cell culture flask for culture. During the first 6 days of culture, replenish the medium every 2 days. Continue to culture for 7 to 12 days, replenish or replace the medium every 2 days depending on the situation.

[0078] The results of flow cytometry detection of surface markers of human umbilical cord mesenchymal stem cells are shown in the figure Figure 1 As shown, the presence of relevant markers was detected, indicating that the flow cytometric detection of umbilical cord mesenchymal stem cells was successfully prepared.

[0079] Step (2): Passaging of umbilical cord mesenchymal stem cells; after culturing for 10 to 12 days, when a large number of cells crawl out from the periphery of the tissue block and the total amount reaches about 40 to 70%, the time for passaging is ripe, and the cells are passaged and inoculated into a 6-well plate, using complete culture medium, and the cells are placed in an incubator at 37° C. and 5% CO2 for 1-3 days.

[0080] The digested mesenchymal stem cells were divided into 1×10 4 cells / cm 2 The cells were seeded at a density of 100 μg / mL in a six-well plate, 3 ml of DMEM complete medium was added to each well, and the cells were cultured in an incubator at 37°C and 5% CO2.

[0081] Step (3): Umbilical cord mesenchymal stem cells are induced to become adipogenic

[0082] Step (3.1): Prepare umbilical cord mesenchymal stem cell adipogenic differentiation medium: DMEM / F12 basal medium, volume concentration 10% fetal bovine serum, 10 μg / ml insulin, 1% penicillin, 1% streptomycin, 1 μmol / L dexamethasone, 0.05 mM 3-isobutyl-1-methylxanthine, 250 μM indomethacin, 12 ng / ml BMP-2, 12 ng / ml FGF-1.

[0083] Step (3.2): When the cell confluence reaches 70%-90%, the complete culture medium is removed, and 2 ml of the mesenchymal stem cell adipogenic differentiation medium of the present invention is added respectively, and the cells are placed in an incubator at 37° C. and 5% CO 2 for further culture.

[0084] Step (3.3): Replace the mesenchymal stem cell adipogenic differentiation medium described above with fresh medium every 2 or 3 days.

[0085] Step (3.4): Continue induction for more than 2 weeks, depending on the cell growth and adipogenesis conditions, and perform Oil Red O staining. The results of Oil Red O staining are shown in Figure 2. Figure 6 As shown, from Figure 6 It can be seen that more lipid droplets have been formed on the 14th day, and their number is much greater than that of commercial reagent group 1 and commercial reagent group 2 after culture until the 21st day.

[0086] Comparative Example 1

[0087] This comparative example is a further improvement of Example 1. This comparative example adopts the same steps (1) separation and preparation of umbilical cord mesenchymal stem cells and step (2) umbilical cord mesenchymal stem cell passage as Example 1. In step (3) umbilical cord mesenchymal stem cell adipogenesis induction step, the following mesenchymal stem cell adipogenesis induction differentiation medium is used:

[0088] DMEM / F12 basal culture medium, volume concentration 10% fetal bovine serum, 10 μg / ml insulin, 1‰ penicillin, 1% streptomycin, 1 μmol / L dexamethasone, 0.05 mM 3-isobutyl-1-methylxanthine, 200 μM indomethacin.

[0089] The remaining steps are the same as those in Example 1.

[0090] On the 14th day of induction, the induced umbilical cord mesenchymal stem cells were stained with Oil Red O. The staining results are shown in Figure 7 As shown, from Figure 7 It can be seen that the number of lipid droplets is small and the size is small, and its effect is far inferior to the adipogenesis induction medium in Examples 1-3.

[0091] Comparative Example 2

[0092] This comparative example is a further improvement of Example 1. This comparative example adopts the same steps (1) separation and preparation of umbilical cord mesenchymal stem cells and step (2) umbilical cord mesenchymal stem cell passage as Example 1. In step (3) umbilical cord mesenchymal stem cell adipogenesis induction step, the following mesenchymal stem cell adipogenesis induction differentiation medium is used:

[0093] DMEM / F12 basal culture medium, volume concentration 10% fetal bovine serum, 10μg / ml insulin, 1‰ penicillin, 1‰ streptomycin, 1μmol / L dexamethasone, 0.05mM 3-isobutyl-1-methylxanthine, 200μM indomethacin, 10ng / mlBMP-2, 10ng / mlTGF-β.

[0094] The remaining steps are the same as those in Example 1.

[0095] On the 14th day of induction, the induced umbilical cord mesenchymal stem cells were stained with Oil Red O. The staining results are shown in Figure 8 As shown, from Figure 8 It can be seen that the number of lipid droplets is small and the size is small, and its effect is far inferior to the adipogenesis induction medium in Examples 1-3.

[0096] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A medium for adipogenic differentiation of umbilical cord mesenchymal stem cells, characterized in that: Substances containing the following components: DMEM / F12 basal medium, fetal bovine serum, insulin, penicillin, streptomycin, dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, 8-15ng / mL BMP-2 and 8-15ng / mL FGF-1.

2. The umbilical cord mesenchymal stem cell adipogenic differentiation medium according to claim 1, characterized in that: Substances containing the following components: DMEM / F12 basal medium, fetal bovine serum, insulin, penicillin, streptomycin, dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, 8-12 ng / mL BMP-2 and 8-12 ng / mL FGF-1.

3. The umbilical cord mesenchymal stem cell adipogenic differentiation medium according to claim 1, characterized in that: The substance comprising the following components: DMEM / F12 basal culture medium, volume concentration 8%-12% fetal bovine serum, 8-10μg / ml insulin, mass concentration 0.8%-1.2% penicillin, mass concentration 0.8%-1.2% streptomycin, 0.8-1.2μM dexamethasone, 0.01-0.05mM 3-isobutyl-1-methylxanthine, 200-250μM indomethacin, 8-15ng / mL BMP-2 and 8-15ng / mL FGF-1.

4. The umbilical cord mesenchymal stem cell adipogenic differentiation medium according to any one of claims 1 to 3 is used in the adipogenic differentiation of adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, amniotic membrane mesenchymal stem cells or placental mesenchymal stem cells.

5. A method for culturing adipogenic differentiation of umbilical cord mesenchymal stem cells, characterized in that: The steps include: Step (1): Isolation and culture of primary umbilical cord mesenchymal stem cells: Umbilical cord tissue was pretreated and inoculated in T175cm 2 In the cell culture flask, add an appropriate amount of mesenchymal stem cell complete medium for culture; Step (2): Passaging of umbilical cord mesenchymal stem cells: After a large number of cells crawl out from the periphery of the tissue block and the total amount reaches about 40-70%, the cells are passaged and inoculated into a 6-well plate. An appropriate amount of complete mesenchymal stem cell culture medium is added and the cells are cultured in a 37°C, 5% CO2 incubator; Step (3): Inducing adipogenesis of umbilical cord mesenchymal stem cells: When the cell confluence reaches 70%-90%, aspirate the complete culture medium, add the umbilical cord mesenchymal stem cell adipogenesis induction differentiation medium as described in any one of claims 1 to 3, and culture the cells in a 37°C, 5% CO2 incubator, replacing fresh mesenchymal stem cell adipogenesis induction differentiation medium every 2-3 days; continue induction.

6. The method for adipogenic differentiation of umbilical cord mesenchymal stem cells according to claim 5, characterized in that: The complete medium for mesenchymal stem cells in step (1) is DMEM / F12 basic medium with a volume concentration of 10% fetal bovine serum, 1.0% penicillin, and 1.0% streptomycin.

7. The method for adipogenic differentiation of umbilical cord mesenchymal stem cells according to claim 5, characterized in that: The continuous induction in step (3) is continuous induction for more than 2 weeks.

8. The method for adipogenic differentiation of umbilical cord mesenchymal stem cells according to claim 5, characterized in that: The mesenchymal stem cells in step (3) are 2nd to 10th generation mesenchymal stem cells.

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