Umbilical cord mesenchymal stem cell culture medium and application thereof

Abstract

The present invention discloses a culture medium for umbilical cord mesenchymal stem cells and its application, and particularly relates to the field of cell culture technology. The culture medium for umbilical cord mesenchymal stem cells comprises the following components: 10-40 ng / mL of human basic fibroblast growth factor, 55-95 ng / mL of rough ragweed, 22-53 ng / mL of sodium porphyrin, 2-5 μg / mL of transferrin, 6-11 mM of L-glutamine, 45-90 μM of β-mercaptoethanol, 8-12 μg / mL of insulin, 7-13 μg / mL of L-glutathione, and DMEM / F12 culture medium. In addition, the culture medium can effectively enhance the proliferation activity and osteogenic differentiation potential of umbilical cord mesenchymal stem cells, and is expected to make important contributions to the development of regenerative medicine and tissue engineering.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to an umbilical cord mesenchymal stem cell culture medium and application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of stem cells with the potential for self-renewal and multidirectional differentiation. They have become a research hotspot in the fields of biomedicine and tissue engineering. They have a very strong proliferation ability and can rapidly expand under appropriate conditions to meet the needs of clinical applications for the number of cells. In addition, they can differentiate into various cell types and have certain immunoregulatory functions. They play an important role in disease treatment. For example, they can provide a large number of bone cells and chondrocytes for bone injury-related diseases.

[0003] Culture medium plays an important role in the in vitro culture of cells, providing appropriate nutrients and ingredients for cell growth and reproduction, and ensuring the multidirectional differentiation potential of cells. However, traditional culture medium often contains fetal bovine serum, which is often complex in composition and varies between different production batches. The quality of the culture medium is unstable, and there are uncertainties and risks of pathogen transmission during use, which make it impossible to meet the growth and differentiation needs of umbilical cord mesenchymal stem cells, limiting their development in clinical applications. With the rapid development of biomedicine and technology, the research and development and application of serum-free culture medium has become a hot topic for researchers.

[0004] In order to improve the in vitro culture effect of umbilical cord mesenchymal stem cells, it is crucial to develop a culture medium that can effectively enhance the proliferation and differentiation ability of umbilical cord mesenchymal stem cells. It can not only provide technical support for the basic research and clinical application of umbilical cord mesenchymal stem cells, but also provide scientific guidance for the culture of other mesenchymal stem cells. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide an umbilical cord mesenchymal stem cell culture medium, which does not contain animal serum, avoids the interference of animal serum, and fully meets the growth requirements of umbilical cord mesenchymal stem cells.

[0006] The second object of the present invention is to provide the use of the above-mentioned umbilical cord mesenchymal stem cell culture medium in culturing umbilical cord mesenchymal stem cells, which can effectively increase the proliferation number of umbilical cord mesenchymal stem cells and improve the in vitro proliferation rate and osteogenic differentiation efficiency of umbilical cord mesenchymal stem cells.

[0007] One of the purposes of the present invention is achieved by the following technical solution:

[0008] A culture medium for umbilical cord mesenchymal stem cells, comprising the following components: 10-40 ng / mL human basic fibroblast growth factor, 55-95 ng / mL ragweed, 22-53 ng / mL sodium chrysoporphyrin, 2-5 μg / mL transferrin, 6-11 mM L-glutamine, 45-90 μM β-mercaptoethanol, 8-12 μg / mL insulin, 7-13 μg / mL L-glutathione, and DMEM / F12 culture medium.

[0009] Furthermore, the culture medium includes the following components: 25 ng / mL human basic fibroblast growth factor, 80 ng / mL rough ragweed, 40 ng / mL sodium porphyrin, 3 μg / mL transferrin, 9 mM L-glutamine, 70 μM β-mercaptoethanol, 10 μg / mL insulin, 10 μg / mL L-glutathione, and DMEM / F12 culture medium.

[0010] Further, the chemical formula of ragweed is shown below:

[0011]

[0012] The second object of the present invention is achieved by adopting the following technical solution:

[0013] The application of the umbilical cord mesenchymal stem cell culture medium of the present invention is to culture umbilical cord mesenchymal stem cells using the umbilical cord mesenchymal stem cell culture medium.

[0014] Furthermore, the specific steps of culturing umbilical cord mesenchymal stem cells are as follows: the umbilical cord tissue is rinsed, cut into pieces, digested with 0.1% collagenase II, and then centrifuged, and the above-mentioned culture medium is added for culturing; after culturing until the cell growth confluence is 80-90%, the primary umbilical cord mesenchymal stem cells are digested to obtain primary umbilical cord mesenchymal stem cells; and then the primary umbilical cord mesenchymal stem cells are inoculated into the above-mentioned culture medium for subculture.

[0015] Furthermore, during the subculture period, the culture medium is replaced every 2-3 days.

[0016] Furthermore, the culture conditions are 37°C, 5% CO 2 .

[0017] Furthermore, the ratio of subculture is 1:2-3.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention provides an umbilical cord mesenchymal stem cell culture medium, which does not contain animal serum, thereby avoiding the interference of animal serum.

[0020] 2. The umbilical cord mesenchymal stem cell culture medium provided by the present invention can significantly increase the in vitro proliferation rate of umbilical cord mesenchymal stem cells, effectively improve their osteogenic differentiation potential, and upregulate the expression of osteogenic differentiation-related proteins when culturing umbilical cord mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A diagram showing the morphological characteristics of umbilical cord mesenchymal stem cells;

[0022] Figure 2 A graph showing the growth curve of umbilical cord mesenchymal stem cells;

[0023] Figure 3 A graph showing the expression results of cyclin in umbilical cord mesenchymal stem cells;

[0024] Figure 4 A graph showing the percentage of positive area of ​​umbilical cord mesenchymal stem cells stained with Alizarin red. DETAILED DESCRIPTION

[0025] Below, in conjunction with the accompanying drawings and specific embodiments, the present invention is further described. It should be noted that, under the premise of no conflict, the embodiments described below or the technical features can be arbitrarily combined to form new embodiments. The specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0026] Example 1

[0027] A culture medium for umbilical cord mesenchymal stem cells is composed of the following components: 25 ng / mL human basic fibroblast growth factor, 80 ng / mL ragweed, 40 ng / mL sodium chrysoporphyrin, 3 μg / mL transferrin, 9 mM L-glutamine, 70 μM β-mercaptoethanol, 10 μg / mL insulin, 10 μg / mL L-glutathione, and DMEM / F12 culture medium.

[0028] Example 2

[0029] The invention discloses an umbilical cord mesenchymal stem cell culture medium, which is composed of the following components: 10 ng / mL human basic fibroblast growth factor, 55 ng / mL ragweed, 22 ng / mL sodium chrysoporphyrin, 2 μg / mL transferrin, 6 mM L-glutamine, 45 μM β-mercaptoethanol, 8 μg / mL insulin, 7 μg / mL L-glutathione, and DMEM / F12 culture medium.

[0030] Example 3

[0031] The invention discloses an umbilical cord mesenchymal stem cell culture medium, comprising the following components: 40 ng / mL human basic fibroblast growth factor, 95 ng / mL ragweed, 53 ng / mL sodium chrysoporphyrin, 5 μg / mL transferrin, 11 mM L-glutamine, 90 μM β-mercaptoethanol, 12 μg / mL insulin, 13 μg / mL L-glutathione, and DMEM / F12 culture medium.

[0032] Comparative Example 1

[0033] The difference between Comparative Example 1 and Example 1 is that the tricholoma glutinosin is omitted from the culture medium, and the rest is the same as Example 1.

[0034] Comparative Example 2

[0035] The difference between this comparative example 2 and example 1 is that sodium chlorophyll is omitted from the culture medium, and the rest is the same as example 1.

[0036] Comparative Example 3

[0037] The difference between this comparative example 3 and example 1 is that both sodium porphyrin and tricholoma bromocriptine are omitted from the culture medium, and the rest is the same as example 1.

[0038] Comparative Example 4

[0039] The difference between this comparative example 4 and example 1 is that the tricholoma bromopoietin is omitted from the culture medium, and the concentration of sodium chlorophyll in the culture medium is adjusted to 120 ng / mL, and the rest is the same as example 1.

[0040] Comparative Example 5

[0041] The difference between this comparative example 5 and Example 1 is that sodium porphyrin is omitted from the culture medium, and the concentration of tricholoma arachnophorin in the culture medium is adjusted to 120 ng / mL, and the rest is the same as Example 1.

[0042] The use of the above umbilical cord mesenchymal stem cell culture medium in culturing umbilical cord mesenchymal stem cells comprises the following steps:

[0043] Soak the umbilical cord in 0.9% saline, then cut the umbilical cord tissue into 2 cm lengths with sterile scissors, wash it three times with 0.9% saline to remove the remaining blood and impurities in the umbilical cord. Then disinfect it with 70% alcohol, remove the Wharton's jelly in the umbilical cord, and cut it into 1 mm lengths with ophthalmic scissors. 2 The precipitate was transferred to a T25 cell culture flask, and the culture medium corresponding to the above Examples 1-3 and Comparative Examples 1-5 was added respectively (the culture medium described in Example 1 was used for culture in Example 1), and the precipitate was placed at 37°C and 5% CO.2 The cells were cultured in a static incubator. After the cells first adhered to the wall, the medium was changed and the non-adherent cells were discarded. The cells were observed under a microscope. When the cell confluence reached 80%, 0.25% Trypsin-EDTA enzyme digestion solution was used for digestion and separation of cells, which were primary umbilical cord mesenchymal stem cells. The digested umbilical cord mesenchymal stem cells were subcultured at a ratio of 1:2. The culture medium was changed every 2 days during the culture period. The culture conditions were 37°C and 5% CO 2 .

[0044] Test Example 1

[0045] The P3 generation umbilical cord mesenchymal stem cells cultured in the above Examples 1-3 and Comparative Examples 1-5 were placed under an inverted microscope to observe the cell morphology and number, and photographed and recorded. The effects of the culture media of Examples 1-3 and Comparative Examples 1-5 on the morphology and number of umbilical cord mesenchymal stem cells were compared. The results are as follows: Figure 1 As shown, Figure 1 Wherein ah corresponds to Examples 1-3 and Comparative Examples 1-5 respectively.

[0046] Depend on Figure 1 It can be observed that the P3 generation umbilical cord mesenchymal stem cells obtained in Examples 1-3 are all long and flat spindle-shaped, the cells are neatly arranged, the distribution is relatively uniform, the growth state is good, the cells are small and the number is large. Compared with the P3 generation umbilical cord mesenchymal stem cells of Example 1, Comparative Examples 1 and 2 respectively omit sodium chrysoporphyrin and rough-haired ambroxan, the number of cells is significantly reduced, and the morphology is still spindle-shaped, Comparative Examples 4 and 5 respectively adjust the concentrations of the above two groups of components in the culture medium, and Comparative Example 3 simultaneously omits sodium chrysoporphyrin and rough-haired ambroxan in the culture medium. Compared with Example 1, the number of cells in each group has decreased to varying degrees, which shows that sodium chrysoporphyrin and rough-haired ambroxan have a synergistic effect in the culture medium and can cooperate with each other to promote the growth of umbilical cord mesenchymal stem cells.

[0047] Test Example 2

[0048] Umbilical cord mesenchymal stem cell proliferation activity assay: First, the P3 umbilical cord mesenchymal stem cells obtained by culture in Examples 1-3 and Comparative Examples 1-5 were collected and prepared into 1×10 4 The cell suspension of 100 cells / mL was inoculated into a 96-well plate, and cultured using the culture medium corresponding to Examples 1-3 and Comparative Examples 1-5, respectively. The 96-well plate inoculated with cells was then placed at 37°C and 5% CO 2 In the incubator, 3 parallel control wells were set for each group. The number of cells was calculated on the 1st, 2nd, 3rd, 4th, 5th, 6th and 7th day of culture. The cell growth curve was plotted with the culture time as the horizontal axis and the average value of the number of cells in Examples 1-3 and Comparative Examples 1-5 as the vertical axis. The results are shown in FIG. Figure 2shown.

[0049] The test results of the umbilical cord mesenchymal stem cell growth curves of Examples 1-3 and Comparative Examples 1-5 are as follows: Figure 2 As shown, it can be intuitively seen that the P3 generation umbilical cord mesenchymal stem cells obtained in Examples 1-3 have a relatively slow proliferation rate on the 1st to 2nd day of culture, and the cell proliferation rate is the highest on the 3rd to 5th day of culture, and gradually stabilizes on the 6th to 7th day. There is no obvious difference in the cell proliferation rate between the examples. Compared with Example 1, Comparative Examples 1-5 changed the components in the culture medium, and the cell proliferation rate showed a downward trend, which was significantly lower than that of Example 1, among which Comparative Example 3 had the largest gap with the above groups. Therefore, the above results illustrate that the trichometurin and sodium cypermethrin in the culture medium of the Example 1-3 groups work together with the components in the culture medium to effectively enhance the proliferation capacity of umbilical cord mesenchymal stem cells.

[0050] Test Example 3

[0051] Western Blot Experiment to Evaluate the Expression of Cyclin Protein Extraction: Collect the P3 mesenchymal stem cells obtained in Examples 1-3 and Comparative Examples 1-5, wash them with PBS, add protein lysis buffer to lyse the cells to release the proteins therein, then centrifuge at 10000 rpm for 10 min to remove cell debris, and collect the supernatant, which is the protein extract.

[0052] Determination of protein concentration and denaturation: Dilute the protein extract into different concentrations according to the instructions of the BCA kit, make a standard curve, and then convert the protein concentration of the sample. Add protein loading buffer according to the ratio (1:4), boil at 100°C for 6 minutes to fully denature the protein, and obtain the protein sample.

[0053] Electrophoresis separation: Add the protein sample to SDS-PAGE gel for electrophoresis separation. When the bromophenol blue dye moves to the bottom of the electrophoresis tank, the electrophoresis ends.

[0054] Membrane transfer: Proteins were transferred to PVDF membrane using wet transfer method.

[0055] Blocking of membrane: Place the membrane in a blocking solution containing 5% skimmed milk powder and shake slowly at room temperature for 1 hour. Then wash with TBST on a shaker for 3 times, 5 minutes each time.

[0056] Antibody binding: Add the primary antibody Cyclin-D1 to react with the protein on the membrane, incubate overnight at 4°C with shaking, and make sure the membrane is fully immersed in the primary antibody solution to avoid uneven binding. Then wash with TBST on a shaker for 3 times, 5 minutes each time. The next day, add the secondary antibody, shake on a shaker at room temperature for 2 hours, and then wash with TBST on a shaker for 3 times, 5 minutes each time.

[0057] Color development: Use a pipette to absorb the chemiluminescent liquid so that it evenly covers the PVDF membrane and develops it. The test results are as follows: Figure 3 shown.

[0058] Immunoblotting is commonly used to detect and analyze the presence and expression levels of specific proteins in a sample. Figure 3 The expression of specific cyclins during the differentiation process is shown. As shown in the figure, the expression level of the cyclin Cyclin-D1 of the P3 generation umbilical cord mesenchymal stem cells of Examples 1-3 is higher, and the gray value is significantly higher than that of Comparative Examples 1-5. The Cyclin-D1 protein plays a key role in each stage of the cell cycle and regulates the proliferation and differentiation of cells. It proves that the P3 generation umbilical cord mesenchymal stem cells obtained in Examples 1-3 of the present invention have good proliferation activity.

[0059] Test Example 4

[0060] Osteogenic differentiation activity assay: First, the P3 generation umbilical cord mesenchymal stem cells obtained by culture in Examples 1-3 and Comparative Examples 1-5 were obtained and the density was 1×10 5 The cells were inoculated into 12-well plates at a density of 100 / mL, and cultured in the corresponding culture medium until the cell confluence reached 80%, and then inoculated into a culture dish containing osteogenic differentiation medium, wherein the osteogenic differentiation medium was DMEM / F12 medium, 0.2 μmol / L dexamethasone, 0.45 mmol / L vitamin C, 10% FBS and 20 mmol / L sodium β-glycerophosphate). After 21 days of induction culture, the cells were fixed with 4% paraformaldehyde for 15 minutes, and then each well was stained with alizarin red dye solution, incubated at room temperature and away from light for 30 minutes, and the area percentage of alizarin red dyeing area was calculated using Image J software to determine the osteogenic differentiation potential of the above cultured umbilical cord mesenchymal stem cells.

[0061] like Figure 4 The percentage result of the alizarin red staining area of ​​the P3 generation umbilical cord mesenchymal stem cells obtained by culture in Examples 1-3 and Comparative Examples 1-5 is shown in the figure. As can be seen from the figure, the calcified nodules of the P3 generation umbilical cord mesenchymal stem cells obtained in Examples 1-3 have the largest staining area, indicating that they have a higher osteogenic differentiation activity. Comparative Examples 1-5 respectively adjusted the concentrations of sodium chrysoporphyrin and ragweed in the culture medium, and the staining intensity was different from that in Example 1 to varying degrees. Among them, after sodium chrysoporphyrin and ragweed were omitted at the same time in Comparative Example 3, the area of ​​the staining positive area was the smallest, and the osteogenic differentiation activity was the worst, while Comparative Examples 4 and 5 increased the dosage of sodium chrysoporphyrin and ragweed, respectively. Compared with Comparative Example 3, the alizarin red staining area of ​​Comparative Examples 4 and Comparative Example 5 increased. Therefore, the above results show that sodium chrysoporphyrin and ragweed can work synergistically in the culture medium to enhance the osteogenic differentiation potential of umbilical cord mesenchymal stem cells.

[0062] The umbilical cord mesenchymal stem cell culture medium provided by the present invention can not only improve the proliferation rate and osteogenic differentiation efficiency of umbilical cord mesenchymal stem cells, but also reduce the culture cost, providing strong support for the large-scale production of umbilical cord mesenchymal stem cells. In short, the above experiments show that the culture medium has broad application prospects in the expansion, differentiation and tissue engineering of umbilical cord mesenchymal stem cells, and is expected to make important contributions to the development of regenerative medicine and tissue engineering.

[0063] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. An umbilical cord mesenchymal stem cell culture medium, characterized in that: The culture medium includes the following components: 10-40 ng / mL human basic fibroblast growth factor, 55-95 ng / mL rough ragweed, 22-53 ng / mL sodium porphyrin, 2-5 μg / mL transferrin, 6-11 mM L-glutamine, 45-90 μM β-mercaptoethanol, 8-12 μg / mL insulin, 7-13 μg / mL L-glutathione, and DMEM / F12 culture medium.

2. The umbilical cord mesenchymal stem cell culture medium according to claim 1, characterized in that: The culture medium includes the following components: 25 ng / mL human basic fibroblast growth factor, 80 ng / mL rough ragweed, 40 ng / mL sodium porphyrin, 3 μg / mL transferrin, 9 mM L-glutamine, 70 μM β-mercaptoethanol, 10 μg / mL insulin, 10 μg / mL L-glutathione, and DMEM / F12 culture medium.

3. The use of an umbilical cord mesenchymal stem cell culture medium according to claim 1 or 2, characterized in that: The application is to culture umbilical cord mesenchymal stem cells using the umbilical cord mesenchymal stem cell culture medium according to claim 1 or 2.

4. The use of an umbilical cord mesenchymal stem cell culture medium according to claim 3, characterized in that: The specific steps of culturing umbilical cord mesenchymal stem cells are as follows: the umbilical cord tissue is washed, cut into pieces, digested with 0.1% collagenase II, centrifuged, added with the culture medium, and cultured; after culturing until the cell growth confluence is 80-90%, the primary umbilical cord mesenchymal stem cells are digested to obtain primary umbilical cord mesenchymal stem cells; and then the primary umbilical cord mesenchymal stem cells are inoculated into the culture medium for subculture.

5. The use of an umbilical cord mesenchymal stem cell culture medium according to claim 4, characterized in that: During the subculture, the culture medium was replaced every 2-3 days.

6. The use of an umbilical cord mesenchymal stem cell culture medium according to claim 4, characterized in that: The culture conditions are 37° C. and 5% CO 2 .

7. The use of an umbilical cord mesenchymal stem cell culture medium according to claim 4, characterized in that: The ratio of the subculture is 1:2-3.

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