Primary culture medium and primary culture method for umbilical cord mesenchymal stem cells
A technology for primary culture medium and stromal stem cells, which is applied to the primary culture medium of umbilical cord mesenchymal stem cells and the field of primary culture, can solve the problems of unclearness and time-consuming primary culture, achieve good effect and shorten the time of primary culture. Effects of culture time, high clinical safety
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Embodiment 1
[0055] Embodiment 1, mixture proportioning
[0056] Mixture working concentration:
[0057] (1) The working concentration of tranexamic acid is 500mg / mL and 10000mg / mL;
[0058] (2) The working concentration of G-CSF is 10ng / mL, 20ng / mL;
[0059] (3) The working concentration of EGF is 10ng / mL and 20ng / mL.
[0060] Prepare two media:
[0061] Experimental group: named group A, which is DMEM / F12+ mixture;
[0062] Control group: named group B, DMEM / F12+10%FBS.
[0063] The specific groups are as follows:
[0064] Table 1 Concentration ratio of each component in medium of group A
[0065]
Embodiment 2
[0066] Embodiment 2, observe and compare primary cell climbing out situation
[0067] (1) peel off the umbilical cord adventitia, 1 vein and 2 arteries to obtain Wharton's jelly;
[0068] (2) Shred Wharton's jelly to 1~2mm 3 , 3ml / 15cm dish was inoculated with Wharton's jelly;
[0069] (3) Cultivate Wharton's jelly with two types of medium in Example 1; culture condition is: 37 ℃, 5% CO 2 Cultured in an incubator, the medium was changed every three days.
[0070] (4) Observe every 3 days, and compare the situation of crawling out of the cells.
[0071] The situation of climbing out of cells is shown in the table below:
[0072] Table 2 Climbing out of cells in each group
[0073] number of days
[0074] It can be seen from the table that, in the case of adding the mixture (A6 / A8), the proliferation speed of primary umbilical cord mesenchymal stem cells is significantly better than that of the medium added with FBS.
Embodiment 3
[0075] Embodiment 3, AB group differentiation ability comparison
[0076] Take the 3rd generation cells of formula A8 and B, press 2.5×10 4 The cells / mL concentration were inoculated in a 24-well plate containing DMEM / F12+20% FBS medium, and when the cells were 80% confluent, they were replaced with 1nmol / L dexamethasone, 10mg / L insulin, 0.5mmol / L 3- The culture solution of isobutyl-1-methylxanthine and 100 μmol / L indomethacin was changed twice a week, fixed with paraformaldehyde after 3 weeks, and stained with Oil Red O. For staining results, see figure 1 . Clearly stained lipid droplets can be seen, which proves that the umbilical cord mesenchymal stem cells cultured in group A8 of the present invention have adipogenic differentiation ability.
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