Culture medium for mesenchymal stem cells and large-scale culture method thereof
A large-scale culture of stem cell technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of high price, cell shape change, cell aging, etc., achieve good proliferation and differentiation ability, delay Aging phenomenon, effect of maintaining characteristics
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Embodiment 1
[0038] Example 1. Primary isolation and culture of umbilical cord mesenchymal stem cells
[0039] Obtain the bovine umbilical cord tissue block, cut it into pieces, add complete medium to the tissue block, pipette evenly and transfer to the cell culture dish; shake the culture dish to make the tissue block evenly distributed in the culture dish, transfer to 5% CO 2 , Cultivate in a cell incubator at 37°C, and observe the growth of the cells regularly; when the cells climb out, discard the old medium and tissue pieces, wash the culture dish with PBS, add complete medium and continue to culture in the cell incubator ; When the cell confluency reaches 80% to 90%, discard the old medium, wash the culture dish with PBS, add the digestion solution containing 0.25v / v% trypsin and 0.04v / v% EDTA to digest for 1 to 2min , after adding complete medium to terminate the digestion, the cells were pipetted into a single cell suspension to obtain umbilical cord MSCs, which were recorded as P1...
Embodiment 2
[0040] Embodiment 2, large-scale culture and cell identification of umbilical cord MSC
[0041] After the P1 generation cells obtained in Example 1 were collected by centrifugation, the Count Star cell counter was used for cell counting, and the complete medium was adjusted to 5 × 10 cells according to the counting results. 4 / mL of the optimal seeding density, and inoculated into a 15cm large Petri dish, placed in 5% CO 2 , 37 ℃ incubator to continue culturing. When the cell fusion degree reaches 80%-90%, it can be subcultured again, and it will be recorded as P2 generation cells. Repeated subculture by analogy for large-scale expansion culture, and when it is passed to P10 generation cells, a large number of cells that meet clinical needs can be obtained. dose of MSC. The complete medium is the medium of the mesenchymal stem cells, including serum-free medium and epidermal growth factor and platelet-derived factor, wherein the concentration of epidermal growth factor is 5 ...
Embodiment 3
[0042] Embodiment 3, the synergistic effect of EGF and PDGF in the culture process of umbilical cord MSC
[0043] Take the P10 generation cells cultivated in Example 2, digest them with trypsin containing 0.25v / v% trypsin and 0.04v / v% EDTA, and press 1×10 with Ultra CULTURE serum-free medium 4 / mL was inoculated onto a six-well plate with 2 mL of the system per well. PDGF and EGF were added according to different addition methods, and the addition schemes are shown in Table 1. Regularly observe the growth state and morphological changes of the cells, change the medium every 3 days, and take pictures of the cells on the 7th day, the results are as follows: figure 1 . The figure below is a comparison picture of cell morphology, in which figure 1 Among them, A, B, C, D, E and F correspond to the addition schemes of groups (1), (2), (3), (4) and (5) in Table 1, respectively. Cells were collected by trypsinization and counted by a cell counter. The results of the cell counts a...
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