Culture medium and cell cultural method
A technology of cell culture and cell culture, applied in the field of cells, can solve the problem of fetal bovine serum increasing allogeneic immune response, etc., and achieve the effect of improving the ability of in vitro expansion
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Embodiment 1
[0036] Take the third-generation frozen GMSCs, take out the cryopreservation tube and immediately put it into a 37°C water bath to dissolve, and shake the cryopreservation tube continuously during the dissolution process. After thawing the liquid for 1-2 minutes, dilute the cell suspension with 10 mL of serum-free medium for mesenchymal stem cells (wash the cryopreservation tube once with the medium), centrifuge at 1000 r / min for 5 minutes, and discard the supernatant. 10mL of complete medium to resuspend the cells, press 5×10 4 Cells / mL density were seeded in 10cm culture dish, placed in 5% CO 2 Culture in an incubator at 37°C. When the cells are 80-90% full, use a straw to discard the old culture medium, add 2-3mL 0.25% trypsin, and digest for 1-3 minutes. When the cells shrink and become round under the microscope, immediately add 5mL complete medium to stop the digestion. Cells were collected for cell counting.
[0037] The formula of the complete medium is: 5ng / mLbFGF+...
Embodiment 2
[0039] Take the third-generation frozen GMSCs, take out the cryopreservation tube and immediately put it into a 37°C water bath to dissolve, and shake the cryopreservation tube continuously during the dissolution process. After thawing the liquid for 1-2 minutes, dilute the cell suspension with 10 mL of serum-free medium for mesenchymal stem cells (wash the cryopreservation tube once with the medium), centrifuge at 1000 r / min for 5 minutes, and discard the supernatant. 10mL of complete medium to resuspend the cells, press 5×10 4 Cells / mL density were seeded in 10cm culture dish, placed in 5% CO 2 Culture in an incubator at 37°C. When the cells are 80-90% full, use a straw to discard the old culture medium, add 2-3mL 0.25% trypsin, and digest for 1-3 minutes. When the cells shrink and become round under the microscope, immediately add 5mL complete medium to stop the digestion. Cells were collected for cell counting.
[0040] The complete medium formula is: 10ng / mLbFGF+5ng / mLEG...
Embodiment 3
[0042] Take the third-generation frozen GMSCs, take out the cryopreservation tube and immediately put it into a 37°C water bath to dissolve, and shake the cryopreservation tube continuously during the dissolution process. After thawing the liquid for 1-2 minutes, dilute the cell suspension with 10 mL of serum-free medium for mesenchymal stem cells (wash the cryopreservation tube once with the medium), centrifuge at 1000 r / min for 5 minutes, and discard the supernatant. 10mL of complete medium to resuspend the cells, press 5×10 4 Cells / mL density were seeded in 10cm culture dish, placed in 5% CO 2 Culture in an incubator at 37°C. When the cells are 80-90% full, use a pipette to discard the old culture medium, add 2-3mL 0.25% trypsin, digest for 1-3 minutes, observe under the microscope that the cells shrink and become round, immediately add 5mL complete medium to stop digestion , collect cells, and perform cell counting.
[0043] The complete medium formula is: 10ng / mLbFGF+1...
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