Isolating stem cells from the umbilical vein
Reagents and materials:
1. Medium: Complete LG-DMEM: low-sugar DMEM containing 2 mmol/L L-glutamine, 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin;
2. PBSA;
3. Trypsin, 0.05%, containing EDTA;
4. Collagenase A, 0.1% formulated with PBSA;
5. Culture the bottle;
6. a catheter;
experimental method:
1. Collect and treat the umbilical cord within 6-12 hours after normal delivery;
2. Insert the catheter into the umbilical vein and wash it twice with PBSA;
3. Clamp the distal umbilical cord;
4. Fill the vein with a 0.1% collagenase solution;
5. Clamp the proximal umbilical cord;
6. Incubate the umbilical cord at 37 ° C for 20 min;
7. Gently massage the umbilical cord, collect a suspension containing endothelial and subendothelial cells, centrifuge at 600g for 10min;
8. Resuspend the cells in medium;
9. After counting, inoculate the cells into a 75 cm2 culture flask at a density of 1 x 103 cells/cm2;
10. After 3 days, the medium was changed, the unattached cells were removed, the adherent cells were allowed to continue to grow, and the fresh medium was changed every 3 days. After about 2 weeks, fibroblast-like cells were observed to grow;
11. In this case, the cells were harvested with 0.25% trypsin/EDTA and passaged into new culture flasks to continue amplification;
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