Enzymatic digestion of stem cells from Wharton gel

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, 2.5%;
4. Collagenase A, 0.1% formulated with PBSA;
5. Culture the bottle;
6. Conical Centrifuge Tube , 50ml;
7. Scissors;
8. Scorpion;

experimental method:
1. After the umbilical cord is taken, it can be stored in PBSA for 1-24 hours;
2. Remove the blood vessel and cut the Wharton gel into small pieces of tissue about 0.5 cm;
3. Transfer the minced tissue block to a 50 ml centrifuge Tube , wash with serum-free DMEM, and centrifuge at room temperature for 250 min at room temperature for 5 min;
4. Pour off the supernatant, scatter the precipitate after centrifugation, soak in 0.1% collagenase (about 3 times the volume of the precipitate), digest at 37 ° C for 16-18h;
5. Add an appropriate volume of PBSA (2 times the volume of the digestive juice), centrifuge at room temperature 250g for 5min;
6. Pour off the supernatant, add 2.5% trypsin (about 2 times the volume of the sediment) at 37 ° C for 30 min, gently agitate;
7. Stop the trypsin by adding an appropriate volume of FBS (10% of the digestive enzyme volume);
8. Wash with medium;
9. Resuspend the isolated cells in medium and inoculate the cells into a culture flask at a density of approximately 1 x 103 cells/cm2;

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