Isolation and Expansion of Umbilical Cord-Derived Cells

UC (n = 12) was obtained from normal full-term-delivery women. Briefly, after disinfection with 75% ethanol, the fresh UC was transferred to PBS where excess blood on the surface was removed. After removal of blood vessels, 2 cm of UC was sliced into very small pieces followed by treatment with 0.1% collagenase II (Gibco, USA), containing antibiotic solution (100 U/mL penicillin and 100 μg/mL streptomycin; CSPC, China) in Dulbecco's Modified Eagle Medium/F12 (DMEM/F12) (Gibco) overnight at 37°C. The cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (Hyclone, China) in 5% CO₂ in a 37°C incubator for 3–4 days to allow cells to adhere. The adherent cells formed colonies and grew rapidly, exhibiting spindle-shaped morphology. The medium was replaced every three days. When the cells reached to 60–80% confluence, they were harvested with trypsin-EDTA solution (Neuronbc, China) and subcultured at a density of 3–6×10³ cells/cm² [38 (link)]. Amplification of pluripotent stem cell markers, such as Oct-4 and Rex-1, was used to demonstrate primitive properties of the cultured cells, as described previously [39 (link)].