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Wharton's jelly stem cells were isolated from umbilical cords collected from human donors.
The mid-region of each umbilical cord was collected in Hank’s balanced salt solution (HBSS) and then cut into 1.5 cm-long pieces. Each piece was slit open with scissors and the inner surface was exposed to enzymatic solution of collagenase type I, collagenase type IV and hyaluronidase in DMEM medium, in Petri dishes (100 mm). The Petri dishes were incubated for 45 min to promote Wharton’s jelly loosening, but not its complete digestion. The cord pieces were then transferred to new Petri dishes containing fresh DMEM medium. Using the blunt surface of a pair of curved forceps, the Wharton’s jelly was gently separated into the fresh medium. The medium containing the Wharton’s jelly was then collected and passed through a needle to release the WJSCs. The suspension of WJSCs was then collected and centrifuged, and cell pellets were resuspended in DMEM (high-glucose) culture medium supplemented with fetal bovine serum (FBS; 20%), bovine fibroblast growth factor (bFGF; 16 ng/ml), L-glutamine (1 mM), insulin-transferrin selenium (ITS, 1:200 dilution) and antimycotic-antibiotic solution. The cells and medium were seeded into plastic tissue culture flasks.