Umbilical tissue-derived cells are isolated from minced and enzymatically digested umbilcal cords.
Umbilical cords are minced and enzymatically digested with Dulbecco's modified Eagle's low-glucose (Lg) medium (DMEM) mixed with collagenase (50 U/ml). The cell suspension is filtered through a 70-μm filter, and the supernatant is centrifuged at 350g. Isolated cells are washed in DMEM-Lg several times and plated at a density of 5,000 cells per cm2 in T75 flasks in DMEM-high glucose (Hg), supplemented with defined fetal bovine serum (FBS; 15%), nonessential amino acids (0.1%) , β-mercaptoethanol, penicillin (100 U/ml), and streptomycin (100 μg/ml). After cryopreservation, cells are thawed and seeded on gelatin-coated flasks, in growth medium (consisting of DMEM-Lg supplemented with FBS (15%) , β-mercaptoethanol ( 0.001%) , penicillin (100 U/ml) and streptomycin (100 μg/ml)). Cells are cultured under standard conditions in atmospheric oxygen with 5% carbon dioxide, at 37°C. When cells reach approximately 70% confluence, they are passaged every 3-4 days, using trypsin/EDTA, and reseeded at a density of 5,000 cells per cm2 onto gelatin-coated flasks. Flasks were gelatin coated by placing a solution of gelatin (2% (wt/vol)) into tissue culture flasks for a minimum of 20 minutes at room temperature. The solution is then aspirated, and the flasks are washed at least two times with phosphate-buffered saline (PBS) prior to cell culture use. Cells are harvested after 10 passages (approximately 20 population doublings) and cryopreserved in supplemented growth medium (Hg) containing DMSO (10%) in a programmable rate freezer.