Mesenchymal stem/progenitor cells are derived from the umbilical cord blood (UCB) samples, which are collected from the umbilical vein upon informed maternal consent.
A 16-gauge needle from a UCB collection bag containing 23 mL of CPDA-1 anticoagulant is inserted into the umbilical vein, and UCB is allowed to flow by gravity. UCB harvests are processed within 24 h of collection, with viability of more than 90%. Mononuclear cells are isolated by centrifugation in a Ficoll/Hypaque gradient (density 1.077 g/cm3). The separated mononuclear cells are washed, suspended in a-MEM, supplemented with FBS (10%), and seeded at a concentration of 5x10^6 cells/cm2. Cultures are maintained at 37oC in a humidified atmosphere containing 5% CO2 with a change of culture medium twice a week. UCB-derived mononuclear cells are set in culture, and the onset of fibroblast-like adherent cells is observed. One to 3 weeks later, when the monolayer of MSPCs colonies reach 80% confluence, cells are trypsinized (0.25% trypsin), washed, resuspended in culture medium (a-MEM supplemented with FBS (10%)) and subcultured at a concentration of 5x10^4 cells/cm2. MSPCs of each UCB harvest are then expanded ex vivo by successive subcultivation under the same conditions.