Mesenchymal stem cells were derived from the bone marrow and tri-lineage differentiation potential (adipocyte, chondrocyte, osteocyte) was confirmed.
Bone-marrow mononuclear cells are separated by density gradient centrifugation in Ficoll-Paque™ PREMIUM. Washed cells are re-suspended in PBS/EDTA and cultured in Dulbecco's modified Eagle's low-glucose medium supplemented with foetal bovine serum (10%) and plated at a density of 1 × 108 cells per cell-factory. Cultures are maintained in cell factories at 37°C in a humidified atmosphere containing 5% CO2. When the cultures are near confluence (>80%), cells are detached by treatment with trypsin-EDTA (0.25%) and re-plated at 3.5 × 106 cells per cell factory. When 2 × 106cells/kg (per participant) or more are obtained, cells are harvested and cryopreserved in human albumin solution (4.5%) with dimethyl sulfoxide, at a final concentration of 10%.