Isolation and cultivation of bone marrow-derived mesenchymal stromal cells

Mononuclear cells (MNC) are isolated from bone marrow aspirates by density gradient centrifugation and washed in Hank's buffered salt solution. MNC are seeded at a cell density of 2 × 10E4 cells/cm² in low glucose DMEM supplemented with 15% (v/v) heat-inactivated FBS, 2 mM L-glutamine and 0.5% (v/v) antibiotic/antimycotic solution. Cells are incubated at 37°C in a 5% (v/v) CO2-enriched humidified atmosphere, cultured up to 90% confluency, trypsinized, centrifuged, and subcultured at a lower density (5 × 10E3 cells/cm²) for all subsequent passages for 2 weeks.