Bone marrow-derived mesenchymal stem cells (University of Torino)

Bone marrow-derived mesenchymal stem cells were layered on a Ficoll gradient (density 1022 g/ml) and centrifuged at 1500 rpm for 30 minutes. The mononucleated cells were cultured in mesenchymal stem cell basal medium. After 5 days in culture, the medium was changed. To expand the isolated cells, the adherent monolayer was detached, by a 5-minute trypsin treatment at 37°C, after 15 days for the first passage and every 7 days for successive passages. Cells were seeded at a density of 1X 100,000 cells/cm2 and used within passage 6.