Bone marrow-derived mesenchymal stem cells (Università degli Studi del Piemonte Orientale)

Bone marrow cells were centrifuged at 900 ×g for 15min to discard the anticoagulant medium and then layered on a Percoll gradient (density: 1.073 g/ml) and centrifuged at 1100 ×g for 30 min. The cells in the interphase were recuperated, washed twice with PBS1X (200 ×g for 10 min) and seeded at a density of 800,000/cm2 in MSC Medium (Cambrex Bioscience) containing fetal bovine serum (FBS, 10%) and maintained at 37 °C in an atmosphere of 5% CO2. After 3 days, the nonadherent cells were removed and refed every 3-4 days. In order to expand the isolated cells, the adhered monolayer was detached with trypsin/EDTA for 5 min at 37 °C, after 15 days for the first passage and every 7 days for successive passages. During in vitro passaging, the cells were seeded at a density of 8000/cm2 and expanded for 2-3 passages.