Mesenchymal stem cells were isolated from patient bone marrow aspirates.
Bone marrow aspirates were collected, and mononuclear cells (MNCs) were separated using a Ficoll-Paque density gradient. The interface was collected and resuspended in low-glucose DMEM/F12 and 40% MCDB-201 medium supplemented with FBS (2% (v/v)), ITS, dexamethasone (1029 M), ascorbic acid 2-phosphate (1024 M), EGF (10 ng/mL) and PDGF BB (10 ng/mL), and then seeded on culture flasks (1x106 cells/ml). The culture was maintained in a humidified incubator containing 5% CO2 at 37°C. After 24-48 hours of culture, nonadherent cells were removed. When reaching 70%-80% confluence, cells were detached with trypsin (0.05%) and EDTA (0.01%) and replated. BM-MSCs were harvested and used at passages 3-6. All cell culture steps were performed in a GMP facility.