Isolation and Expansion of Adipose-Derived Mesenchymal Stem Cells

Fat tissues (obtained from hip or abdomen) were cut into mm-size pieces using a scalpel. The samples were washed several times with phosphate buffered saline (PBS). To release Ad-MSCs from the tissue samples were incubated in a 0.7% collagenase II solution (Biochrom, Berlin, Germany) for approximately 30 minutes at 37 °C. The addition of culture medium (DMEM 4.5 g/L glucose, 10% FCS, 100 U/mL penicillin, 100 µg/mL Streptomycin) stopped the collagenase II activity. After centrifugation at 600 g for 10 min the Ad-MSC pellet was re-suspended in culture medium for expansion (37 °C, 5% CO₂, humidified atmosphere). Experiments were performed in passage 3 or 4 when Ad-MSCs were negative (determined by RT-PCR) for CD14 and positive for CD73, CD90, and CD105, as reported earlier [21 (link),39 (link)].