Cultivation of ASCs

ASCs were expanded essentially as described [33]. The cells were washed with PBS and trypsinized using 0.05% trypsin-EDTA 1x solution (Sigma). Trypsin was inactivated by addition of ASC medium plus 10% FBS and removed by centrifugation at 300 g for 5 min and the cells seeded in a density of 5000 cells/cm² in ASC medium plus 10% FBS and maintained at 37°C with 5% CO₂. Sixteen hours later, the medium was replaced by PM4 medium (ASC medium containing 2.5% FBS, 10 ng/ml EGF (Immuno Tools Friesoythe, Germany), 1 ng/ml bFGF (Immuno Tools, Germany), 500 ng/ml insulin (Sigma). ASC were passaged at a ratio of 1: 2, medium was changed every third day and the cells were grown to 70% confluence before splitting. Population doublings (PDL) were calculated using the following equation: 1 PDL = Log₁₀ (N/N°) × 3.33 (N = number of cells at the end of a passage, N° = number of cells that were seeded at the beginning of a passage) [39]. For storage cells were pelleted as described above, diluted in cryomedium (DMEM/F-12 medium (1:1) (1x) with HEPES and L-glutamine (Sigma), with 20% FBS and 7.5% DMSO), at a density of 10⁶ cells/ml, slowly brought to – 80°C and then stored in frozen nitrogen. After thawing, cryomedium was immediately removed by centrifugation. ASCs cultivated to P6 were used in this study.