Joris van Dongen

107 tSVF acts anti-inflammatory on chondrocytes in vitro Colony formation unit capacity of ASCs Ten thousand viable cells enzymatically processed from tSVF passage 0 as well as thousand cells from passage 1 and 2 (n = 11 and six technical replicates) were seeded and cultured for twelve days to assess the colony forming capacity. Afterwards, cells were fixed in 4% formalin and stained with 5% Crystal Violet (Sigma-Aldrich, St. Louis, MO). Colony frequency was calculated as the mean number of colonies / total seeded cells x 100%. Differentiation capacity of tSVF and ASCs tSVF-derived cells from passage 0, 1, 2, 3 (n = 11) were collected and used for chondrogenic, adipogenic and osteogenic differentiation assays. For osteogenic differentiation the cells were seeded at a density of 128.000 viable nucleated cells for passage 0 and 32.000 viable nucleated cells for passages 1, 2 and 3 perwell of a 12-wells plate in expansion medium. The P0 were cultured for 72 hours and P1, 2 and P3 cells for 24 hours before the medium was changed to osteogenic differentiation medium. The osteogenic differentiationmedium consisted of alpha-MEM (Gibco) supplemented with 10% FBS, 10 nM dexamethasone, 0.2 mM ascorbic acid-2-phosphate, 10 mM of β -glycerophosphate and pen/strep. The cells were cultured in osteogenic medium for 3 weeks and medium was changed three times a week. After 3 weeks, the cells were fixed with formalin and washed with PBS and stained for mineralization with 3% Alizarin Red S and washed 6 times with PBS where after microphotographs were taken. For adipogenic differentiation the cells were seeded at 960.000 for P0 and 240.000 for P1, P2 and P3 cells per well of a 24-wells plate. The P0 were cultured for 72 hours and P1, 2 and P3 cells for 24 hours before the medium was changed to adipogenic differentiation medium that consisted of alpha-MEM supplemented with 10% FBS, 500 µM 3-isobutyl-1-methylxanthine (IBMX), 1 µM dexamethasone, 0.2 mM indomethacin, 1.72 µM insulin and pen/strep. The cells were cultured in osteogenic medium for 3 weeks and medium was changed three times a week. After culture, the cells were fixed in formalin, washed with PBS followed by 60% isopropanol, stained with Oil red O, washed with 60% isopropanol and washed with PBS whereafter they were microphotographed. For chondrogenic differentiation 1 million P0 or 250.000 P1, P2 and P3 viable nucleated cells were seeded in a 15 mL falcon tube that was centrifuged for 5 minutes at 300xg to pellet the cells. After 3 days, the culture medium was replaced by chondrogenic medium consisting of DMEM supplemented with 1% ITS+ premix (354352; BD Bioscienes), 10−7 M dexamethasone (D8893; Sigma), 0.2mM L-ascorbic