Joris van Dongen

314 Chapter 13 Colony formation assay Tagged ASCs were seeded at respectively hundred and thousand cells per well of a six- well culture plate in duplicate and were cultured for 14 days. Cell were washed with PBS and fixed with 2%PFA in PBS for 15 mins. Cells were washed well with PBS and stained with Crystal Violet (Sigma‐Aldrich). Plated were scanned and the ability to form colonies was assessed by determining the area of the colonies. Histological characterization of acellular matrix by haematoxylin & eosin Cryo-sections of 12 µm were made from snap-frozen adipose tissue, SVF and ECM derived from the same donor (n =17). Samples were stained with haematoxylin solution for 5 min. After staining, samples were washed with tap water for 5 min. Afterwards, samples were stained with eosin solution for 10 min. and subsequently washed with tap water for 5 min. Finally, samples were mounted with Aquatex and visualized under light microscope (Leica Microsystems, DM IL). MTT conversion assay for cell viability Hydrogels (n=3) in triplicate were prepared in a 24 well plate with a volume of 0.5 ml. One ml of culture medium (basal DMEM) was placed on top of each hydrogel and collected every 24h for four days. Conditioned medium from each hydrogel for each time point was used in triplicate. A serial twofold dilution series of the hydrogel- derived medium was used. Human dermal fibroblasts (PK84) were cultured in 96 well plates in culture medium (as previously described) until confluency. Culture medium was replaced with the dilution series of hydrogel-derived medium for 48h. As a positive control for cytotoxicity, a twofold serial dilution series of puromycin (Gibco, 10mg/ ml) in medium was used, starting at 10 ul per well. Normal culture medium served as negative control. After 48h, 5 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) in PBS was added to each well. Plates were incubated at 37 º C for 3h. Afterwards, the medium was removed and the purple formazan crystal dissolved in 200 µl of dimethyl sulfoxide (DMSO) by careful mixing. Optical density was measured at 650 nm and 585 nm. The difference in optical density between 650 nm and 585 nm was plotted against the log dilution to generate cytotoxicity graphs. Results were analysed with GraphPad Prism 7 using a nonlinear regression for a dose- response inhibition. The half maximal inhibitory concentration (IC 50 ) was used as an indicator of cytotoxicity. The IC 50 value indicates howmuch of the substance is needed to induce 50% of cell death.