Joris van Dongen

311 Adipose ECM hydrogels to release paracrine factors the aim of this research was to assess the influence of human adipose tissue-derived ECM hydrogels on viability, proliferation and migration of therapeutic cells i.e. ASCs and fibroblasts. MATERIALS AND METHODS Fractionation of adipose tissue procedure The fractionation of adipose tissue (FAT) procedure was performed as previously described. 15 Briefly, adipose tissue was harvested during normal liposuction procedures and transferred cooled from the operation room. Informed consent was obtained according to the local ethical committee of the University Medical Center of Groningen. For the FAT procedure, lipoaspirates were warmed to room temperature (RT) and divided into 50 ml tubes. Then, lipoaspirate was centrifuged at 956xg for 3 min. at RT to separate adipose tissue into three layers: oil, adipose tissue and infiltration fluid. The oily fractions as well as the infiltration fluid were discarded. Next, the centrifuged adipose tissue was placed in a 10 ml syringe and connected to the fractionator (a luer to luer connector with three holes of 1.4 mm inside). An empty 10 ml syringe was connected on the other side of the fractionator. Adipose tissue was pushed 30 times forwards and back and the adipocytes were mechanically disrupted. Finally, adipose tissue was centrifuged again at 956xg at RT for 3 min. The second round of centrifugation yielded four fractions: oily fraction, SVF and infiltration fluid containing a small pellet. SVF was collected and washed with phosphate-buffered saline (PBS) and stored at -20 º C until further use (Fig. 1). Decellularization of stromal vascular fraction SVF was frozen with 50% ethanol/water in a -80 º C fridge for 2h and thawed for 30-60 min. for four cycles. 26 After thawing, the 50% ethanol/water mixture was replaced every time. Next, the SVF was incubated with 0.05% trypsin/ 0.05 mM ethylenediaminetetraacetic acid (EDTA) (1:1 v/v) under constant stirring (Bambino machine) at 37 º C for 90 min. Then, samples were washed with phosphate buffered saline (PBS) and sonicated (70W) with 0.5% sodium dodecyl sulphate (SDS) at 46 º C for 20 min. Samples were centrifuged and washed with PBS to completely remove SDS. Finally, samples were lyophilized and subsequently immersed in xylene and placed on a rolling bench for 17 min. Afterwards, samples were washed with PBS and subsequently washed with 100% ethanol until the solution became clear. Samples were incubated with DNAse solution (1:1 v/v) (LS002007, Worthington, final concentration of 30 μg/ ml DNAse in 1.3 mMMgSO4 and 2 mM CaCl2) overnight at 37 º C. Next day, samples