Joris van Dongen

283 Adipose ECM hydrogels to release paracrine factors In this study, we interrogated uptake and release of ASC-released paracrine factors by human adipose tissue-derived ECM hydrogels in vitro . The prepared adipose ECM hydrogels were first incubated with factors released by ASC i.e. conditioned culture medium and subsequently studied for wound healing purposes. Additionally, structural differences between diabetic and non-diabetic ECM regarding binding of factors was investigated. MATERIAL & METHODS Processing and decellularization of adipose tissue Adipose tissue was harvested from the abdomen of non-diabetic (n=5) and diabetic (n=5) patients during regular liposuction procedures and processed anonymously using the FAT procedure, as previously described by van Dongen et al. 22 . Informed consent was obtained according to the local ethical committee of the UniversityMedical Center of Groningen. Briefly, 50 ml tubes containing lipoaspirate were centrifuged at 960 xg at roomtemperature (RT) for2.5min.Next, 10ml of centrifuged adipose tissuewas pushed forward and back thirty times through a Luer-to-Luer connector with three holes of 1.4 mm. The fragmented adipose tissue was again centrifuged at 960 xg at RT for 2.5 min. This procedure resulted in 1 ml of tSVF, which was then decellularized, as previously described by Roehm et al. 23 . Briefly, 10 ml of tSVF was mixed with 30 ml of a 50% ethanol/water mixture and frozen at -80 °C and thawed at RT in 30 min. for four cycles. Then, tSVFwas incubated in 0.05% trypsin/ 0.05 mM ethylenediaminetetraacetic acid (EDTA) (1:1 v/v) for 1.5h at 37 °C. Afterwards, samples were sonicated (70W) in 0.5% SDS at 46 °C for 20 min. Samples were then lyophilized and immersed in xylene (1:10 w/v) for 17 min. Next, samples were washed with 96% ethanol and incubated in DNAse solution (LS002007, Worthington) (containing a final concentration of 30 µg/ml DNAse in 1.3 mMMgSO 4 and 2 mM CaCl 2 ) for 24h. Finally, samples (acellular matrix) were lyophilized again and homogenized with the use of an UltraTurrax fragmer (PM Tamson Instruments) and stored at -80°C before used in experiments. Histological characterization of acellular adipose matrix One-time centrifuged adipose tissue and tSVF (controls) as well as acellular matrix of non-diabetic (NAM) and diabetic patients (DAM) (n=3) were formalin-fixed and embedded in paraffin. Four µm thickness sections were cut, deparaffinized, then incubated overnight with 0.1 M Tris/HCL buffer (pH 9.0) at 80 °C and stained with antibody against Perilipin A (1 : 200, Abcam) to visualize adipocytes as previously described. 22 Samples were visualized under a light microscope Leica Microsystems,