Joris van Dongen

315 Adipose ECM hydrogels to release paracrine factors Live/dead staining of ASCs on hydrogels Hydrogels (n=3) in triplicate were prepared in a 24 well plate with a volume of 0.5 ml Hydrogels were incubated with one ml of basal medium (as previously described in 2.5) for eight days and replaced every day. A suspension of 300 µl of culture medium containing 200,000 immortalized ASCs was added on top of each hydrogel. Immortalization was performed with the large T antigen of SV40 and lentivirally tagged (Harmsen lab, described in 2.4) with enhanced GFP (green, iASC13EGFP) or with Dtomato (red, iADSC13Dtomato). Cells were evaluated for seven days. Culture medium was changed every day and micrographs were taken every day. After 7 days a live/dead staining was performed with CFDA-SE (1:2000), PI (1:500) and DAPI (1:5000) for iASC13GFP. For iADSC13dTOMATO only CFDA-SE and DAPI were used, PI staining was not used as it has the same wavelength as already pre-stained cells, therefore they are both the same colour and it cannot be differentiated. As a control, 1,300 µl of culture medium containing 200,000 iASC13EGFP in a 24 well plate was used. Samples were visualised with an immunofluorescence microscope (EVOS® FL Cell Imaging System). After evaluation, samples were fixed with 2% paraformaldehyde in PBS for 30 min. Finally, samples were embedded in paraffin for immunohistology staining. Immunohistology staining of ASCs inside hydrogels From the above-mentioned paraffin embedded hydrogels (2.6), 4 µm sections on adhesive slides were made with Leica Reichert-Jung 2055 microtome. Samples were deparaffinised and incubated overnight with 0.1 M Tris/HCl solution (pH 9.0) at 80 º C. Next day, samples were stained with haematoxylin for 4 min. and mounted with Aquatex. Results were evaluated with a light microscope (Leica Microsystems, DM IL). RESULTS Characterization of immortalized ASC Immortalized ASC retain ASC characteristics Amean of 98.9% , 99.0%, 95.9% and 23% of the immortalizedASCs showed expression of CD29, CD44, CD90 and CD105. Endothelial marker CD31 and common leukocyte marker CD45 was not detected. These results show that the lentivirally tagged immortalized ASC have a similar surface marker expression as ASC (Fig.2). 15