Joris van Dongen

285 Adipose ECM hydrogels to release paracrine factors humidified incubator in Dulbecco’s Modified Eagle’s Medium (DMEM) (BioWhittaker Walkersville, MD: 10% fetal bovine serum (FBS), 1% L-Glutamine (L-Glut), 1% Penicillin/Streptomycin (P/S)). Medium was refreshed twice a week. After culture, cells were characterized for ASC characteristics. Cells were characterized based on CD marker surface expression using flow cytometry (CD29, CD31, CD44, CD45, CD90 and CD105), adipogenic, osteogenic and smooth muscle cell differentiation capacity as well as colony formation capacity. Pooled non-diabetic ASC (n=3) of passage 4 – 6 were used to prepare ASC conditioned medium (ASC-CMe) (containing: DMEM, 1% L-Glut, 1% P/S) or RPMI 1% L-Glut, 1% P/S). Medium was collected and filtered through a 0.22 µm filter after 24h of culture. ASC characterization was confirmed in accordance with the International Federation of Adipose Therapeutics and Science (IFATS)/International Society of Cellular Therapy (ISCT) criteria 25 . Generation of hydrogels incubated with released factors by ASCs NAM and DAM samples (n=3) were digested with 2 mg/ml of porcine pepsin (3,200 I.U. Sigma-Aldrich) in 0.01 M hydrochloric acid. 1 mg of porcine pepsin was used to digest 10 mg of lyophilized acellular matrix. Acellular matrix was digested under constant stirring for 6h at RT. Afterwards, the porcine pepsin was inactivated by pH neutralization (pH 7.4) with 0.1 M sodium hydroxide to reach 0.01M final concentration (Fig. 1). Then, ASC-CMe was mixed in order to allow released factors to bind to acellular matrix and finally, salt was added in order to allow for self-assembly gelation. To maintain the appropriate concentration of acellular matrix in hydrochloric acid to facilitate gelation, ASC-CMe and PBS were added in a concentrated form. ASC- CMe was concentrated with the use of 3 kDa cut-off Amicon® Ultra-Centrifugal filters (Sigma-Aldrich), respectively twenty-, forty- and eighty-times. Twenty-times, forty- times or eighty-times concentrated ASC-CMe was added and carefully mixed to reach final dilutions of respectively one-time (ASC-CMe1), two-times (ASC-CMe2) and four-times (ASC-CMe4) concentrated ASC-CMe (Fig. 1). The released factors bind to the acellular matrix pre-gel solutions at 4 °C for 24h. Next, the pre-gel solutions were brought to physiological conditions i.e. 1x PBS by adding twenty times concentrated PBS. Finally, the pre-gel solution was placed in an incubator to allow for self-assembly gelation for 1h at 37 °C (Fig. 1). Released factors from the incubated hydrogels byASC- CMe1, ASC-CMe2, ASC-CMe4 were harvested in serum free medium after 24h and stored at -80 °C for immunoassaying. The respective concentrated ASC-CMe served as input controls (2.8). Released factors from the incubated hydrogel by ASC-CMe1 were harvested in serum free medium after respectively 24h, 48h and 96h and stored at -80 °C for biological assays. For fibroblast migration and endothelial angiogenesis, the respective concentrated ASC-CMe1 as well as DMEM serum free incubated hydrogels