Joris van Dongen

289 Adipose ECM hydrogels to release paracrine factors for the controls (serum free culture medium and ASC-CMe1) and stiffness as well as relaxation data of the hydrogels. Data of the controls as well as stiffness and relaxation of the hydrogels was expressed as mean with standard deviation. A one-tailed t -test was used to determine the statistical difference between the hydrogels and hydrogels vs controls with the use of Graphpad, version 7.0c (Graph Pad Software Inc., Los Angeles, CA). RESULTS Fractionation and decellularization of adipose tissue results in an acellular matrix The fractionation of adipose tissue and its subsequent decellularization process resulted in a completely acellular adipose matrix. Macroscopically, acellular matrix was white of color, while one-time centrifuged adipose tissue and tSVF were yellow colored (Fig. 1, left panels, supplemental content). Microscopically, the H&E staining together with the absence of perilipin staining showed that the acellular matrix contained neither adipocytes nor other remaining cells. In contrast, the cell number in tSVF was higher as compared to one-time centrifuged adipose tissue as observed by dense clusters of stromal cells (Fig. 1, second series of panels, supplemental content). In accordance with our previous experience, fractionation of adipose tissue disrupted adipocytes while other cell types and extracellular matrix were preserved 22 . The absence of perilipin staining confirmed the disruption of adipocytes (Fig. 1, third series of panels, supplemental content). Furthermore, Masson’s Trichrome staining demonstrated visibly higher amounts of collagen (blue) per unit area in acellular matrix as compared to one-time centrifuged adipose tissue and tSVF (Fig. 1, right panels, supplemental content). Low amount of DNA left in acellular adipose matrix NAM and DAM samples contained negligible amounts of DNA with 12.83 +/-2.62 ng/mg per dry weight tissue for NAM and 60 +/53.01 ng/mg per dry weight tissue for DAM (p>0.05) (Fig. 2A, supplemental content). The amount of DNA in DAM is, however, higher than the amount of DNA in NAM. All samples, except for one DAM sample, contained a lower amount of DNA than 50 ng/mg per dry weight tissue, which is the standard for successful decellularization. DAPI staining could not reveal nuclei in both NAM as well as DAM as compared to the high number of nuclei after the fractionation of adipose tissue procedure (Fig. 2B, supplemental content).