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Optimization and Characterization of Decellularized Adipose Tissue Malik J. Snowden1; Christopher M. Mahoney, M.S.1; Kacey G. Marra, Ph.D.1-4; J. Peter Rubin, MD1-4 Dept. of Bioengineering1, Dept. of Plastic Surgery2, School of Medicine3, McGowan Institute of Regenerative Medicine4, University of Pittsburgh, Pittsburgh, PA 15213 INTRODUCTION There is currently a clinical need for the improvement of soft tissue repair. Soft tissue repair is required after soft tissue loss which can be caused by congenital deformities, traumatic wounds and tumor resections. A common type of soft tissue repair is autologous fat grafting.1 However, the problem with grafted fat is that it tends to succumb to fat ischemia caused by a lack of vascular integration with the implant site. This fat ischemia leads to adipocyte necrosis and the reabsorption of the graft back into the body.2 In this, the graft volume will be reduced 40-60% within the first six months of the grafting procedure.3 In order to combat the volume reduction that is associated with autologous fat grafting, studies have shown that a suitable regenerative technique may be to develop scaffolds derived from the extracellular matrices (ECM) of human adipose tissue.4 Adipose tissue is used to produce the extracellular matrices because it contains high concentrations of proteins that contribute to adipogenesis.4 The approach taken in the laboratory requires two protocols, one being to decellularize human adipose tissue and the other being to derive a hydrogel from the decellularized adipose tissue. The purpose of the decellularization protocol is to remove all of the cell, DNA, and lipid content from the tissue. However, after decellularization, there is still remaining cell and lipid content in the ECM. The purpose of the gelation protocol is to form a thermosensitive hydrogel derived from the decellularized adipose tissue, however hydrogels formed do not always maintain thermosensitive properties. Steps have been taken towards optimizing these protocols so that the goal of an off-the-shelf biomaterial scaffold can be achieved. METHODS Non-diabetic abdominal whole fat was donated from patients undergoing elective abdominoplasty at the University of Pittsburgh Medical Center. The decellularization process used to produce the ECM includes four main stages, consisting of alcohol rinses, delipidization, and disinfection of the adipose matrix. After processing, the matrix is to be snap frozen through the use of liquid nitrogen, or frozen overnight in a -80C freezer, and then lyophilized. Following the freeze drying process, the matrix is broken down into a powder in preparation for digestion by pepsin through the use of a Mini Wiley Mill. A 1:10 ratio of pepsin to ECM is used. The pH of the ECM-pepsin solution is neutralized (7.4) through the use of sodium hydroxide and gelation is tested by heating the hydrogel to body temperature (37°C). In order to begin optimizing the protocol used for tissue decellularization, familiarization with the protocol first occurred, in which the protocol was repeated multiple times. Following this, observations were made that could potentially be reasons for why end results of the decellularization process still have lipid and cell content. The alteration that was made in attempt to optimize the protocol was changing the volumes and concentrations of the alcohols and detergents used so that the tissue would be fully covered during the decellularization rinses. To optimize the protocol used for the gelation of a hydrogel derived from adipose tissue, the ratio of pepsin to ECM was changed from 1:10 to 2:15, so that more pepsin would be present to digest the ECM. RESULTS By altering the concentrations and volumes of the alcohols and detergents used during the decellularization protocol, the protocol was effectively optimized as there was a decrease in the lipid and cell content. As seen in figure 1 below, Hematoxylin and Eosin staining indicates decreased cell content following the optimization of the decellularization, as seen by the decreased amount of purple dots and the decreased amount of overall purple color. Figure 2 indicates decreased lipid content following the optimization of the decellularization protocol. This is indicated by the lack of brown color in the matrix, as the brown color indicates lipid content. Figure 1: Hematoxylin and Eosin stains nucleic acid to detect cells. Left image represents post-optimization and right image represents preoptimization. ECM sample on the right indicates high cell content, characterized by the higher concentration of purple. Figure 2: ECM prior to optimization (left) and after optimization (right). The brown color of the ECM indicates high lipid content while the white color of the right ECM indicated minimal lipid content. By altering the ratio of pepsin to ECM in the hydrogel gelation protocol, the protocol was effectively optimized. The ratio was changed from 1:10 to 2:15 in order to increase the amount of pepsin present to digest the ECM. This allowed the hydrogel to successfully gel at 37°C (the font here isn’t the same as the rest of the paper). DISCUSSION The goal of this project is to optimize two protocols used throughout the laboratory, one being to decellularize adipose tissue and the other to derive an injectable thermosensitive hydrogel form the decellularized adipose tissue. The problem faced when using the decellularization protocol is that tissue still contains cell and lipid content after decellularization. The problem faced when using the hydrogel gelation protocol is that the hydrogels made don’t always possess the thermosensitive properties that we expect them to. The problems initially faced with the decellularization protocol were caused by a lack of interaction between the reagents and the tissue during the decellularization rinses. In order to solve this problem, the surface area of the tissue was increased by adding in additional manual processing of the tissue, as well as by increasing the amount of the reagents used. The problems faced with the hydrogel gelation protocol were caused by a lack of ECM digestion by the pepsin. This was combated by increasing the pepsin concentration to increase the digestion of the pepsin. CONCLUSION The decellularization protocol and the hydrogel gelation protocol were both successfully optimized. This is important as they are necessary to reaching the project’s end goal, which is to develop an injectable biomaterial. Optimizing these protocols allows for ECM suitable for hydrogel gelation and a hydrogel suitable to be an injectable biomaterial. ACKNOWLEDGEMENTS I would like to acknowledge my lab’s primary investigators Dr Kacey Marra, PhD and Dr Peter Rubin, MD, FACS, as well as my graduate mentor Christopher Mahoney and the other members of the Adipose Stem Cell Center as well as our funders. REFERENCES 1. Choi, JH et al. “Adipose Tissue Engineering for Soft Tissue Regeneration.” Tissue Engineering (2010) 413-428. 2. Kelmendi-Doko, A et al. “Adipogenic FactorLoaded Microspheres Increase Retention of Transplanted Adipose Tissue.” Tissue Engineering (2014) 1-8. 3. Wang, W et al. “Adipose tissue engineering with human adipose tissue-derived adult stem cells and a novel porous scaffold.” Society For Biomaterials (2012) 68-75. 4. Sano, H et al. “Acellular adipose matrix as a natural scaffold for tissue engineering.” Journal of Plastic, Reconstructive & Aesthetic Surgery (2014) 99-106.