Development of soft tissue equivalents - build up with mature adipocytes in a gelatin hydrogel

Introduction: Ther is an urgent need of large fatty tissue constructs for clinical applications. They are necessary if the subcutis is damaged or lacking due to high-graded burns, congenital deformities, tumor resection or trauma. To date, no suitable replacement is available; autologous fatty tissue transfer results in a high shrinkage of the tissue1. Also the use of adipogen differentiated stem cells has not shown the requested long term functionality and differentiation2. Our strategy is to build up a subcutis equivalent by using mature adipocytes in a chemically modified gelatin matrix, which allows an easy and fast tissue composition Materials and Methods: For the generation of 3D constructs, bovine gelatin was methacrylated3. Cytotoxicity of methacrylated gelatin and the non-cured and cured photoinitiator lithium acylphosphinate (LAP) was determined. An isolation method for mature adipocytes from human fatty tissue was established. These mature adipocytes were characterized in a 2D culture by comparison to differentiated adipose-derived stem cells (ASCs) regarding cell morphology, size and lipid accumulation. Adipocytes were encapsulated into methacrylated gelatin and cultured for several weeks. The elasticity of the constructs was adapted to that of native fatty tissue which was evaluated performing rheological measurements. Viable cells were detected by staining of lipid droplets with Nile Red, nuclei with Hoechst 33342 and cytoplasm with fluorescein diacetate (FDA). Furthermore, H&E staining and the staining of perilipin A and collagen IV were performed on paraffin sections. Cell functionality was determined by glycerol and leptin release. Results and Discussion / References: Methacrylated gelatin as well as the non-cured and cured photoinitiator LAP showed no cytotoxicity tested with adipocytes. Isolated mature adipocytes showed a different morphology, cell size and lipid accumulation pattern compared to differentiated ASCs. A method for the encapsulation of mature adipocytes into methacrylated gelatin was successfully established. The elasticity of the subcutis equivalents was comparable to that of native fatty tissue which was seen in rheological measurements. Cells stayed viable and functional for up to 14 days, which was shown by FDA staining, glycerol and leptin release as well as perilipin A and collagen IV stainings. Conclusion: We were able to show that human adipocytes are a suitable cell type for buildup 3D subcutis equivalents. Methacrylated gelatin is an excellent matrix due to its biocompatibility and tunable properties. Our long-term goal is the composition of large subcutis constructs supplied by a vascular system.