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A variety of skin substitutes that restore epidermal and dermal structures are currently available on the market. However, the main focus in research and clinical application lies on dermal and epidermal substitutes whereas the development of a subcutaneous replacement (hypodermis) is often disregarded. In this study we used fibrin sealant as hydrogel scaffold to generate a three-layered skin substitute. For the hypodermal layer adipose-derived stem cells (ASCs) and mature adipocytes were embedded in the fibrin hydrogel and were combined with another fibrin clot with fibroblasts for the construction of the dermal layer. Keratinocytes were added on top of the two-layered construct to form the epidermal layer. The three-layered construct was cultivated for up to 3 weeks. Our results show that ASCs and fibroblasts were viable, proliferated normally, and showed physiological morphology in the skin substitute. ASCs were able to differentiate into mature adipocytes during the course of four weeks and showed morphological resemblance to native adipose tissue. On the surface keratinocytes formed an epithelial-like layer. For the first time we were able to generate a three-layered skin substitute based on a fibrin hydrogel not only serving as a dermal and epidermal substitute but also including the hypodermis.