A variety of skin substitutes that restore epidermal and dermal structures are currently available on the market. While the main focus in research and clinical application lies in dermal and epidermal substitutes, the development of a subcutaneous replacement, the hypodermis, is often neglected. This chapter describes the use of fibrin sealant as a hydrogel scaffold to generate a three-dimensional skin substitute. For the hypodermal layer adipose-derived stem cells (ASCs) and mature adipocytes are seeded within a fibrin hydrogel. On top, another fibrin clot with incorporated fibroblasts is placed for the construction of the dermal layer. Keratinocytes are added on top of the two-layered construct to form the epidermal layer. The three-layered construct is cultivated for up to 3 weeks with keratinocytes being exposed to air according to the air-liquid interface cultivation model.
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