In vitro 3D full-thickness reconstituted human skin has high physiological relevance due to the presence of differentiation features often lacking in 2D cell cultures. Here, we present a protocol to reconstruct a 3D skin model using human fibroblasts, keratinocytes, and rat-tail collagen I. We describe steps for cell expansion, the casting of cellular and acellular layers, seeding keratinocytes, the air lifting of culture, and incubation. We also demonstrate the use of chitosan to prevent tissue contraction in 12-well inserts. For complete details on the use and execution of this protocol, please refer to Robinson et al..
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