Protocol for reconstructing 3D human skin equivalents on endogenous dermal matrix with optional pigmentation

Previous in vitro 3D skin models are limited by their reliance on animal-derived collagen, a poor representation of the human dermis, and low throughput. Here, we present a protocol for reconstructing 3D human skin equivalents on an endogenous dermal matrix, in 12- and 24-well format, with optional pigmentation. We describe the expansion and seeding of fibroblasts, keratinocytes, and melanocytes in non-contractile polystyrene scaffolds. We then detail air-lifting and epidermal maturation, resulting in a fibroblast-derived dermal matrix with a fully stratified epidermis. For complete details on the execution of this protocol, please refer to Tham et al. 1