Pigmented Full-Thickness Human Skin Model Based on a Fibroblast-Derived Matrix for Long-Term Studies

Reconstructed human skin models are a valuable tool for drug discovery, disease modeling, and basic research. In the past decades, major progress has been made in this field leading to the development of full-thickness skin models (FTSms) better representative of the native human skin by including the cellular cross talk between the dermal and epidermal layers. However, current available FTSms still present important limitations since they are only suitable for short-term studies, include nonhuman extracellular matrix (ECM) components and have a weak skin barrier function compared with in vivo human skin. In this study, a fibroblast-derived matrix was combined with the use of an inert polystyrene scaffold for the development of a fully human dermis capable of supporting a differentiated epidermis. To produce a pigmented FTSm, a coculture with keratinocytes, melanocytes, and fibroblasts was established. The structure and functionality of the developed FTSms were studied for short- and long-term cultivation using histological and immunofluorescence staining. The integrity of the skin barrier was evaluated using transepithelial electrical resistance (TEER) measurements. It was possible to obtain a mature dermis capable of supporting an epidermis without keratinocyte infiltration in only 6 days. ECM components (collagen IV and fibrin) were secreted by the fibroblasts and accumulated in the scaffold structure, recreating the microenvironment of the native human dermis. Moreover, the use of a scaffold resulted in a structure with mechanical stability due to its noncontracting nature. The coculture of primary human keratinocytes resulted in a terminally differentiated skin equivalent that could maintain its architecture and homeostasis up to 50 days. Melanocytes were correctly integrated within the epidermal basal layer and made it possible to reproduce constitutive pigmentation. TEER levels increased during culture time, reaching values of 1.1 ± 0.2 kΩ.cm(2) for the FTSm, indicative of a functional skin barrier. IMPACT STATEMENT: The developed fully human full-thickness skin model has the potential to reduce the dependence on animal models for long-term studies of skin diseases and safety and efficacy assessment of novel drugs. Its longevity and robustness allow the experimental testing phase to be lengthened. The presence of active melanocytes at the dermal–epidermal junction makes this model the ideal platform to study skin pigmentation disorders.