Standardized, Defined Serum-free Culture of a Human Skin Equivalent on Fibroblast-populated Collagen Scaffold

Abstract

The defined, serum-free media used in the cultivation of skin equivalents are liable to inter-laboratory variations, require the preparation of multiple additives, and are potentially difficult to replicate. In this study, we assessed the use of standardized, serum-free and bovine pituitary extract-free keratinocyte culture media in the development of a human skin equivalent. After culture at the air-liquid interface for 3 weeks on a fibroblast-populated collagen lattice, an orthokeratinized, pluristratified epithelium was produced which expressed cytokeratins, cornified cell envelope precursors (involucrin, transglutaminase 1, filaggrin) and desmosomal components (desmoglein and desmocollin 1 and 3, plakophilin 1) in a differentiation-specific manner. There was also evidence of basement membrane reconstitution with collagen IV/VII, laminin 5, α6 and β4 integrin subunit expression at the epithelial-matrix junction. Overall, our findings demonstrate that readily available, defined organotypic culture media can be used to generate a reproducible skin equivalent with hallmarks of epidermal differentiation.