Establishment of 3D organotypic cultures using human neonatal epidermal cells

Abstract

This protocol describes an ex vivo three-dimensional coculture system optimized to study the skin regenerative ability of primary human keratinocytes grown at the air-liquid interface on collagen matrices embedded with human dermal fibroblasts. An option for enrichment of keratinocyte stem cells and their progeny using fluorescence-activated cell sorting is also provided. Initially, dermal equivalents, comprising human passaged fibroblasts seeded in a collagen matrix, are grown on porous filters (3 μm) placed in transwells. After 1 week, primary human keratinocytes are seeded on this base. One week later, an air-lift transition is performed, leading to the differentiation of the keratinocyte..