Establishment of 3D organotypic cultures using human neonatal epidermal cells

Nat Protoc. 2007;2(1):178-86. doi: 10.1038/nprot.2006.448.


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 mum) 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 keratinocytes, which are macroscopically visible as artificial skin after a couple of days. The cultures can be harvested 1 week after the air-lift and processed for immunohistochemistry or gene expression analysis. The overall procedure can be completed in 3 weeks, including the preparation of the dermal equivalent and the seeding of the primary keratinocytes.

MeSH terms

  • Cell Culture Techniques / methods*
  • Cell Differentiation / physiology
  • Collagen
  • Epidermal Cells*
  • Fibroblasts
  • Flow Cytometry / methods
  • Humans
  • Infant, Newborn
  • Keratinocytes / cytology*


  • Collagen