Organotypic 3D Skin Models: Human Epidermal Equivalent Cultures from Primary Keratinocytes and Immortalized Keratinocyte Cell Lines

Methods Mol Biol. 2020;2154:45-61. doi: 10.1007/978-1-0716-0648-3_5.


The three-dimensional culturing of human keratinocytes at the air-liquid interface yields a fully stratified epidermis including a functional stratum corneum and thus enables the study on epidermal structure and function in the context of biomedical, toxicological and pharmaceutical sciences. Here we provide a step-by-step detailed protocol for the isolation of human primary keratinocytes and the development of human epidermal equivalents generated from primary keratinocytes or immortalized keratinocytes (N/TERT-1; N/TERT-2G), including widely accepted procedures for the analysis of barrier function, tissue morphology, cell proliferation, and gene expression.

Keywords: Epidermis; Keratinocyte isolation; Laboratory protocol; Proliferation; Skin barrier; Skin tissue engineering.

MeSH terms

  • Animals
  • Biomarkers
  • Cell Culture Techniques*
  • Cell Differentiation
  • Cell Line, Transformed
  • Cell Separation / methods
  • Cells, Cultured
  • Epidermal Cells / cytology*
  • Fluorescent Antibody Technique
  • Gene Expression
  • Humans
  • Immunohistochemistry
  • Keratinocytes / cytology*
  • Keratinocytes / metabolism
  • Mice
  • Skin / cytology*


  • Biomarkers