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

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.