Validated Step-by-Step Protocols for Human Keratinocyte Isolation and the Construction of Organotypic 3D Epidermal...

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 of epidermal structure and function in the context of biomedical, toxicological, and pharmaceutical sciences. Here, we provide an updated step-by-step, detailed protocol for the isolation of human primary keratinocytes and the development of human epidermal equivalents (HEEs) generated from primary keratinocytes or immortalized keratinocytes (N/TERT-1; N/TERT-2G). This chapter includes information on widely accepted procedures for the analysis of epidermal equivalent tissue morphology, keratinocyte proliferation and differentiation, and gene expression. In addition, this chapter handles qualitative and quantitative procedures for measuring epidermal barrier function, including the more recently developed electrical impedance spectroscopy (EIS).