The Simplest Protocol for Rapid and Long-Term Culture of Primary Epidermal Keratinocytes from Human and Mouse

Abstract

Although mouse models have been used as an essential tool for studying the physiology and diseases of the skin, propagation of mouse primary epidermal keratinocytes remains challenging. In this chapter, we introduce the simplest, at least to our knowledge, protocol that enables long-term expansion of p63⁺ mouse epidermal keratinocytes in low Ca²⁺ media without the need of progenitor cell-purification steps or support by a feeder cell layer. Pharmacological inhibition of TGF-β signaling in crude preparations of mouse epidermis robustly increases proliferative capacity of p63⁺ epidermal progenitor cells, while preserving their ability to differentiate. Suppression of TGF-β signaling also permits p63⁺ epidermal keratinocytes to form macroscopically large clones in 3T3-J2 feeder cell co-culture. Suppression of TGF-β signaling also enhances the clonal growth of human keratinocytes in co-culture with a variety of feeder cells. This simple and efficient approach will not only facilitate the use of mouse models by providing p63⁺ primary epidermal keratinocytes in quantity but also significantly reduce the time needed for preparing the customized skin grafts in Green method.