Primary mouse keratinocyte culture.
Mouse epidermal keratinocytes have traditionally been difficult to grow in vitro. In this chapter, we present a method for isolating epidermal keratinocytes from a single, newborn mouse pup for long-term culture. The protocols we describe will be especially useful for the isolation and analysis of cells harvested from transgenic or knockout mice. We explain how to use a supplemented fibroblast-conditioned medium, along with mouse collagen IV-coated culture dishes, to establish and subculture these fastidious cells for multiple passages. We describe how to induce expression of markers of the late stages of epidermal differentiation in cultured cells and how to ship whole mouse skins for culture at a site removed from the mice, should it be required. This chapter also contains a method of cryopreservation that ensures high cell viability after periods of storage over liquid nitrogen. The techniques described here in detail should be of interest to investigators currently producing transgenic or null mice with epidermal defects.
Pirrone
Annalisa
A
Department of Medicine, University of Seattle, WA, USA.
Hager
Barbara
B
Fleckman
Philip
P
D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000737:chemistry; D000078404:Epidermal Cells; D015603:Keratinocytes / Q000166:cytology*; D051379:Mice
D017077:Culture Media, Conditioned