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Updated: Aug 21, 2026
Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin
Published on: July 14, 2017
Primary mouse keratinocyte culture
Annalisa Pirrone1, Barbara Hager, Philip Fleckman
1Department of Medicine, University of Seattle, WA, USA.
Abstract:
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.
Insights
This study details a new method for culturing mouse epidermal keratinocytes, enabling long-term cell growth and analysis. This technique is crucial for research involving transgenic or knockout mice with skin conditions.
Area of Science:
- Dermatology
- Cell Biology
- Developmental Biology
Background:
- Mouse epidermal keratinocytes are challenging to culture in vitro.
- Existing methods limit long-term studies and analysis of genetically modified mice.
Purpose of the Study:
- To present a robust protocol for isolating and culturing mouse epidermal keratinocytes.
- To facilitate long-term cell culture and analysis, particularly for transgenic and knockout mouse models.
- To provide detailed methods for inducing differentiation, shipping skins, and cryopreserving cells.
Main Methods:
- Isolation of epidermal keratinocytes from single newborn mouse pups.
- Utilization of supplemented fibroblast-conditioned medium and collagen IV-coated dishes.
- Establishment of protocols for subculturing, inducing differentiation markers, shipping skins, and cryopreservation.
Main Results:
- Successful long-term culture and multiple passages of mouse epidermal keratinocytes.
- Demonstrated induction of late-stage epidermal differentiation markers in vitro.
- High cell viability achieved after cryopreservation and storage in liquid nitrogen.
Conclusions:
- The presented method overcomes challenges in culturing mouse epidermal keratinocytes.
- This protocol is valuable for researchers studying epidermal development and defects in transgenic or knockout mice.
- The techniques support diverse research applications, including remote sample processing and long-term cell banking.
