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Updated: Jan 20, 2026
Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture
Published on: August 10, 2019
The Simplest Protocol for Rapid and Long-Term Culture of Primary Epidermal Keratinocytes from Human and Mouse
Filipa Pinto1, Daisuke Suzuki1, Makoto Senoo2
1Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, USA.
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.
Insights
Researchers developed a simple method to grow mouse epidermal keratinocytes long-term. Inhibiting TGF-β signaling boosts progenitor cell proliferation, aiding skin research and graft preparation.
Area of Science:
- Dermatology
- Cell Biology
- Regenerative Medicine
Background:
- Mouse models are crucial for skin research but culturing primary epidermal keratinocytes is difficult.
- Existing methods often require complex progenitor cell purification or feeder cell layers.
Purpose of the Study:
- To present a simplified protocol for long-term expansion of mouse epidermal keratinocytes.
- To investigate the role of TGF-β signaling in keratinocyte proliferation and differentiation.
Main Methods:
- Utilized low calcium media for keratinocyte culture.
- Pharmacologically inhibited TGF-β signaling in crude mouse epidermis preparations.
- Assessed proliferative capacity and differentiation potential of p63+ keratinocytes.
- Evaluated clonal growth in co-culture with feeder cells.
Main Results:
- Developed a protocol for long-term expansion of p63+ mouse epidermal keratinocytes without feeder cells or purification.
- TGF-β signaling inhibition significantly enhanced the proliferative capacity of epidermal progenitor cells.
- Inhibited TGF-β signaling preserved keratinocyte differentiation potential.
- Suppression of TGF-β signaling improved clonal growth of both mouse and human keratinocytes in co-culture.
Conclusions:
- Pharmacological inhibition of TGF-β signaling offers a simple and efficient method for expanding primary mouse epidermal keratinocytes.
- This approach facilitates the use of mouse models in skin research and accelerates the preparation of customized skin grafts.
- The method also shows promise for enhancing human keratinocyte expansion.
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