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Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice

Nature Protocols volume 3, pages 799–810 (2008)Cite this article

Abstract

Protocols for preparing and culturing primary keratinocytes from newborn and adult mouse epidermis have evolved over the past 35 years. This protocol is now routinely applied to mice of various genetic backgrounds for in vitro studies of signaling pathways in differentiation and cell transformation, and for assessing the in vivo phenotype of altered keratinocytes in grafts of cells on immunodeficient mice. Crucial in the development and application of the procedure was the observation that keratinocytes proliferate in media of low calcium concentration, but rapidly commit to differentiation at calcium concentrations >0.07 mM after the initial attachment period. Preparing primary keratinocytes from ten newborn mice requires 2–3 h of hands-on time. Related procedures are also provided: preparing immature hair follicle buds, developing dermal hair follicles and fibroblasts from newborn mice, preparing primary keratinocytes from adult mice and grafting cell mixtures on athymic nude mice.

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Figure 1: Steps in the procedure for obtaining epidermis and dermis from newborn mice.
Figure 2: Phase contrast images of freshly prepared and cultured cell fractions from epidermis and dermis of newborn BALB/c mice.
Figure 3: Phase contrast images of freshly prepared and cultured hair follicle fractions from epidermis and dermis of newborn BALB/c mice.
Figure 4: Steps in the procedure for grafting cell mixtures on the backs of athymic nude mice to determine their in vivo phenotype.

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Acknowledgements

Many current and former members of the laboratory have been instrumental in refining the techniques described. Several of them are acknowledged by citing their publications in the text. Special thanks are owed to Christophe Cataisson, a current member of the laboratory, for contributing the images comprising Figure 4.

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  1. NIH/NCI/CCR, Laboratory of Cancer Biology and Genetics, In vitro Pathogenesis Section, 37 Convent Drive, Building 37/4068, Bethesda, 20892-4255, Maryland, USA

    Ulrike Lichti, Joanna Anders & Stuart H Yuspa

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  1. Ulrike Lichti
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  2. Joanna Anders
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Correspondence to Ulrike Lichti.

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Lichti, U., Anders, J. & Yuspa, S. Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. Nat Protoc 3, 799–810 (2008). https://doi.org/10.1038/nprot.2008.50

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