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.
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References
Brennan, J. K., Mansky, J., Roberts, G., and Lichtman, M. A. (1975) Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: application to studies of lymphoblast proliferation in vitro. In Vitro 11, 354–360.
Dulbecco, R. and Vogt, M. (1954) Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99, 167–182.
Tsao, M. C., Whithall, B. J., and Han, R. G. (1982) Clonal growth of normal human epidermal keratinocytes in a defined medium. J. Cell Physiol. 110, 219–229.
Hager, B., Bickenbach, J. R., and Fleckman, P. (1999) Long-term culture of murine epidermal keratinocytes. J. Invest. Dermatol. 112, 971–976.
Kang, S. J. and Fleckman, P. (1995) Convenient computer colony image analysis using an ordinary flatbed scanner for evaluating keratinocyte cryopreservation methods (abstract). J. Invest. Dermatol. 104, 612.
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© 2005 Humana Press Inc.
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Pirrone, A., Hager, B., Fleckman, P. (2005). Primary Mouse Keratinocyte Culture. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology™, vol 289. Humana Press. https://doi.org/10.1385/1-59259-830-7:003
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DOI: https://doi.org/10.1385/1-59259-830-7:003
Publisher Name: Humana Press
Print ISBN: 978-1-58829-267-4
Online ISBN: 978-1-59259-830-4
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