Abstract
This chapter provides methods suitable for the culture of primary human keratinocytes in serum-free culture conditions, starting from very small skin biopsies. It also explains procedures required for reconstruction of a stratified epidermis on polycarbonate filter, starting from keratinocytes cultured in serum-free conditions. Tissues reconstructed according to this method have been proven suitable for characterization of epidermal morphogenesis and for in vitro studies of the epidermal barrier. Utilization of the same method for successful isolation of keratinocytes from a patient suffering from Darier’s disease and the reconstruction of a pathological epidermis which displays the same histological features as in vivo are also presented.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Pruniéras M, Régnier M, Woodley D (1983) Methods for cultivation of keratinocytes with an air-liquid interface. J Invest Dermatol 81:28s–33s
Rosdy M, Clauss LC (1990) Terminal epidermal differentiation of human keratinocytes grown in chemically defined medium on inert filter substrates at the air-liquid interface. J Invest Dermatol 95:409–414
Coquette A, Berna N, Vandenbosch A, Rosdy M, Poumay Y (1999) Differential expression and release of cytokines by an in vitro reconstructed human epidermis following exposure to skin irritant and sensitizing chemicals. Toxicol In Vitro 13:867–877
Poumay Y, Coquette A (2007) Modelling the human epidermis in vitro: tools for basic and applied research. Arch Dermatol Res 298:361–369
Poumay Y, Dupont F, Marcoux S, Leclercq-Smekens M, Hérin M, Coquette A (2004) A simple reconstructed human epidermis: preparation of the culture model and utilization in in vitro studies. Arch Dermatol Res 296:203–211
Frankart A, Coquette A, Schroeder K-R, Poumay Y (2012) Studies of cell signalling in a reconstructed human epidermis exposed to sensitizers: IL-8 synthesis and release depends on EGFR activation. Arch Dermatol Res 304:289–303
Frankart A, Malaisse J, De Vuyst E, Minner F, Lambert de Rouvroit C, Poumay Y (2012) Epidermal morphogenesis during progressive in vitro 3D reconstruction at the air-liquid interface. Exp Dermatol 21:871–875
Minner F, Herphelin F, Poumay Y (2010) Study of epidermal differentiation in human keratinocytes cultured in autocrine conditions. Methods Mol Biol 585:71–82
Lambert de Rouvroit C, Charlier C, Lederer D, De Glas V, De Vuyst E, Dargent J-L, Grammatico P, Binni F, Rousseau C, Hennecker J-L, Nikkels AF, Poumay Y (2013) In vitro reconstruction of epidermis from primary Darier’s disease keratinocytes replicates the histopathological phenotype. J Dermatol Sci 71:138–140
Acknowledgements
The valuable technical help from Daniel Van Vlaender is gratefully acknowledged. This work was financially supported by FRFC grants 2.4.506.01 and 2.4.522.10F and by FNRS grant 1.5.033.06F to Y.P. E.D.V. and S.G. are supported by a grant from the Région Wallonne.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
De Vuyst, E., Charlier, C., Giltaire, S., De Glas, V., de Rouvroit, C.L., Poumay, Y. (2013). Reconstruction of Normal and Pathological Human Epidermis on Polycarbonate Filter. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology, vol 1195. Springer, New York, NY. https://doi.org/10.1007/7651_2013_40
Download citation
DOI: https://doi.org/10.1007/7651_2013_40
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4939-1223-0
Online ISBN: 978-1-4939-1224-7
eBook Packages: Springer Protocols