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Establishment of 3D organotypic cultures using human neonatal epidermal cells

Abstract

This protocol describes an ex vivo three-dimensional coculture system optimized to study the skin regenerative ability of primary human keratinocytes grown at the air–liquid interface on collagen matrices embedded with human dermal fibroblasts. An option for enrichment of keratinocyte stem cells and their progeny using fluorescence-activated cell sorting is also provided. Initially, dermal equivalents, comprising human passaged fibroblasts seeded in a collagen matrix, are grown on porous filters (3 μm) placed in transwells. After 1 week, primary human keratinocytes are seeded on this base. One week later, an air-lift transition is performed, leading to the differentiation of the keratinocytes, which are macroscopically visible as artificial skin after a couple of days. The cultures can be harvested 1 week after the air-lift and processed for immunohistochemistry or gene expression analysis. The overall procedure can be completed in 3 weeks, including the preparation of the dermal equivalent and the seeding of the primary keratinocytes.

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Figure 1: The assembly of organotypic cultures requires the casting of acellular and cellular collagen layers, which serve as a dermal equivalent (DE) containing fibroblasts.
Figure 2: Dot plots of 7-AAD (a) and α6/CD71 (b) staining on human primary keratinocytes.
Figure 3: H&E staining of organotypic cultures harvested at days 2, 7 and 14 after air-lift and embedded for paraffin sections.
Figure 4: Immunostaining of frozen sections of organotypic cultures generated from the α6bri CD71dim stem-cell-enriched fraction obtained by FACS of freshly isolated primary human keratinocytes, harvested at day 14 after air-lift.

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Correspondence to Sophie Paquet-Fifield.

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Gangatirkar, P., Paquet-Fifield, S., Li, A. et al. Establishment of 3D organotypic cultures using human neonatal epidermal cells. Nat Protoc 2, 178–186 (2007). https://doi.org/10.1038/nprot.2006.448

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