Establishment of 3D organotypic cultures using human neonatal epidermal cells.
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 mum) 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.
Gangatirkar
Pradnya
P
Epithelial Stem Cell Biology Laboratory, Peter MacCallum Cancer Centre, St. Andrew's Place, East Melbourne, Victoria 3002, Australia.
Paquet-Fifield
Sophie
S
Li
Amy
A
Rossi
Ralph
R
Kaur
Pritinder
P
D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology; D003094:Collagen; D000078404:Epidermal Cells; D005347:Fibroblasts; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007231:Infant, Newborn; D015603:Keratinocytes / Q000166:cytology*
D003094:Collagen