title: Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.
abstract: This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.
authors.affiliation: Division of Gastroenterology, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Jiri
authors.initials: J
authors.lastname: Kalabis
authors.forename: Gabrielle S
authors.initials: GS
authors.lastname: Wong
authors.forename: Maria E
authors.initials: ME
authors.lastname: Vega
authors.forename: Mitsuteru
authors.initials: M
authors.lastname: Natsuizaka
authors.forename: Erle S
authors.initials: ES
authors.lastname: Robertson
authors.forename: Meenhard
authors.initials: M
authors.lastname: Herlyn
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Anil K
authors.initials: AK
authors.lastname: Rustgi
mesh_terms: D000818:Animals; D002460:Cell Line; D004847:Epithelial Cells; D004947:Esophagus / Q000166:cytology*; D006801:Humans; D051379:Mice; D046509:Tissue Culture Techniques; D023822:Tissue Engineering / Q000379:methods*