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Nat Protoc. 2012 Jan 12;7(2):235-46. doi: 10.1038/nprot.2011.437.

Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.

Author information

1
Division of Gastroenterology, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

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.

PMID:
22240585
PMCID:
PMC3505594
DOI:
10.1038/nprot.2011.437
[Indexed for MEDLINE]
Free PMC Article

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