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

被引：131

作者：

Kalabis, Jiri ^{[1
,2
]}

Wong, Gabrielle S. ^{[1
,2
]}

Vega, Maria E. ^{[1
,2
]}

Natsuizaka, Mitsuteru ^{[1
,2
]}

Robertson, Erle S. ^{[2
,3
]}

Herlyn, Meenhard ^{[4
]}

Nakagawa, Hiroshi ^{[1
,2
]}

Rustgi, Anil K. ^{[1
,2
,5
]}

机构：

[1] Univ Penn, Dept Med, Div Gastroenterol, Perelman Sch Med, Philadelphia, PA 19104 USA

[2] Univ Penn, Abramson Canc Ctr, Perelman Sch Med, Philadelphia, PA 19104 USA

[3] Univ Penn, Dept Microbiol, Perelman Sch Med, Philadelphia, PA 19104 USA

[4] Wistar Inst Anat & Biol, Philadelphia, PA 19104 USA

[5] Univ Penn, Dept Genet, Perelman Sch Med, Philadelphia, PA 19104 USA

来源：

NATURE PROTOCOLS | 2012年 / 7卷 / 02期

基金：

美国国家卫生研究院;

关键词：

STRATIFIED SQUAMOUS EPITHELIA; IN-VITRO; DIFFERENTIATION; KERATINOCYTES; EXPRESSION; INVASION; GENE; IDENTIFICATION; CARCINOMA; NETWORK;

D O I：

10.1038/nprot.2011.437

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

T his 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.

引用

页码：235 / 246

页数：12

共 31 条

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