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

Nature Protocols volume 7, pages 235–246 (2012)Cite this article

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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.

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Figure 1: Establishment of OTC on inserts placed on plates (organogenesis).
Figure 2: The use of OTC for LCM.
Figure 3: Detection of side population of esophageal epithelial cells.
Figure 4: Morphology of esophageal OTC derived from mouse cells and immunohistological detection of proliferation and differentiation markers.
Figure 5: Morphology of OTC derived from immortalized human keratinocytes (EPC2-hTERT).

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Acknowledgements

We acknowledge support from the US National Institutes of Health (NIH)/National Cancer Institute (NCI) P01-CA098101, NIH/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) P30-DK050306 and NIH/NCI U01-CA143056. The Molecular Pathology and Imaging, Molecular Biology/Gene Expression, Cell Culture, Transgenic and Chimeric Mouse Core Facilities of the P30-DK050306 NIH/NIDDK Center for Molecular Studies in Digestive and Liver Diseases at the University of Pennsylvania support this work. We also thank the Penn Flow Cytometry and Microarray Array Core Facilities for supporting this work. Additional sources of support include the American Cancer Society Research Professorship (A.K.R.), NIH/NIDDK R01DK077005 (H.N.), NIH/NCI T32-CA115299 (G.W.) and NIH T32-CA009140-37 (M.E.V.). We thank S. Naganuma for the photomicrographs given in Supplementary Figure 1.

Author information

Authors and Affiliations

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

    Jiri Kalabis, Gabrielle S Wong, Maria E Vega, Mitsuteru Natsuizaka, Hiroshi Nakagawa & Anil K Rustgi

  2. Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Jiri Kalabis, Gabrielle S Wong, Maria E Vega, Mitsuteru Natsuizaka, Erle S Robertson, Hiroshi Nakagawa & Anil K Rustgi

  3. Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Erle S Robertson

  4. Wistar Institute, Philadelphia, Pennsylvania, USA

    Meenhard Herlyn

  5. Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Anil K Rustgi

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  1. Jiri Kalabis
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Contributions

J.K., G.S.W., M.E.V. and M.N. contributed to the experimental results; E.S.R., M.H., H.N. and A.K.R. contributed to the experimental design; all authors contributed to the manuscript preparation and writing.

Corresponding author

Correspondence to Anil K Rustgi.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Representative ESCC cell lines in organotypic 3D culture TE9 (a), TE10 (b) and KYSE70 (c) ESCC cells were grown in organotypic 3D culture and subjected to hematoxylin-eosin staining for morphological assessment (x200). (a) TE9 cells grow in a downward fashion toward the stromal compartment without apparent invasion. (b) TE10 cells invade aggressively into the underlying matrix. (c) KYSE70 cells invade as well into the underlying matrix. Scale bar = 50 µm. (TIFF 24680 kb)

Supplementary Tables 1, 2 and Supplementary Information

Supplementary Table 1: Human fibroblasts used in organotypic 3D culture Supplementary Table 2: EPC2 cell derivatives used extensively in organotypic culture Supplementary information and cell culture protocol for EPC2-hTERT and its derivatives (DOCX 28 kb)

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Kalabis, J., Wong, G., Vega, M. et al. Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture. Nat Protoc 7, 235–246 (2012). https://doi.org/10.1038/nprot.2011.437

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