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A Human Colonic Crypt Culture System to Study Regulation of Stem Cell-Driven Tissue Renewal and Physiological Function

  • Alyson Parris
  • Mark R. Williams
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1212)

Abstract

The intestinal epithelium is one of the most rapidly renewing tissues in the human body and fulfils vital physiological roles such as barrier function and transport of nutrients and fluid. Investigation of gut epithelial physiology in health and disease has been hampered by the lack of ex vivo models of the native human intestinal epithelium. Recently, remarkable progress has been made in defining intestinal stem cells and in generating intestinal organoid cultures. In parallel, we have developed a 3D culture system of the native human colonic epithelium that recapitulates the topological hierarchy of stem cell-driven tissue renewal and permits the physiological study of native polarized epithelial cells. Here we describe methods to establish 3D cultures of intact human colonic crypts and conduct real-time imaging of intestinal tissue renewal, cellular signalling, and physiological function, in conjunction with manipulation of gene expression by lentiviral or adenoviral transduction. Visualization of mRNA- and protein-expression patterns in cultured human colonic crypts, and cross-validation with crypts derived from fixed mucosal biopsies, is also described. Alongside studies using intestinal organoids, the near-native human colonic crypt culture model will help to bridge the gap that exists between investigation of colon cancer cell lines and/or animal (tissue) studies, and progression to clinical trials. To this end, the near native human colonic crypt model provides a platform to aid the development of novel strategies for the prevention of inflammatory bowel disease and cancer.

Keywords:

Human Intestine Colon Crypt Stem cells Culture Signalling Immunocytochemistry In situ hybridization Imaging Proliferation Migration Tissue renewal 

Notes

Acknowledgements

We are grateful to all the past and present members of the Williams laboratory for their input into the development and application of the human colonic crypt culture system. The work in the Williams laboratory has been funded by grants from the Biotechnology and Biological Sciences (BB/F015690/1, BB/D018196/1), the Humane Research Trust, Big C cancer charity, John and Pamela Salter Charitable Trust.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.School of Biological SciencesUniversity of East AngliaNorwich, NorfolkUK

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