Chapter 10. Isolation and Culture of Intestinal Epithelial Cells

  1. R. Ian Freshney and
  2. Mary G. Freshney
  1. Catherine Booth and
  2. Julie A. O'Shea

Published Online: 28 APR 2002

DOI: 10.1002/0471221201.ch10

Culture of Epithelial Cells, Second Edition

Culture of Epithelial Cells, Second Edition

How to Cite

Booth, C. and O'Shea, J. A. (2002) Isolation and Culture of Intestinal Epithelial Cells, in Culture of Epithelial Cells, Second Edition (eds R. I. Freshney and M. G. Freshney), John Wiley & Sons, Inc., New York, USA. doi: 10.1002/0471221201.ch10

Editor Information

  1. CRC Beatson Laboratories, Glasgow, Scotland

Author Information

  1. EpiStem Ltd., Incubator Building, Grafton St., Manchester M13 9XX, UK

Publication History

  1. Published Online: 28 APR 2002
  2. Published Print: 12 APR 2002

Book Series:

  1. Culture of Specialized Cells

Book Series Editors:

  1. R. Ian Freshney

Series Editor Information

  1. CRC Beatson Laboratories, Glasgow, Scotland

ISBN Information

Print ISBN: 9780471401216

Online ISBN: 9780471221203

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Keywords:

  • intestinal crypts;
  • epithelial cell culture;
  • colon;
  • dispase;
  • collagenase;
  • 3T3 feeder layers;
  • differential sedimentation;
  • colony-forming assays;
  • labeling index;
  • transplantation;
  • acutase

Summary

A disaggregation technique is described, based on collagenase and dispase, which allows the isolation of crypts from mouse colon. The crypts may be purified by selective sedimentation and plated out in 24-well plates. Application of the technique to small intestine, neonatal tissue, polyps, and tumors is also described. A separate isolation and sedimentation protocol is provided for primary culture of human colonic crypts which may be plated on to collagen or 3T3 feeder layers, but which do not subculture readily. Chelating agents give a higher yield when isolating crypts, but the viability tends to be lower. Two colony-forming assays are provided, one of which is clonogenic, and other quantitative assays include labeling index with [3H]-thymidine and estimation of cell number with crystal violet staining. Differentiation has been studied by implanting collagenase/dispase- or Acutase-generated cell suspensions in Matrigel into immune-deprived mice.