The isolation and culture of adult mouse colonic epithelium.
A technique is described for the reproducible primary culture of colonic epithelium from adult mice. A collagenase-dispase digestion technique (adapted form Evans et al. 1992) is used to release the epithelium, followed by differential sedimentation to produce a high purity crypt preparation with maintained structural integrity and minimal mesenchymal contamination. The crypt units attach to collagen coated plastic within 24 h and the epithelial cells quickly begin to migrate outwards producing a monolayer surrounding the attached crypts. Electron microscopy revealed that the migrating epithelial cells possessed both desmosomes and microvilli. Proliferation in the colony supports the outward migration of cells until the migratory cells of adjacent colonies connect and a confluent monolayer begins to form. Proliferation is routinely maintained for 10 days (although cultures have now been maintained without subculturing for 35 days) and is demonstrated by increased cell numbers in spite of continuous cell loss into the culture media. Culture growth is enhanced by increasing concentrations of fetal calf and mouse serum and EGF but does not appear to respond significantly to added transferrin. Growth is also stimulated by a murine small intestinal extract thought to contain a potentially novel growth factor or cocktail of factors. This culture model has considerable potential for studies on growth factor control of this carcinoma susceptible tissue and its differentiated function as well as studies into the mechanisms of carinogenesis.
Booth
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CRC Department of Epithelial Biology, Paterson Institute, Christie Hospital (NHS) Trust, Withington, Manchester, UK.
Patel
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Bennion
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GR
Potten
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CS
D000818:Animals; D001798:Blood Proteins; D002448:Cell Adhesion / Q000502:physiology; D002452:Cell Count; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured / Q000166:cytology*; D003106:Colon / Q000166:cytology* / Q000648:ultrastructure; D003470:Culture Media; D004847:Epithelial Cells; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008854:Microscopy, Electron; D016196:S Phase / Q000502:physiology; D013936:Thymidine / Q000378:metabolism; D014316:Tritium / Q000378:metabolism; D014357:Trypsin
D001798:Blood Proteins; D003470:Culture Media; D014316:Tritium; D014357:Trypsin; D013936:Thymidine