title: A method for the isolation and culture of human colonic crypts in collagen gels.
abstract: Little is known concerning the biological factors that control the proliferation of the stem cells of the colonic mucosa. In part this is due to a lack of systems suitable for studying the proliferation of this mucosa in vitro. We describe a simple technique for the isolation of single viable intact crypts which are free of stroma and which can then be cultured for periods of at least 16 d using a collagen gel culture method. This method of crypt isolation was efficient with the mean yield of viable intact crypts being 1.4 +/- 1.2 X 10(4) (mean +/- 1 SD) crypts/cm2 of mucosa. In culture, mucosal cells only survived for extended periods when the crypts were cultured in collagen gels over a feeder layer of bovine aortic endothelial cells. Cells containing mucus were present in the cultured crypts at all stages of the culture; however we have not been able to demonstrate alkaline phosphatase activity in these crypts. Studies of DNA synthesis after 7 d in culture, using a 18-h pulse label with bromodeoxyuridine (BUdR) has shown that DNA synthesis, as measured by incorporation of BUdR into nuclei, is still occurring in these cultured crypts.
mesh_terms: D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D003106:Colon / Q000473:pathology*; D003110:Colonic Neoplasms / Q000473:pathology*; D003470:Culture Media; D004247:DNA / Q000096:biosynthesis; D005260:Female; D006651:Histocytochemistry; D006801:Humans; D007413:Intestinal Mucosa / Q000473:pathology*; D008297:Male; D008854:Microscopy, Electron; D008875:Middle Aged; D013234:Stem Cells / Q000473:pathology
chemical_list: D003470:Culture Media; D004247:DNA