Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes

Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from fres hly slaughtered animals. After digestion with collagenase I, the isolated t issue was centrifuged on a 2% D-sorbitol gradient to separate epithelial cr ypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibrobla sts was prevented. The cells proliferated under the chosen culture conditio ns and formed monolayer cultures which were maintained for several weeks, i ncluding subcultivation steps. A population doubling time of about 21 hr wa s estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatical ly. By using antibodies against cytokeratine 7 and 13 the isolated cells we re identified as cells of epithelial origin. Antibodies against vimentin se rved as negative control. Morphological features such as microvilli, desmos omes and tight junctions, which demonstrated the ability of the cultured ce lls to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydr ogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells whi ch was stable over 5 days in cultured cells. Then activity decreased comple tely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon epithelium in toxicological studi es bt vitro. (C) 2000 Elsevier Science Ltd. All rights reserved.