Isolation of pig colonic crypts for cytotoxic assay of luminal compounds: effects of hydrogen sulfide, ammonia, and deoxycholic acid.

Some colonic luminal molecules resulting from bacterial metabolism of alimentary or endogenous compounds are believed to exert various effects on the colonic epithelial cell physiology. We isolated surface epithelial cells and intact colonic crypts in order to test bacterial metabolites in the pig model, which is often considered relevant for extrapolation to the physiopathology of the human gastrointestinal tract. Using colonocytes isolated with EDTA, we found that the initial cell viability, estimated by the membrane integrity and oxidative capacity measurement, fell rapidly despite several experimental attempts to preserve it such as the use of a medium designed to increase the adherence of epithelial cells and of a coated extracellular matrix, the presence in the culture medium of the oxidative substrate butyrate, and the use of an inhibitor of the caspases involved in cell apoptosis. In contrast, using dispase and collagenase as proteolytic agents, we were able to obtain pig colonic crypts that maintain an excellent membrane integrity after 4 h. Using this preparation, we were able to test the presumably cytotoxic luminal compounds hydrogen sulfide, ammonia, and deoxycholic acid on colonic crypt viability. Of these, only deoxycholic acid was found to significantly alter the cellular membrane integrity. It is concluded that pig colonic crypts can be useful for the in vitro appraisal of the cytotoxic properties of luminal compounds.