Submitted to: In Vitro Cellular And Developmental Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 17, 1998
Publication Date: N/A
Interpretive Summary: We report a relatively simple technique for the isolation and in vitro culture of pig bile duct cells of high purity & of in vivo-like character. Bile duct cells make up about 5 % of the liver and function in the secretion and recycling of bile, a multi-component substance produced by the liver that aides in the digestion of fats. Bile duct cells have been nvery difficult to grow in vitro so that their biology could be directly manipulated and studied. Pig bile duct cells were grown free of liver cell (hepatocytes) by passaging the culture several times so as to dilute our or destroy the more fragile and non-replicating hepatocytes. Liver macrophages were eliminated from the culture by brief treatment with a toxic lectin, ricin A. An alternative isolation technique was to freeze the relatively crude liver cell preparation. Freezing destroyed the relatively fragile hepatocytes and macrophages and left the bile duct cells intact to be cultured in a nearly pure state. Biochemical assays of specific enzymatic activities confirmed the bile duct nature of the cultures. The cells spontaneously formed ductal structures in culture and these multi cellular structures appeared "organotypic", i.e., in vivo-like as assessed by transmission electron microscopy. The culture system provides for the study an important component cell of the liver and helps confirms the character of a previously isolated liver stem cell line. The ability to manipulate the various cells comprising the liver in culture may help in the rational design of an artificial liver device or the production of liver tissue in vitro.
Technical Abstract: Secondary culture of non-transformed bile duct epithelium (BDE) has been difficult to achieve. STO feeder cell-dependent secondary cultures of adult pig bile duct cells were established from primary cultures of adult pig liver cells. Adult pig hepatocytes exhibited limited or no replication, and were lost from the secondary culture at passage 3 or 4. In contrast, adult pig bile duct cells replicated, and were carried for 4 passages in secondary culture. A simple method to produce nearly pure pig intrahepatic BDE cultures was to first freeze a relatively crude liver cell preparation. Upon subsequent thawing, all hepatocytes and most macrophages were lysed. BDE comprised 95% of the surviving cells after the freeze/thaw, and they grew out rapidly. The bile duct cells grew on top of the STO feeder cells as closely knit epithelial colonial outgrowths. Histocytochemical and biochemical analyses demonstrated high levels of gamma-glutamyltranspeptidase activity and low levels of P450 activity in the bile duct cultures. The bile duct cells spontaneously adopted a multicellular ductal morphology after 7-10 days in static culture which was similar to that found in in vivo pig liver. Polarized fluid transport activity was evidenced by dome-like formations. Transmission electron microscopic examination revealed complex junctions and desmosomes typical of epithelium, and lumenally projecting cilia typical of in vivo intrahepatic bile ductules. This simple method for the co-culture of pig intrahepatic bile duct cells which adopt in vivo like structure may facilitate biological studies of this important, but difficult to culture, cell type.