Selective and organotypic culture of intrahepatic bile duct cells from adult pig liver

Secondary culture of nontransformed bile duct epithelium 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 f rom the secondary culture at Passage 3 or 4. In contrast, adult pig bile du ct cells replicated and were carried for 4-8 passages in secondary culture. A simple method to produce nearly pure pig intrahepatic bile duct cultures was first to freeze a relatively crude liver cell preparation. Upon subseq uent thawing, all hepatocytes and most macrophages were lysed. Bile duct ce lls composed 95% of the surviving cells after the freeze/thaw, and they gre w out rapidly. The bile duct cells grew on top of the STO feeder cells as c losely knit epithelial, colonial outgrowths. Histocytochemical and biochemi cal analyses demonstrated high levels of gamma-glutamyltranspeptidase activ ity and low levels of P450 activity in the bile duct cultures. The bile duc t cells spontaneously adopted a multicellular ductal morphology after 7-10 d in static culture which was similar to that found in in vivo pig liver. T ransmission electron microscopic examination revealed complex junctions and desmosomes typical of epithelium, and lumenally projecting cilia typical o f in vivo intrahepatic bile ductules. This simple method for the coculture 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.