A new rat bile ductular epithelial cell culture model characterized by the appearance of polarized bile ducts in vitro.

The rat treated with bile duct ligation (BDL) and furan is a unique animal model of massive bile ductular hyperplasia in which normal liver parenchyma is largely replaced with well-differentiated proliferated bile ductules. We have now developed a simple cell isolation procedure to obtain and culture viable bile ductular epithelial cells in high numbers and with a high degree of purity from the livers of BDL/furan-treated rats. Primary monolayer cell cultures were readily established when the isolated bile ductular epithelial cells were cultured in plastic tissue culture wells coated with rat tail tendon type I collagen plus bovine plasma fibronectin. Under these conditions, epidermal growth factor (EGF) was mitogenic for the cultured cells, and they retained phenotypic features typical of hyperplastic bile ductular epithelium but did not show evidence of ductal morphogenesis in vitro. In contrast, when the isolated bile ductular cells were cultured for 7 to 16 days in the presence of 25 ng EGF/mL and 10% fetal bovine serum on type I collagen gels, they formed into branching ductal structures whose ultrastructural features very closely resembled those of polarized hyperplastic bile ductules/ducts in vivo. Histological preparations of these gel cultures further showed that numerous ductal structures with defined lumens were present. Phenotypically, these ductal structures were completely surrounded by a thickened basement membrane that was strongly immunoreactive for laminin. Like their in vivo biliary cell counterparts, the epithelial cells comprising these ductal structures in culture also exhibited strong immunocytochemical staining reactions for cytokeratins 8 and 19, for glutathione S-transferase pi 7, and for luminal gamma-glutamyl transpeptidase, but they did not express immunoreactive albumin or alpha-fetoprotein. Occasional epithelial cells of the ductal structures, when examined in 10-day-old primary gel culture, showed strong nuclear staining for incorporated 5-bromo-2'deoxyuridine, indicating active cell proliferation. Our results support the development of a novel biliary epithelial cell culture model that has the potential of serving as a powerful tool for investigation of factors that regulate hyperplastic bile ductular morphogenesis, cell proliferation, and polarized cell functions in a structural form in vitro that mimics that of hyperplastic bile ductules induced in vivo.

Ductular hepatocytes. Evidence for a bile ductular cell origin in furan-treated rats.

Cholangiolar-like structures composed of biliary epithelial cells and typically a single ductular hepatocytic cell in various stages of maturation appeared in association with an extensive bile ductular reaction within the atrophied right liver lobe of young adult Fischer 344 rats that had been subjected to a severely hepatotoxic treatment with furan. In contrast to normal cholangioles, these cholangiolar-like structures were completely surrounded by a basement membrane, which gave strong immunohistochemical staining reactions for both laminin and type IV collagen. All of the biliary epithelial cells and ductular hepatocytic cells exhibited a strongly positive immunohistochemical staining for gamma-glutamyl transpeptidase and for cytokeratin 8. Cytokeratin 19 was also strongly expressed in all of the biliary epithelial cells, but in just some of the ductular hepatocytic cells, whereas only the latter cells were immunohistochemically positive for albumin and contained peroxisomes in their cytoplasm. Cell junctions were formed between individual ductular hepatocytic cells and adjacent biliary epithelial cells of the same cholangiolar-like structures. In this novel model of severe hepatic injury, the range of cell and nuclear diameters characterizing the ductular hepatocytic cells, together with their phenotypic features, are consistent with a differentiation of rare bile ductular-like cells to transitional ductular cells to more mature ductular hepatocytes.