Isolation and Characterization of Biliary Epithelial Cells

Abstract

The epithelial cells that line the intrahepatic bile ducts make up less than 2% of the total liver cell mass. Methods for the isolation and purification of biliary epithelial cells (BEC) with good yield have been hampered by the similarity of these cells with other non-parenchymal cells in size and anatomical organization as they are dispersed throughout the liver and are ensheathed in connective tissue. BEC were isolated by a two-step enzyme digestion procedure. Hepatocytes were mechanically isolated from collagenase perfused rat liver and the separated biliary tissue strands were macerated and digested with pronase. After filtering the digest over 60 um Nybolt the cells were centrifuged at 300 g and the resuspended pellet was recentrifuged over a Percoll density gradient at 25000 gav for 20 min at 20°C. The cells which separated in bands between median densities 1.03 and 1.08 g/ml contributed more than 90% of the loaded cell population and more than 80% were ultrastructurally similar to the epithelial cells of intrahepatic bile ducts. Approximately 5 x 107 cells were isolated by this method and more than 90% excluded Trypan blue. The viability of the isolated cells was not significantly decreased by storage in an oxygen gassed atmosphere in Hepes buffered medium at 37°C for 60 min. SDS polyacrylamide gel electrophoresis of isolated BEC, hepatocytes and liver lysates showed that only low molecular weight proteins below 45 kd were detected in the BEC fraction. Antibodies were raised in rabbits and mice against isolated hepatocytes (anti-hepatocyte) and isolated BEC (anti-BEC) Dot blot assays were performed for semiquantitative titration of these sera. A rabbit antiserum to bovine hoof prekeratin which reacted with the intermediate filaments of biliary epithelia was also used Liver sections incubated with antiserum to bovine hoof prekeratin, rabbit anti-BEC serum or mouse anti-BEC serum exhibited preferential staining of biliary epitheliumin the portal tracts and larger intrahepatic bile ducts. Isolated cell fractions were also stained and the binding of the antibodies to isolated BEC was demonstrated. Western blot assays were performed to determine the specific antigenic determinants recognized by the antisera. The rabbit antiserum to isolated BEC was found to recognize a biliary epithelial cell protein of approximately 45 kd whereas the antiserum to bovine hoof prekeratin recognized different proteins in BEC and hepatocytes. The glycolipids of isolated BEC, hepatocytes and liver homogenates were separated using thin layer chromatography. One glycolipid band was seen in BEC but liver and hepatocyte extracts contained three glycolipid bands. The anti-BEC serum cross reacted by immunoblotting with hepatocyte and whole liver glycolipids but did not react with major BEC glycolipids. The anti-prekeratin serum did not react with BEC glycolipids but did react with the glycolipids of isolated hepatocytes and liver homogenates. Isolated BEC and biliary tissue strands were cultured in Dulbecco's MEM. The cultured cells were found to be ultrastructurally similar to freshly isolated BEC and during the first 1-2 days of culture contained proteins similar to those of isolated cells. The effects of the fungal mycotoxins sporidesmin and cytochalasin B on the morphology of freshly isolated BEC were examined. Sporidesmin appeared to act as a membrane-perturbing agent while the effects of cytochalasin were consistent with the known role of depolymerizing microfilaments.