A method for culturing and transplanting biliary epithelial cell from syrian golden hamster.

Abstract

The present paper describes the establishment of a method for simultaneous culturing of biliary epithelial cells (BECs) from the gall bladder (GB), extrahepatic bile duct (EBD) and intrahepatic bile duct (IBD) of the hamster. GB, EBD and IBD were cut from the biliary tree after collagenase perfusion of the liver. These biliary segments were minced into fragments. The fragments were embedded in collagen gel and cultured in Dulbecco's modified Eagle medium/HamF12 medium containing 10% fetal bovine serum. The various cells subsequently spread from the fragments and formed cellular sheets. After the fragments and flattened cells were removed with the aid of a Pasteur pipette under phase-contrast microscopy, the sheets remaining were found to be composed of cuboidal cells. These cuboidal cells were shown to express gamma glutamyl transpeptidase and cytokeratin 7, which are known to be specific markers of BECs. Ultrastructurally, a large number of microvilli were observed on the luminal surface and junctional complex and interdigitation was identifiable on the lateral surfaces. BEC cultures were subcultured by digestion with collagenase and dispase and then dissociated by subsequent digestion in trypsin and ethylenediaminetetraacetic acid and then maintained on collagen gel for up to 8 weeks. After several passages, the BECs in culture eventually increased in size and showed vacuoles in the cytoplasm. They demonstrated irreversible growth arrest at 9 weeks. The BECs tended to form cystic structures when the BECs with collagen gel were transplanted into the interscapular fat pads of syngeneic hamsters. We established a method for culturing and transplanting biliary cells from syrian golden hamsters. This method may help to clarify the mechanism of hepatobiliary diseases.