ISOLATION AND CULTURE OF INTRAHEPATIC BILE-DUCTS AND ITS APPLICATION IN ASSESSING PUTATIVE INDUCERS OF BILIARY EPITHELIAL-CELL HYPERPLASIA

  • 1 August 1987
    • journal article
    • research article
    • Vol. 68  (4) , 501-510
Abstract
A method for the isolation and culture of intact intrahepatic bile ducts from normal rats, and its use in studying putative inducers of biliary epithelial cell (BEC) hyperplasia was developed. Ducts were isolated by sequential perfusion of the liver with EGTA and collagenase-hyaluronidase followed by mild mechanical agitation. The resultant fraction, consisting of numerous small bile ducts within a connective tissue framework, was collected and embedded in a collagen gel and cultured on a raft assembly in Medium 199 supplemented with 15% newborn calf serum and antibiotics. Following 10-15 days in culture, the tissue consisted of dilated bile ducts lined by large cuboidal to elongated BEC. At day 15, the BEC3H-thymidine-labelling index was 5.56 .+-. 0.66% (mean .+-. s.e.m.) which is nine times that observed in normal rat BEC in situ and similar to the rate of cell division of BEC lining hyperplastic ductules following bile duct ligation in the rat. Putative cholangiotrophic factors, proline, lithocholic acid and extracts of liver and small intestinal mucosa from normal rats and rats after 3 weeks'' total biliary obstruction (TBO), were added to the culture medium for the last 5 days of a 15-day culture. With the exception of the extract of liver following TBO which had a growth inhibitory effect and lithocholic acid which was toxic, these treatments did not result in any alteration in the rate of BEC replication.