Purification and Monolayer Cell Culture of Murine Extrahepatic Biliary Epithelial Cells

The preparation of biliary epithelial cells is fundamental for their biologic and pathophysiologic evaluation. A new method for the isolation of extrahepatic biliary epithelial cells, using explants of murine extrahepatic bile ducts and gallbladder, is presented. First, small tissue pieces of the extrahepatic biliary tree and the gallbladder were plated as primary explants on collagen gel. The epithelial cells spread from the explants toward the periphery as a monolayer sheet, while the nonepithelial mesenchymal cells of the explants migrated into the gel below the epithelial layer. These different growth patterns created areas in epithelial cell sheets in which no mesenchymal cells were present. These areas were cut with scissors and then cultured on another collagen gel as secondary explants. From these explants, epithelial cells without nonepithelial mesenchymal cells spread out as a monolayer on the collagen gel. At the front and periphery of both the primary and secondary monolayer cell cultures, epithelial cells exhibited a polygonal flattened morphology, and BrdUpositive S-phase cells were densely distributed. In the central parts of both explants, the cells were cuboidal or low columnar with a luminal mucin positive layer and BrdU-positive S-phase cells were sparsely distributed, suggesting that the in vivo morphologic characteristics of the epithelial cells of the extrahepatic biliary tree were retained. This method provides purification and a monolayer cell culture or extrahepatic biliary epithelial cells that can be employed in various types of studies