Isolation of a nonparenchymal liver cell fraction enriched in cells with biliary epithelial phenotypes

In the present study we have isolated and purified fractions of nonparenchymal liver cells enriched in biliary epithelial cells. Nonparenchymal liver cells were isolated by collagenase-pronase digestion of the biliary and connective hepatic tissue, which remained undissociated after collagenase perfusion of the liver. Fractionation of the nonparenchymal fractions was then achieved by centrifugal elutriation. Both normal rats and rats with proliferated bile duct-like structures, which were induced either by a 14-day bile duct ligation or by feeding 0.1% α-naphthylisothiocyanate for 28 days, were used in these studies. Using a normal rat liver, the fraction richest in biliary epithelial cells was that obtained at a pump flow rate of 36–40 ml/min. In this fraction 1.8–3.8 × 106 cells per liver were recovered and up to 55% of them were positive for γ-glutamyl transpeptidase and cytokeratins 7 and 19, all of which were histochemically or immunohistochemically detected solely in the biliary structures in the intact rat liver. When the nonparenchymal cells were isolated from hyperplastic livers, the number of cells recovered in such a fraction ranged from 12 to 19 × 106 per liver, and as many as 60%–85% of the cells expressed phenotypes of biliary epithelial cells. These results indicate that (a) by centrifugal elutriation a fraction of nonparenchymal cells enriched in cells with biliary epithelial phenotypes can be obtained from rat liver and (b) the hepatic hyperplasia induced by biliary obstruction or α-naphthylisothiocyanate feeding is a useful and valid strategy for improving both the yield and the purity of the isolated biliary epithelial cells.