ISOLATION AND CHARACTERIZATION OF BILIARY EPITHELIAL-CELLS FROM RAINBOW-TROUT LIVER

Lectin binding and density gradient centrifugation were explored for i solating epithelial cells from trout liver. Hepatocytes exhibited pref erential attachment to coverslips coated with Phaseolus vulgaris eryth roagglutinin. Biliary epithelial cells attached with glycine max agglu tinin; however, significant attachment of cellular debris limited the use of glycine max agglutinin. Percoll-density gradient centrifugation separated liver cells into two distinct populations with biliary cell s and hepatocytes banding at densities of 1.04 and 1.09, respectively. A discontinuous gradient composed of 13% Ficoll (wt/wt) separated bil iary cells from hepatocytes. The recovery of highly enriched biliary e pithelial cells from trout liver using Ficoll gradients yielded approx imately 8 million cells (0.1 ml packed cells) from 10 g liver. Western blot analysis demonstrated that the cytokeratin profile for extracts from biliary epithelial cell-enriched populations differ significantly from those seen with whole liver extracts or with extracts from hepat ocyte-enriched populations. Ficoll-gradient purified biliary cells and hepatocytes attached to culture plates coated with trout skin extract and carried out linear incorporation of leucine into protein and thym idine into DNA for 24 h. A mixture of growth hormones (insulin, epider mal growth factor, and dexamethasone) stimulated thymidine incorporati on into DNA; however, long-term culture of dividing biliary epithelial cells was not achieved. Chemical analysis of neutral and acidic glyco lipids indicated that hepatocytes and biliary cells have similar glyco lipid profiles with an exception in the region of GM3 mobility, which is attributable to differences in the ceramide moiety. These studies p rovide a starting point for further characterization of unique cell ty pes of the trout liver that may be important in their response to toxi c and carcinogenic agents.