In orthotopic liver transplantation, extended cold ischemia of the graft may induce cell damage, particularly in biliary epithelium. We have investigated the effects of a cold University of Wisconsin (UW) solution on cultured human gallbladder biliary epithelial cells (GBEC) exposed or not exposed to stagnant bile. In UW solution, morphological alterations of cultured GBEC were not prominent under light microscopy after 16 hours at 4°C, being more striking after 24 to 48 hours. Ultrastructural examination of GBEC showed a condensation of chromatin at the periphery of the nuclei after 16 hours in cold UW solution. Both protein and DNA syntheses were strikingly reduced in these cells. After rewarming in standard Williams' medium at 37°C for 24 hours, cultured GBEC exhibited both normal morphology and function. As in both freshly isolated and routinely cultured GBEC, rewarmed cells expressed various mucin genes, namely MUC1, MUC3, MUC4, MUC5AC, and MUC5B genes, whereas MUC2 mRNAs were barely detectable. A dramatic decline in the steady-state mRNA levels of both MUC3 and MUC5B was found in cultured GBEC versus freshly isolated cells. Addition of bile into UW solution at 4°C had no significant effect on GBEC morphology and DNA and protein syntheses. When bile was added during the rewarming period, both protein and DNA syntheses were strongly reduced. Addition of bile during either storage in UW solution or rewarming period induced increased steady-state MUC2, MUC3 and MUC5AC mRNA levels. These results show that UW is a reliable cold storage solution for GBEC and the presence of stagnant bile within the culture medium during the rewarming period leads to deleterious phenotypic alterations of these cells. This suggests changes in the management of liver graft during orthotopic liver transplantation. (Hepatology 1995;21:223–231).