Optimization of culture conditions for determining hepatobiliary disposition of taurocholate in sandwich-cultured rat hepatocytes

This study was undertaken to examine the influence of time and volume of co llagen overlay, type of media, and media additives on taurocholate (TC) acc umulation and biliary excretion in hepatocytes cultured in a collagen-sandw ich configuration. Hepatocytes were isolated from male Wistar rats by in si tu perfusion with collagenase, seeded onto collagen-coated 60-mm dishes, ov erlaid with gelled collagen, and cultured for 4 d. Experiments to examine t he influence of Lime and volume, of collagen overlay were conducted in Dulb ecco's modified Eagle's medium (DMEM) + 1.0 LM dexamethasone (DEX) + 5% fet al bovine serum (FBS). Hepatocytes were overlaid at 0 h with 0.1 or 0.2 ml collagen, or at 24 h with 0.1 or 0.2 ml collagen. The influence of media ty pe and additives was examined in hepatocytes overlaid at 0 It with 0.2 ml c ollagen and incubated in DMEM + 0.1 muM DEX, DMEM + 0.1 WW DEX + 5% FBS, Wi lliams' medium E + 0.1 muM DEX + 1% ITS(R)+, DMEM + 1.0 muM DEX, DMEM + 1.0 muM DEX + 5% FBS, or modified Chee's medium (MCM) + 0.1 muM DEX + 1% ITS(R )+. [H-3] TC accumulation by hepatocytes in Hank's balanced salt solution ( HBSS) and Ca2+-free HBSS was measured, and the biliary-excretion index (BEI : percentage of accumulated TC localized in the canalicular compartment) wa s calculated. Light microscopy and carboxydichlorofluorescein fluorescence were employed to examine the cellular and canalicular morphologies. The vol ume of collagen used for both the substratum and the overlay did not affect TC accumulation or biliary excretion. The BEI tended to be higher in cells overlaid at 24 h (BEI 0.649 [0.1 ml collagen] BEI 0.659 [0.2 ml collagen]) compared with those overlaid at 0 It after seeding (BEI 0.538 [0.1 ml coll agen]; BEI 0.517 [0.2 ml collagen]), although the differences were not stat istically significant. Hepatocytes cultured in MCM produced consistently th e lowest BEI of TC (BEI = 0.396). Differing DEX concentrations (0.1 muM ver sus 1.0 muM) with or without 5% FBS did not appear to have a significant ef fect on the BEI of TC.