PARTIAL MAINTENANCE OF TAUROCHOLATE UPTAKE BY ADULT-RAT HEPATOCYTES CULTURED IN A COLLAGEN SANDWICH CONFIGURATION

Purpose. This study was designed to characterize taurocholate uptake p roperties in primary cultures of rat hepatocytes maintained under diff erent matrix conditions. Methods. Hepatocytes isolated from male Wiste r rats (230-280 g) were cultured on a simple collagen film, on a subst ratum of gelled collagen or between two layers of gelled collagen (san dwich configuration). Hepatocyte morphology, taurocholate uptake prope rties, and expression of the sinusoidal transport protein, Na+/tauroch olate-cotransporting polypeptide (Ntcp) were examined in these culture s at day 0 and day 5. Results. By day 5, monolayer integrity had deter iorated in simple collagen cultures. In contrast, cell morphology was preserved in hepatocytes maintained in a sandwich configuration. At da y 5, taurocholate accumulation at 5 min in hepatocytes cultured on a s imple collagen film, on a substratum of gelled collagen, and in a sand wich configuration was similar to 13%, 20% and 35% of day-0 levels, re spectively, and occurred predominately by a Na+-dependent mechanism. T he initial taurocholate uptake rate vs. concentration (1-200 mu M) pro file was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent K-m values were compar able for day-0 and day-5 hepatocytes (32-41 mu M). In contrast, the V- max values of hepatocytes cultured on a simple collagen film, on gelle d collagen and in a sandwich configuration were similar to 5, 6 and 14 % of the values at day 0, respectively; values for the first-order rat e constant were 5-, 3- and 2-fold lower, respectively. Immunoblot anal ysis indicated that at day 5 Ntcp expression in hepatocytes cultured i n a sandwich configuration was greater than in hepatocytes cultured on a simple collagen Aim. Conclusions. A collagen sandwich configuration reestablishes normal morphology and partially restores bile acid upta ke properties in primary cultures of rat hepatocytes.