Partial Maintenance of Taurocholate Uptake by Adult Rat Hepatocytes Cultured in a Collagen Sandwich Configuration

Purpose. This study was designed to characterize taurocholate uptake properties in primary cultures of rat hepatocytes maintained under different matrix conditions. Methods. Hepatocytes isolated from male Wistar rats (230–280 g) were cultured on a simple collagen film, on a substratum of gelled collagen or between two layers of gelled collagen (sandwich configuration). Hepatocyte morphology, taurocholate uptake properties, and expression of the sinusoidal transport protein, Na⁺/taurocholate-cotransporting polypeptide (Ntcp) were examined in these cultures at day 0 and day 5. Results. By day 5, monolayer integrity had deteriorated in simple collagen cultures. In contrast, cell morphology was preserved in hepatocytes maintained in a sandwich configuration. At day 5, taurocholate accumulation at 5 min in hepatocytes cultured on a simple collagen film, on a substratum of gelled collagen, and in a sandwich configuration was 13%, 20% and 35% of day-0 levels, respectively, and occurred predominately by a Na⁺-dependent mechanism. The initial taurocholate uptake rate vs. concentration (1-200 M) profile was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent K_m values were comparable for day-0 and day-5 hepatocytes (32–41 M). In contrast, the V_max values of hepatocytes cultured on a simple collagen film, on gelled collagen and in a sandwich configuration were 5, 6 and 14% of the values at day 0, respectively; values for the first-order rate constant were 5-, 3- and 2-fold lower, respectively. Immunoblot analysis indicated that at day 5 Ntcp expression in hepatocytes cultured in a sandwich configuration was greater than in hepatocytes cultured on a simple collagen film. Conclusions. A collagen sandwich configuration reestablishes normal morphology and partially restores bile acid uptake properties in primary cultures of rat hepatocytes.