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 μM) profile was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent Km values were comparable for day-0 and day-5 hepatocytes (32−41 μM). In contrast, the Vmax 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.