Partial Maintenance of Taurocholate Uptake by Adult Rat Hepatocytes Cultured in a Collagen Sandwich Configuration
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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 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.
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- 1.E. L. LeCluyse, P. L. Bullock, and A. Parkinson. Strategies for restoration and maintenance of normal hepatic structure and function in long-term cultures of rat hepatocytes. Adv. Drug Deliv. Rev. 22:133-186 (1996).Google Scholar
- 8.L. M. Reid, M. Narita, M. Fujita, Z. Murray, C. Liverpool, and L. Rosenberg. Matrix and hormonal regulation of differentiation in liver cultures. In Guillouzo, A, and C Guguen-Guillouzo (eds.), Research in “Isolated and cultured hepatocytes.” John Libbey Eurotext Ltd/INSERM, London/Paris, 1986, pp. 225-258.Google Scholar
- 14.E. L. LeCluyse, P. Bullock, A. Parkinson, and J. H. Hochman: Cultured rat hepatocytes. In R. T. Borchardt, G. Wilson, and P. Smith (eds.), Model Systems for Biopharmaceutical Assessment of Drug Absorption and Metabolism. Plenum Press, New York, 1996. pp. 121-159.Google Scholar
- 15.X. Liu, K. R. Brouwer, E. L. LeCluyse, L-S. L. Gan, and K. L. R. Brouwer. Prediction of in vivo biliary excretion of model compounds from hepatocytes cultured in a sandwich configuration, Pharm. Res. 14:S-459 (1997).Google Scholar
- 16.R. P. J. Oude Elferink, D. K. F. Meijer, F. Kuipers, P. L. M. Jansen, A. K. Groen and G. M. M. Groothuis. Hepatobiliary secretion of organic compounds; molecular mechanisms of membrane transport. Biochim. Biophys. Acta 1241:251-268 (1995).Google Scholar
- 20.H. Akaike. An information criterion (AIC). Math Sci. 14:5-9 (1976).Google Scholar