FORMATION OF EXTENSIVE CANALICULAR NETWORKS BY RAT HEPATOCYTES CULTURED IN COLLAGEN-SANDWICH CONFIGURATION

Rat primary hepatocytes were cultured under different extracellular ma trix configurations and evaluated for the acquisition and maintenance of structural and functional cell polarity. De novo repolarization of the plasma membrane was variable in rate and extent in hepatocyte cult ures maintained on a conventional single layer of either gelled or ung elled collagen. However, cultures maintained in a collagen-sandwich co nfiguration initiated uniform formation of a contiguous anastomosing n etwork of bile canaliculi throughout the entire culture. Localization of apical membrane markers demonstrated normal distribution at the can alicular membrane. A marked rearrangement of the intracellular microfi laments to the cell periphery was observed and coincided with the deve lopment of the bile canaliculi. Acquisition of normal bile canalicular function and integrity was observed within 3-4 days postoverlay as in dicated by the concentration and retention of carboxyfluorescein withi n the canalicular network. These results demonstrate that cultures of hepatocytes maintained in a sandwich configuration may serve as a more reliable and representative model in which to study the physiology of hepatic function as well as the morphogenesis of polarized membrane d omains in vitro.