ABSTRACT
Spheroids of adult rat hepatocytes are spherical cell aggregates which retain three-dimensional architecture and hepatocyte specific functions. In this study, we investigated the detailed structure and function of bile canaliculi in spheroids. Hepatocytes were prepared from adult rat liver and cultured with epidermal growth factor (50 ng/ml). Hepatocytes formed floating spheroids 4 days after inoculation. The morphology of hepatocyte spheroids was investigated after fluorescent staining for actin using confocal laser scanning microscopy and electron microscopy. To study the function of bile canaliculi, the transcellular transport of fluorescein diacetate was observed. These experiments were performed in a control group and in a group treated with the actin inhibitor cytochalasin B. In a control group, spheroids contained bile canalicular structures which were surrounded by actin filaments. Added fluorescent dye was secreted and pooled in bile canaliculi. Cytochalasin B caused marked distention of bile canaliculi and prominent accumulation of secreted fluorescent dye in dilated bile canaliculi. This phenomenon was based on the impairment of contractile movement of bile canaliculi. These results demonstrate that hepatocyte spheroids maintain functional and morphological peculiarity, and therefore this model may be useful in investigation of the mechanism of bile formation and intrahepatic cholestasis.