A study on mechanisms of intrahepatic cholestasis using neonatal rat hepatocytes cultured in three-dimensions.

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We have investigated on the mechanisms of intrahepatic cholestasis to elucidate developmental changes of liver-specific functions in the immature liver using neonatal rat hepatocytes in primary culture. In this study, we developed a spheroidal culture model of neonatal hepatocytes which resembles three-dimensional structure in vivo and examined hepatocellular functions such as protein secretion and formation of bile canaliculi in the model.Neonatal hepatocytes formed semispheroids after 5 days cultured on dishes coated with polyhydroxyethyl methacrylate. They formed complete spheroids on the dishes coated with BSA.Spheroidal cultured hepatocytes maintained higher levels of albumin secretion than plain cultured hepatocytes after 14 days in culture. Hepatocytes cultured in collage gel sandwich culture system, which provides three-dimensional structures as in vivo, also maintained cuboidal shape of hepatocytes and high levels albumin secretion for 14 days. Bile canaliculilike structure was observed between adjacent hepatocytes by actin staining using rhodamine-phalloidin. Cholestatic agents caused dilatation of these canaliculi.Thus, three-dimensional culture of neonatal hepatocytes was established. As hepatocytes maintain higher of liver-specific function and morphology, these culture systems should be useful for studying the mechanisms of hepatic cholestasis in the neonatal period.