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

本研究利用新生大鼠肝细胞原代培养，探讨了肝内胆汁淤积的发生机制，以阐明未成熟肝脏中肝脏特异性功能的发育变化。在本研究中，我们建立了一种类似于体内三维结构的新生肝细胞球形培养模型，并在该模型中检测了肝细胞的功能，如蛋白分泌和胆小管的形成。在BSA包被的培养皿上形成完整的球状体，培养14天后，球状体培养的肝细胞比普通培养的肝细胞保持更高的白蛋白分泌水平。在胶原凝胶夹心培养系统中培养的肝细胞，提供了在体内的三维结构，也保持了肝细胞的立方体形状和高水平的白蛋白分泌14天。用罗丹明-鬼笔环肽肌动蛋白染色观察到相邻肝细胞之间的胆汁小管样结构。胆汁淤积剂引起这些小管扩张。因此，新生肝细胞的三维培养建立。由于肝细胞保持较高的肝脏特异性功能和形态，这些培养系统将有助于研究新生期肝胆汁淤积症的机制。

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.