Objective: To investigate the nature of the restoration of cell polarity and bile excretion function in Sandwich-cultured hepatocytes. Methods: Freshly isolated hepatocytes from male Sprague-Dawley rats were cultured in a double layer collagen gel Sandwich configuration. Morphological changes were observed under a inverted microscope. The domain specific membrane associated protein DPPⅣwas tested by immunofluorescence. and the bile excretion function was determined by using fluorescein diacetate. Hepatocytes cultured on a single layer of collagen gel were taken as control. Results: Adult rat hepatocytes cultured in a double layer collagen gel sandwich configuration regained its morphological and functional polarity and maintained polygonal morphology for at least 4 weeks. Immunofluorescence studies using antibodies against DPPⅣshowed polarity restoration as early as 48 h. After cultured in the double layer collagen gel Sandwich configuration for 96 h the hepatocytes began to excrete bile; while hepatocytes cultured on a single layer collagen gel had no bile excretion. Conclusion: Hepatocytes cultured in a double layer collagen gel Sandwich configuration are able to regain their morphological and functional polarity given certain conditions. Hepaotcyte culture is a useful tool for the study of polarity restoration.
