Development of bio-artificial liver by the mass transfer in the small hepatocytes

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The morphogenesis and movement of bile canaliculi (BC) are not well understood. This is because culture of hepatocytes that maintain polarity of cell membranes and possess highly differentiated functions has never been successful. We found that small hepatocytes (SHs), which are known to be hepatic progenitor cells, could proliferate and differentiate into mature hepatocytes and that BC-like structures developed between rising/piled-up cells. In the present experiment we examined how SHs could form BC-like structures in the process of their maturation, whether the structures could be functionally active as BC, how BC-like structures contract to expel substances, and whether A23187 and Endothelin-1 caused BC contractions or not.The results of immunocytochemistry, immunoblots, and immunoelectron micrographs revealed that BC proteins localized in the intercellular space that coincided with BC-like structures formed between rising/piled-up cells. Fluorescein was secreted into BC and accumulated without leakage. Time-lapse microscopy showed that continuous contraction and dilatation occurred in the BC-like structures, and fluorescent dye was secreted into the reformed BC and unidirectionally carried through the structures. We also found out BC contractions occurred in a coordinated manner, which at least a few cells length of the BC-like structures contract synchronously. It is also found out that both A23187 and Endothelin-1 caused BC contractions. The functional abilities of reformed BC may be equivalent to those of in vivo BC.