A new culture technique for hepatocyte organoid formation and long-term maintenance of liver-specific functions.

To develop a useful hybrid artificial liver, it is important to use cultured hepatocytes that maintain liver-specific functions for a long time. These requirements were achieved recently by the use of a hepatocyte multicellular aggregate (organoid) with a tissue-like structure. In this study, we developed a three-dimensional culture of hepatocytes that formed an organoid. Primary rat hepatocytes were immobilized inside hollow fibers (for plasma separation) by centrifugation. Hepatocytes formed a cylindrical organoid (cylindroid) of 200 mum in diameter by day 2 of culture. We used two types of culture media, medium A (Williams' medium E containing insulin and epidermal growth factor) and medium B (Dulbecco's modified Eagle's medium containing insulin, epidermal growth factor, and hydrocortisone). In medium A, the hepatocyte cylindroid diminished after 14 days of culture and liver-specific functions of the hepatocyte cylindroid nearly disappeared after 1 month of culture. In contrast, hepatocyte cylindroid cultured in medium B maintained its morphology and liver-specific functions for 2-5 months. These results indicate that a combination of the new culture technique and suitable culture medium is effective for expression and maintenance of liver-specific functions of hepatocytes. This culture technique will be helpful in the development of a hybrid artificial liver.