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With a view toward furthering the development of artificial liver systems, we have been culturing hepatocytes in vitro. The object of this research was to investigate the ideal conditions of oxygen tension for the efficient functioning of hepatocytes. Viable hepatocytes isolated from rat livers were cultured under five different oxygen tensions: 5, 10, 20, 50 and 90% O2. DNA contents, gluconeogenesis, urea synthesis, adenosine triphosphate (ATP) levels, and lipid peroxidation of hepatocytes were evaluated. Under the 5% oxygen conditions, the function of hepatocytes was very inferior and was accompanied by a low ATP level. However, hepatocytes cultured under 90% oxygen tension functioned less effectively than the control (20% O2) with elevated lipid peroxidation. The data in this study suggest that the optimum oxygen condition for cultured hepatocytes is 10 approximately 50%, and that especially under conditions of 20% oxygen tension, i.e., that of the ordinary atmosphere, hepatocytes can function most effectively.

Since 1987, we have been developing a bioartificial liver (BAL) using multiplated cultured hepatocyte monolayers. With the goal of promoting hepatic functions of cultured hepatocyte monolayers, we combined the use of a collagen gel layer over the monolayers of hepatocytes and/or cocultured hepatocytes with nonparenchymal cells (NPCs). The study was divided into four groups according to culture configurations: Group 1: hepatocyte monolayer culture (control); Group 2: coculture of hepatocytes and NPCs; Group 3: hepatocyte monolayer with a overlaid collagen gel layer; and Group 4: coculture with a overlaid collagen gel layer. The culture continued for 14 days. Morphological changes and hepatic functions were evaluated by urea and albumin syntheses. The morphological status of the hepatocytes remained for 2 weeks in Groups 3 and 4. Deterioration and detachment of hepatocytes and/or NPCs started in Group 1 and 2 on the third day in culture. Significantly high urea synthesis was noted in Group 4 (p < 0.001 compared with Group 1 and 2: p = 0.0014 compared with Group 3). Although there was no significant difference in albumin synthesis among the four groups, those hepatocytes covered by the collagen gel (Groups 3 and 4) tended to secrete albumin throughout the observation period. These results indicted that the environment, although artificial (but close to the in vivo state), supplied with collagen gel and the coculture, enhanced the activities of the cultured hepatocyte monolayers. We suggest that use of cocultured hepatocytes under a collagen gel is a promising candidate for a bioreactor of multiplated BAL.

We have been developing a multiplate type of bioartificial liver (BAL) using primary cultured hepatocyte monolayers since 1987. This BAL has been shown to prolong the survival time of anhepatic dogs and rabbits. Initially, hepatocytes were cultivated on collagen-coated plates. To increase the multiplated BAL function, a sandwich cultivation method was employed in which rat hepatocytes were cultured between two collagen gel layers and were then evaluated in both stationary and perfusion cultures. In the stationary culture, the sandwich method showed a higher activity in urea synthesis than in the other culture methods (culture on a collagen coating, culture on a collagen gel, and culture between a collagen coating and a collagen gel) for 14 days. In the perfusion culture, a BAL housing cultured hepatocytes (6.5 x 10(7) cells) in the sandwich system showed urea synthesis activity ranging from 17.5 to 22.6 micrograms/2 x 10(6) cells/90 min. This activity was maintained for 5 days in the perfusion culture. The sandwich type of cultivation is applicable to the multiplated BAL.