Tyrosine aminotransferase (TAT) induction and albumin secretion abilities were examined in rat hepatocytes immobilized within calcium alginate; the immobilized hepatocytes lost these abilities within a week. An attempt was then made to immobilize multicellular spheroids of hepatocytes for the purpose of stabilizing the liver functions. Although it takes at least 4 days to form spheroids in the conventional method using monolayer‐cultured cells, in this study we developed a new method for rapid spheroid formation. Isolated hepatocytes were seeded into a polystyrene dish and incubated on a rotary shaker. Hepatocytes started to aggregate after 6 h of the rotation culture, and spheroids approximately 100 μm in diameter formed within 24 h. The immobilized spheroids had higher TAT induction and albumin secretion abilities, which were maintained for a longer time, than the immobilized nonaggregated cells. Further stabilization was observed in immobilized heterospheroids formed in the presence of non‐parenchymal liver cells. This method for the rapid formation of spheroids consisting of hepatocytes and non‐parenchymal liver cells could be utilized in the construction of a bioartificial liver support system.

Hepatocytes and non-parenchymal liver cells were isolated from adult rat liver and co-cultured for 48 hours as a monolayer on polystyrene culture dishes. The ability of tyrosine aminotransferase (TAT) induction in hepatocytes was examined in the presence of dexamethasone and dibutyryl cAMP. Non-parenchymal cells greatly enhance the ability of TAT induction of hepatocytes. A soluble factor with molecular weight of more than 10,000 is responsible for this enhancement, because conditioned medium prepared from non-parenchymal cells is also stimulatory. Non-parenchymal cells restored the ability in hepatocytes damaged with the addition of D-galactosamine. Conditioned medium prepared from non-parenchymal cells treated with D-galactosamine had higher activity of enhancement than the medium from normal cells. The soluble factor might be released in response to some signal of injury. Hepatocytes and non-parenchymal cells were immobilized within Ca-alginate, and although immobilized hepatocytes rapidly lost the ability to induce TAT, hepatocytes co-immobilized with non-parenchymal cells maintained the ability during 4 days of culture. These results indicated that non-parenchymal liver cells, as well as hepatocytes, could be used to construct a bioartificial liver support system.

Multicellular spheroids of hepatocytes are known to maintain liver functions for a long period. Rat hepatocytes were isolated to form spheroids by rotation culture and immobilized within calcium alginate. Immobilized spheroids had a much higher extent of tyrosine aminotransferase induction, which is one of the liver-specific differentiated functions, than immobilized non-aggregated cells, while the spheroids secreted significantly less prothrombin than non-aggregated cells. Co-culture of hepatocytes and non-parenchymal liver cells in a monolayer enhanced tyrosine aminotransferase induction and suppressed prothrombin secretion, while conditioned medium prepared from non-parenchymal cells greatly stimulated tyrosine aminotransferase induction and suppressed the prothrombin secretion and DNA synthesis in monolayer-cultured hepatocytes. Prothrombin secretion in hepatocytes was subjected to cell-density-dependent regulation. In a similar manner to other growth-related functions, prothrombin secretion was stimulated at low cell density. It has been reported that thrombin activates the zymogen of hepatocyte growth factor activator [Shimomura, T., Kondo, J., Ochiai, M., Naka, D., Miyazawa, K., Morimoto, Y. & Kitamura, N. (1993) J. Biol. Chem. 268, 22,927-22,932]. Therefore, prothrombin secretion could be one of the growth-related functions and involved in wound healing and liver regeneration.

Multicellular spheroids of hepatocytes are known to maintain liver functions for a long period. Rat hepatocytes were isolated to form spheroids by rotation culture and immobilized within calcium alginate. Immobilized spheroids had a much higher extent of tyrosine aminotransferase induction, which is one of the liver‐specific differentiated functions, than immobilized non‐aggregated cells, while the spheroids secreted significantly less prothrombin than non‐aggregated cells. Co‐culture of hepatocytes and non‐parenchymal liver cells in a monolayer enhanced tyrosine aminotransferase induction and suppressed prothrombin secretion, while conditioned medium prepared from non‐parenchymal cells greatly stimulated tyrosine aminotransferase induction and suppressed the prothrombin secretion and DNA synthesis in monolayer‐cultured hepatocytes. Prothrombin secretion in hepatocytes was subjected to cell‐density‐dependent regulation. In a similar manner to other growth‐related functions, prothrombin secretion was stimulated at low cell density. It has been reported that thrombin activates the zymogen of hepatocyte growth factor activator [Shimomura, T., Kondo, J., Ochiai, M., Naka, D., Miyazawa, K., Morimoto, Y. & Kitamura, N. (1993) J. Biol. Chem. 268, 22927–229321. Therefore, prothrombin secretion could be one of the growth‐related functions and involved in wound healing and liver regeneration.

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