Development of artificial liver using macroporous support

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1. To establish a three dimensional culture of mammalian cell, anchorage dependent or independent cell culture was carried out by using some macroporous cell supports, such as a support linked with heparin or cell adhesion factor and with positive or negative charged groups. In the culture using a cellulose macroporous support, relatively high cell density was obtained. It was found that the highest value of the maximum cell density and the production of biochemicals was obtained in the cellulose support with positive charged group.2. To construct a bioreactor packed with a artificial liver for clinical use, long term cultivation of rat hepatocyte with hepatocyte specific function should be established. For this purpose, the effect of additives on albumin secretion rate as a hepatocyte specific function was studied. The albumin secretion rate as a specific hepatocyte function was checked in dish culture of rat hepatocyte. Although the hepatocyte function including an albumin secretion rate decrease gradually during cultivation, it was found that 2 to 3 times higher rate was obtained by addition of anti-oxidative agent such as proline and histidine among amino acids, catalase and superoxide dismutase among enzymes, ascorbic phosphate ester and transferrine.3. The effect of glucose concentration on hepatocyte specific function was studied. Gulucose was added at the variable levels from 0.1 to 3.0 mM in the dish culture. It was found that the secretion rate in the early stage increased with decrease of glucose concentration. When glucose concentration was shifted up from low level (0.1mM) to high level (10 mM) at various culture days by changing the medium, high secretion rate was also obtained. It was found that when the medium was changed after 4 days, 2 times higher rate was kept even through after 14 days. This result means that the glucose level controlled culture become one of the effective strategy for long term hepatocyte culture.