A study on p450-related detoxication of hepatocytes in primary culture.

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We have previously found that isolated rat hepatocytes assembled into multicellular spheroids in the presence of liver-derived proteoglycans and various differentiated liver-specific functions were retained in the spheroids. The present research project aimed to solve the following questions, 1) What molecule in the liver-derived proteoglycans was responsible for the spheroid formation, 2) Whether the ability of p450-related detoxication was retained in the spheroid, and 3) Whether a module that can utilize liver specific functions of spheroids can be constituted.1) Molecules responsible for spheroid formationCharacterization of liver-derived proteoglycans by analyses of glycosaminoglycan and core proteins revealed that decorin (108kD) and biglycan (200kD), both small chondroitin/dermatan sulfate proteoglycans, were the molecules responsible for the spheroid formation. It also appeared that decorin and biglycan were effective only when they were immobilized via core protein to the surf … More ace of culture wares. Glycosaminoglycan specificity for the spheroid formation was analyzed by using synthetic neoproteoglycan which were constituted of various types of glycosaminoglycans and phosphatidyl ethanolamine. The result indicated that chondroitin sulfate including dermatan sulfate were effective as a sugar chain of neoproteoglycan for the spheroid formation, but heparin and heparan sulfate were much less.2) Preservation of p450-related detoxicating ability in spheroidDetoxication ability of cultured hepatocytes ability in spheroid the following procedures ; measuring p450 proteins by western blotting using specific monoclonal antibodies, measuring transcritional signal of p450 reductase, and measuring microsomal proteins. Results indicated that detoxication ability was better retained in spheroid hepatocytes than monolayer hepatocytes. Among p450 proteins, phenobarbital inducible type was best preserved ; 60% of the initial level was retained at day 5 in spheroid hepatocyte in contrast only 20% was in monolayer hepatocytes. However the total enzyme activity of p450 retained in spheroid was only a small percent of liver tissue in vivo.3) A module utilizing liver specific functions retained in spheroidsTo utilize liver specific functions of hepatocytes, the spheroids were encapsulated with calcium arginate gel droplet, and the droplets were entrapped in a bioreactor chamber which was inserted in the medium circuit. The liver specific functions of encapsulated spheroids were assessed by measuring the albumin and urea in medium samples periodically collected. Both albumin and urea accumulated in a linear fashion in the circulation ; the production rates obtained with encapsulated were equivalent to those with non-capsulated spheroids. Although we had aimed to utilize the bioreactor system for assessment of drug metabolism, drug was introduced in the system since p450-related detoxicating ability was not well retained in spheroids as described above. Less