IMMUNOPHARMACOLOGICAL STUDIES ON FULMINANT HEPATITIS MODEL USING THE SPHEROID CULTURE OF RAT LIVER CELLS

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An experimental acute liver injury model can be produced by the injection of bacterial lipopolysaccharide (LPS) into Propionibacterium acnes (P.acnes) pretreated rats. The massive liver cell necrosis is estimated by elevation of serum transaminase activities. In this study, we produced this necrosis in an in vitro model by using primary co-cultured rat liver cells. A novel method for the preparation of spheroids consisting of P.acnes pretreated parechymal and nonparechymal liver cells has been successfully developed quickly by the rotary culture system within 24 h although it takes 7 days to form the spheroid using a collagen-conjugated thermo-responsive polymer such as a cell substratum. Clear elevations of transaminase activities, TNF-a and CINC-1/gro/KC (IL-8) leaked from these spheroids into the medium caused by the exposure of 10 mug/ml LPS for 48 h were observed. These results suggest that this rotary co-culture system of rat liver cells is useful model as an alternative to animal tests for fulminant hepatitis. Therefore, several hepato-protective agents, tiopronin, bucillamine, catechin, gomisin A,and zinc porphyrins, were screened. These agents showed obvious hepato-protective activities in this hepatitis model as well as in vivo.On the other hand, the distribution of copper in spheroids was examined using multicellular spheroids of LEC rat liver cells. When LEC rat spheroids were exposed to CuC12 at concentrations of more than 50 muM for 24h, copper contents in LEC rat spheroids were maintained for 24h after the withdrawal of CuC12 and higher than those of the SD rat spheroid.