Co-culture of primary rat hepatocytes with rat liver epithelial cells enhances interleukin-6-induced acute-phase protein response.

Three different primary rat hepatocyte culture methods were compared for their ability to allow the secretion of fibrinogen and albumin under basal and IL-6-stimulated conditions. These culture methods comprised the co-culture of hepatocytes with rat liver epithelial cells (CC-RLEC), a collagen type I sandwich culture (SW) and a conventional primary hepatocyte monolayer culture (ML). Basal albumin secretion was most stable over time in SW. Fibrinogen secretion was induced by IL-6 in all cell culture models. Compared with ML, CC-RLEC showed an almost three-fold higher fibrinogen secretion under both control and IL-6-stimulated conditions. Induction of fibrinogen release by IL-6 was lowest in SW. Albumin secretion was decreased after IL-6 stimulation in both ML and CC-RLEC. Thus, cells growing under the various primary hepatocyte cell culture techniques react differently to IL-6 stimulation with regard to acute-phase protein secretion. CC-RLEC is the preferred method for studying cytokine-mediated induction of acute-phase proteins, because of the pronounced stimulation of fibrinogen secretion upon IL-6 exposure under these conditions.

Arginase release by primary hepatocytes and liver slices results in rapid conversion of arginine to urea in cell culture media.

Precision-cut liver slices and primary hepatocytes constitute suitable model systems for studying liver function. Frequently, urea cycle activity is used as a parameter to determine hepatocyte viability. Liver cells contain high levels of the urea cycle enzyme arginase, which converts arginine into urea and ornithine. Arginase can leak from the cells into the supernatants, converting arginine directly to urea and in this way circumventing the urea cycle. In this study, a hepatocellular cell line (HepG2 cells), a primary rat hepatocyte culture, and precision-cut rat liver slices were compared with respect to arginase leakage in the media by determining arginine conversion into urea. HepG2 cells did not show arginine conversion to urea during 24h incubations. In contrast, in both precision-cut liver slices and primary rat hepatocytes all arginine was converted to urea. Arginase activity was confirmed by showing that freshly added arginine to the cell-free supernatants again was converted to urea. In conclusion, when choosing urea production of primary hepatocytes cultures as a viability indicator, one has to take into account that arginase can leak from the cells into the supernatant. This can lead to an overestimation of the viability of the cells, since arginase converts arginine into urea without involvement of the urea cycle. We suggest using an extra incubation in an arginine-free buffer supplemented with ornithine and NH4Cl. In addition, arginase leakage can lead to depletion of the supernatant of arginine in primary hepatocytes cell cultures. This might have implications for studying cellular activities where arginine is involved, like, e.g. nitric oxide (NO) production.