In vitro toxicology in hepatocyte bioreactors-extracellular acidification rate (EAR) in a target cell line indicates hepato-activated transformation of substrates

Abstract

In this article we introduce an in vitro model for hepato-mediated toxicity testing consisting of a Hepatocyte-Bioreactor connected to a microphysiometer system for monitoring of the extracellular acidification rate (EAR) of cells. The EAR in this system represented the metabolic activity of a tested cell line under the influence of bioreactor supernatant. Cyclophosphamide (CYCL), a well-known hepato-activated cytostatic drug was used as a model substrate because of its widespread clinical use. The predrug CYCL needed CYP 450 dependent activation to its active cytotoxic metabolite 4-OH cyclophosphamide. Primary pig hepatocytes from slaughterhouse organs were cultured in a collagen sandwich configuration in specially designed flasks and after 3 days introduced into a 50 ml recirculating perfusion system including 30 μg/ml CYCL. In a parallel open circuit, this bioreactor was connected to three perfusion chambers of a microphysiometer system housing 1.5×10 5 ZR 751 cells (breast tumor cell line). Bioreactor supernatant including CYCL was pumped at 150 μl/min into the microphysiometer. The recorded EARs under CYCL influence were correlated to controls, which were set to be 100%. After 1 and 7 h of bioreactor supernatant perfusion, including activated CYCL, the ZR 751 cell line showed an EAR of 98.99%±3.15 (mean±SD) and 81.32%±10.18 ( P<0.05), respectively, as compared to controls (bioreactor supernatant from the identical set-up without CYCL). The inactivated predrug CYCL showed no effect on the EAR: Perfusion of medium with 30 μg/ml CYCL alone, excluding the bioreactor activation, resulted in an EAR of 100. 11%±4.74 (mean±SD) after 7 h. Thus the presented model of hepato-activated toxicity showed an EAR decrease in the ZR 751 cell line that reflected the toxic activation of the predrug by the bioreactor.