Development of a small-scale biohybrid simulator for human metabolism and its application as a newcytotoxicity testing system

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Conventional cytotoxicity tests cannot usually include various metabolic processes in humans. We therefore developed a physiologically-based multi-compartment perfusion coculture system (bio-hybrid simulator) using a Caco-2 cell monolayer on a semipermeable membrane and microcarrier-based three-dimensional culture of Hep G2 cells to mimic the absorption across the small intestine and biotransformation of the small intestine and the liver. Stable operations enabled to maintain various activities of the both cells for at least four days.Cocultivation improved the growth of Hep G2 cells and enhanced the cytochrome P450 lAl/2 capacities of both cell lines. When benzo[a]pyrene (BaP) was, loaded to the apical side of the Caco-2 cell layer, the enhanced P450 capacities produced a larger amount of BaP-7,8-hydrodiol, an immediate precursor to the highly-reactive ultimate toxicant of BaP, BaP-7,8-dihydrodiol-9,l0-epoxide (BPDE). This led to initially retarded and later stronger expression of BaP toxicity in the coculture system than in pure culture, which agreed well with the largest time integral of the concentration (area under curve, AUC) of BaP-7,8-hydrodiol in the Hep G2 cells compartment of the coculture system.Because this kind of system can reproduce such complicated phenomena including those derived from Organ-to-organ interactions, it is useful as a new in vitro experimental system when we understand unknown mechanisms involved in final toxicity in humans and develop physiologically-based pharmacokinetic (PBPK) numerical simulation models.