Development of a multi-compartment cell culture system for quantitative evaluation of chemical impacts to humans

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The final goal of this research is to develop a human body simulator that mimics various responses to added chemicals by combining compartments havingorgan-derived cells in a physiologically-relevant manner.First, we developed a simple double-layered culture system consisting of a model small intestine (cultured Caco-2 cell layer) and a model target organs (cultured human diploid fibroblasts, TIG-1 cells). All ED50 values for four model chemicals in the double-layered system changed into higher concentration ranges compared those obtained in a single-layered culture system (without the Caco-2 cell layer), according to the in vitro absorbability of the model chemicals. This resulted in enhancement of predictivity of in vivo toxicity because in vivo absorbability is considered and affects the final toxicity in the double-layered culture system. An interesting phenomenon was that detoxification and active transport mechanisms were postulated for some chemicals in the Caco-2 cell layer (AA … More TEX, published).Second, in the similar double-layered culture system using human hepatoma, Hep G2 cells, instead of the TIG-1 cells, we focused on involvement of detoxification and active transport of the Caco-2 cell layer in the toxicity expression mechanisms of benzo[a]pyrene used as a model chemical that exhibits strong toxicity through bioactivation by cytochrome P450 enzymes in humans. Quantification of benzo[a]pyrene metabolites (some of them are procarcinogens) showed that only less than one-tenth amount could permeate the Caco-2 membrane. P450 1A1/2 was strongly induced not only in the Hep G2 cells but also in the Caco-2 cells. These observations indicate that such a double-layered culture system is advantageous over conventional single-population-based cytotoxicity tests because it can closely mimic very complex toxicity expression mechanism occurring in in vivo humans.Although such simple culture systems are effective in screening or ranking of chemical toxicities in vivo, they are not suitable for quantitative or kinetic analyses of toxicity expressions in humans, because of their very low cell density. Therefore, we developed a perfusion culture system consisting of Caco-2 cells and Hep G2 cells combined with a physiologically-relevant circuit. In a series of experiments using acetaminophen as a model chemical that is well absorbed across the small intestine, biologically-activated in the liver, and expresses specific toxicity in the liver. Unexpectedly, observed toxicity was higher in the Caco-2 cell-containing system than in the Caco-2-cell-free system. Measurement of cytochrome P450 3A that is responsible for acetaminophen toxicity in humans showed the very high enzymatic activity in the Caco-2 cells. We therefore concluded that acetaminophen was transformed into more toxic metabolites when it permeates across the Caco-2 cell layer.To improve duration of culture system, we are developing a new perfusion culture system in which cells are continuously shaken to meet oxygen consumption of the cells in the system. Less