Kinetic modelling of in vitro cell-based assays to characterize non-specific bindings and ADME processes in a static and a perfused fluidic system.

Recently, physiologically based perfusion in vitro systems have been developed to provide cell culture environment close to in vivo cell environment (e.g., fluidic conditions, organ interactions). In this work, we model and compare the fate of a chemical, benzo[a]pyrene (B[a]P), in a perfusion and a standard (static well-plate) system. These in vitro systems are composed of Caco-2 and HepG2 cells so as to mimic absorption across the small intestine and intestinal and hepatic metabolism. Compartmental models were developed and calibrated with B[a]P kinetics data in the culture medium to estimate the apparent permeability of Caco-2 cells, the in vitro biotransformation of B[a]P, as well as the different routes of loss by non-specific adsorption. Our results show that non-specific binding is the main process responsible for the depletion of B[a]P in the culture media: at steady state, only 40% and 24% of the total concentration of B[a]P are bioavailable in the static and perfused systems, respectively. We also showed that Caco-2 permeability in the perfused culture system is closer to in vivo conditions than the one obtained in the static system and that higher cellular metabolic activities are observed in static conditions. Perfused in vitro systems combined with kinetic modelling are promising tools for studying in vitro the different processes involved in the toxicokinetics of xenobiotics.

Enhanced performance of a surface plasmon resonance immunosensor for detecting Ab-GAD antibody based on the modified self-assembled monolayers.

To enhance the feasibility of surface plasmon resonance (SPR) immunosensor as a tool for diagnosing type I diabetes, we enhanced the sensitivity of immunoresponse for detecting the monoclonal anti-glutamic acid decarboxylase (GAD) antibody by modification of mixed self-assembled monolayers (SAMs). The effects of the different mixed SAMs were evaluated with respect to the degree of streptavidin immobilization, the degree of biotin-GAD immobilization, and the immunoresponse sensitivity. Consequently, the sensitivity of the immunoresponse for the detection of anti-GAD antibody was enhanced as a result of the reduction in steric hindrance brought about by using SAMs of heterogeneous lengths. The immunoresponse for detecting the monoclonal anti-GAD antibody was also enhanced with the reduction of the excess immobilization of biotin-GAD and the minimization of non-specific binding that resulted from the simple substitution of the spacer from a carboxylic-terminated SAM for the hydroxyl-terminated SAM.

Use of a perfusion co-culture system consisting of Caco-2 and Hep G2 cell compartments for the kinetic analysis of benzo[a]pyrene toxicity.

Conventional cytotoxicity tests cannot usually include various metabolic processes in humans. We therefore developed a physiologically based, multi-compartment perfusion co-culture system, using a Caco-2 cell monolayer on a semi-permeable membrane and a microcarrier-based, three-dimensional culture of Hep G2 cells to mimic permeation across the small intestine and biotransformation of the small intestine and the liver. Stable operations allowed us to maintain various activities of both cells for at least 4 days. Cocultivation improved the growth of Hep G2 cells and enhanced the cytochrome P450 1A1/2 capacities of both Hep G2 and Caco-2 cells. When benzo[a]pyrene (BaP) was loaded on the apical side of the Caco-2 cell layer, the enhanced P450 capacities produced a higher amount of BaP-7,8-hydrodiol, a precursor of the ultimate carcinogen of BaP, BaP-7,8-dihydrodiol-9,10-epoxide (BPDE). These phenomena led to the initially retarded, but later stronger, expression of BaP toxicity in the co-culture system than in pure cultures, which agreed with the actual load of BaP-7.8-hydrodiol to the Hep G2 cells. Because this kind of system can reproduce such complicated phenomena, including those influenced by organ-organ interactions, it is useful as a new in vitro experimental system, for understanding the unknown mechanisms involved in final toxicity in humans and thereby improving physiologically based pharmacokinetic (PBPK) simulation models.

Development of a biohybrid simulator for absorption and biotransformation processes in humans based on in vitro models of small intestine and liver tissues.

The scope of conventional cytotoxicity tests does not usually include various metabolic processes in humans. We therefore developed a physiologically based multicompartment perfusion coculture system (biohybrid simulator) using a Caco-2 cell monolayer on a semipermeable membrane and a microcarrier-based three-dimensional culture of Hep G2 cells to mimic absorption across the small intestine and biotransformation in the small intestine and the liver. Stable operation enabled the maintenance of various activities of both cell types for at least 4 days. Cocultivation improved the growth of Hep G2 cells and enhanced the cytochrome P450 1A1/2 capacities of both cell lines. When benzo[a]pyrene (BaP) was introduced 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-9,10-epoxide. 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) of BaP-7,8-hydrodiol in the Hep G2 cell 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 to help elucidate unknown mechanisms involved in final toxicity in humans and to develop physiologically based pharmacokinetic numerical simulation models.

A portable toxicity biosensor using freeze-dried recombinant bioluminescent bacteria.

A portable biosensor has been developed to meet the demands of field toxicity analysis. This biosensor consists of three parts, a freeze-dried biosensing strain within a vial, a small light-proof test chamber, and an optic-fiber connected between the sample chamber and a luminometer. Various genetically engineered bioluminescent bacteria were freeze-dried to measure different types of toxicity based upon their modes of action. GC2 (lac::luxCDABE), a constitutively bioluminescent strain, was used to monitor the general toxicity of samples through a decrease in its bioluminescence, while specific toxicity was detected through the use of strains such as DPD2540 (fabA::luxCDABE), TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2511 (katG::luxCDABE). These inducible strains show an increase in bioluminescence under specific stressful conditions, i.e. membrane-, protein-, DNA-, and oxidative-stress, respectively. The toxicity of a sample could be detected by measuring the bioluminescence 30 min after addition to the freeze-dried strains. In an attempt to enhance the sensitivity of the freeze-dried cells, glucose and Tween 80 were tested as additives. It was found that the addition of glucose had a negative effect on the viability of the freeze-dried cells, while samples having Tween 80 showed an increase in their viability. On the other hand, the addition of either Tween 80 or glucose decreased the final bioluminescent response of DPD2540 in response to 4-chlorophenol. Using these strains, many different chemicals were tested and characterized. This portable biosensor, with a very simple protocol, can be used for field sample analysis and the monitoring of various water systems on-site.