An on-chip small intestine-liver model for pharmacokinetic studies.

Testing of drug effects and cytotoxicity by using cultured cells has been widely performed as an alternative to animal testing. However, the estimation of pharmacokinetics by conventional cell-based assay methods is difficult because of the inability to evaluate multiorgan effects. An important challenge in the field is to mimic the organ-to-organ network in the human body by using a microfluidic network connecting small-scale tissues based on recently emerging MicroTAS (Micro Total Analysis Systems) technology for prediction of pharmacokinetics. Here, we describe an on-chip small intestine-liver coupled model for pharmacokinetic studies. To construct an in vitro pharmacokinetic model that appropriately models in vivo conditions, physiological parameters such as the structure of internal circulation, volume ratios of each organ, and blood flow ratio of the portal vein to the hepatic artery were mimicked using microfluidic networks. To demonstrate interactions between organs in vitro in pharmacokinetic studies, Caco-2, HepG2, and A549 cell cultures were used as organ models of the small intestine, liver, and lung, respectively, and connected to each other through a microporous membrane and microchannels to prepare a simple model of a physiological organ-to-organ network. The on-chip organ model assay using three types of substrate-epirubicine (EPI), irinotecan (CPT-11), and cyclophosphamide (CPA)-were conducted to model the effects of orally administered or biologically active anticancer drugs. The result suggested that the device can replicate physiological phenomena such as activity of the anticancer drugs on the target cells. This microfluidic device can thus be used as an in vitro organ model to predict the pharmacokinetics of drugs in the human body and may thus provide not only an alternative to animal testing but also a method of obtaining parameters for in silico models of physiologically based pharmacokinetics.

Image-based evaluations of distribution and cytotoxicity of Irinotecan (CPT-11) in a multi-compartment micro-cell coculture device.

We recently developed a polydimethylsiloxane (PDMS)-based three-compartment microfluidic cocultivation device enabling real-time interactions of different cell populations as an advanced physiologically-relevant cell-based assay. This device had valves and small magnetic stirrer-based internal pumps for easy and flexible perfusion operations. In this study, we applied this device for the evaluation of Irinotecan (CPT-11) toxicity to the lung, because it is detoxified by the liver and accumulated in the fat in humans. We successfully cultured representative three different tissue model cells in each compartment under the individual culture conditions and also in entire perfusion. Growth inhibition of rat lung epithelial cell line L-2, was measured when administered with 50 µM CPT-11 under various cocultivation conditions with respect to the presences and absence of primary rat hepatocytes (liver tissue model) and adipocyte-like cells (fat tissue model) induced from a mouse fibroblast cell line, 3T3-L1. Although CPT-11 showed moderate toxicity to the pure culture of L-2 cells in the device after 72 h of perfusion culture, this was lowered mainly in the presence of the liver tissue. Inhibition of the L-2 cell growth agreed with the area under curve (AUC) values obtained from fluorescent image-based analyses in each compartment. These results demonstrate that developed simple and flexible microfluidic cocultivation device, with appropriate image-based analyses, can be used in evaluating toxicokinetic behaviors of drug candidates in systemic levels.

Cytotoxicity evaluation of reactive metabolites using rat liver homogenate microsome-encapsulated alginate gel microbeads.

We present an improved cytotoxicity test for reactive metabolites, in which the S9 microsomal fraction of rat liver homogenate is encapsulated in alginate gel microbeads to avoid cytotoxic effects of S9-self-generated toxicants, microsomal lipid peroxides. The S9-encapsulated gel microbeads were prepared by a coaxial two-fluid nozzle and surfaces of the microbeads were coated with poly-L-lysine (PLL). Although the initial metabolic rate of the S9-encapsulated gel microbeads was about 20% slower than that of bare S9, the microbeads prevented the leakage of microsomal lipid peroxides thanks to the dense alginate and PLL polymer networks. In fact, the half maximal effective concentration of the indirect mutagen cyclophosphamide on NIH3T3 cells in the presence of the S9-encapsulated gel microbeads was about 5 times higher than that in the presence of bare S9. Use of the S9-encapsulated gel microbeads enabled the more accurate evaluation of the cytotoxicity of the reactive metabolites without the S9-based cytotoxicity.

Evaluation of the ecotoxicity of solid wastes using rapid leaching test and bioassays.

A rapid leaching test (RLT) involving the use of ultrasonic irradiation was developed. The applicability and suitability of different leaching procedures for solid wastes were evaluated in terms of the leached concentration of heavy metals and total organic carbon and the ecotoxicity potential of the solid wastes. Solid wastes, including incineration ash and sludge, were used. We performed the RLT and the Japanese standard leaching test (JLT-13) to evaluate the ecotoxicity of the solid wastes. The leaching test samples were analyzed using 3 different bioassays. A higher concentration of heavy metals was obtained by the RLT than by JLT-13. The concentrations of organic compounds leached by the RLT correlated well with those of the organic compounds leached by JLT-13. These results indicate that the RLT developed in this study is a useful method to estimate the leachability of heavy metals and organic compounds in solid waste samples. Although the 50% effective concentration (EC(50)) values of the RLT and JLT-13 solid waste eluates derived by a human cell-based bioassay were slightly different, the EC(50) values of these eluates derived by 2 other ecotoxicity tests correlated well.