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

人体新陈代谢小型生物混合模拟器的开发及其作为新型细胞毒性测试系统的应用

基本信息

批准号：
13555222
负责人：
SAKAI Yasuyuki
金额：
$ 8.45万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

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.

传统的细胞毒性试验通常不能包括人体的各种代谢过程。因此，我们开发了一种基于生理学的多室灌注共培养系统（生物混合模拟器），使用半透膜上的Caco-2细胞单层和基于微载体的Hep G2细胞三维培养来模拟小肠吸收和小肠和肝脏的生物转化。结果表明，两种细胞均能保持稳定的生物学活性，共培养可促进Hep G2细胞的生长，并提高细胞色素P450 1A 1/2的活性。当苯并[a]芘（BaP）被加载到Caco-2细胞层的顶侧时，增强的P450容量产生更大量的BaP-7，8-hydrodiol，其是BaP的高反应性最终毒物BaP，BaP-7，8-dihydrodiol-9，10-epoxide（BPDE）的直接前体。这导致在共培养系统中BaP毒性的最初延迟和随后比在纯培养中更强的表达，这与浓度的最大时间积分很好地吻合。共培养系统中Hep G2细胞区室中BaP-7，8-hydrodiol的曲线下面积（area under curve，AUC）。由于这种系统可以重现包括器官间相互作用在内的复杂现象，当我们理解涉及人体最终毒性的未知机制和开发基于生理学的药代动力学（PBPK）数值模拟模型时，它作为一种新的体外实验系统是有用的。