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.

常规的细胞毒性测试通常不能包括人类的各种代谢过程。因此，我们在半透明的膜和基于微载体的Hep G2细胞的三维三维培养物上使用CACO-2细胞单层开发了一种基于生理的多室灌注共培养系统（Bio-Hybrid模拟器）小肠和肝脏的生物转化。稳定的操作使两个细胞的各种活性至少四天。分类改善了HEP G2细胞的生长，并增强了两个细胞系的细胞色素P450 LAL/2能力。当苯并[a] pyrene（BAP）被加载到Caco-2细胞层的顶端时，增强的P450容量产生了更大量的BAP-7,8-氢二醇，这是高度反应性终极的直接前体BAP，BAP-7,8-二氢二醇-9，L0-环氧（BPDE）的毒物。这导致最初迟缓，随后在共培养系统中的BAP毒性表达比在纯文化中更强，这与HEP中BAP-7,8-二醇的浓度（曲线下AUC，AUC，AUC）的最大时间一致G2细胞隔室的共培养系统。因为这种系统可以重现这种复杂的现象，包括从器官到器官相互作用的现象，当我们理解在人类最终毒性中涉及的未知机制时，它作为一种新的体外实验系统非常有用并开发基于生理的药代动力学（PBPK）数值模拟模型。