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

常规的细胞毒性试验通常不能包括人体内的各种代谢过程。因此，我们开发了一种基于生理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的吸收以及小肠和肝脏的生物转化。稳定的操作使两种细胞的各种活性至少维持了4天，共培养促进了Hep G2细胞的生长，并提高了两种细胞系的细胞色素P450 Lal/2能力。当苯并[a]芘(BaP)被加载到Caco-2细胞层的顶侧时，增强的P450能力产生了更多的BaP-7，8-羟基二醇，这是高活性最终毒物BaP-7，8-二氢二醇-9，10-环氧化物(BPDE)的直接前体。这导致BaP毒性在共培养系统中的表达开始延迟，后来比纯培养系统中表达更强，这与共培养系统Hep G2细胞室中BaP-7，8-羟基二醇浓度(曲线下面积，AUC)的最大时间积分很好地吻合。由于这种系统可以再现包括器官与器官相互作用在内的复杂现象，因此它可以作为一种新的体外实验系统，有助于我们理解人类最终毒性的未知机制，并开发基于生理的药代动力学(PBPK)数值模拟模型。

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.