Development of a multi-compartment cell culture system for quantitative evaluation of chemical impacts to humans

这项研究的最终目标是开发一种人体模拟器，通过结合具有器官来源细胞的隔室，以生理相关的方式模拟对添加的化学物质的各种反应。首先，我们建立了一个简单的双层培养系统，包括一个模型小肠（培养的Caco-2细胞层）和一个模型靶器官（培养的人二倍体成纤维细胞，TIG-1细胞）。根据模型化学物质的体外可吸收性，与单层培养系统（不含Caco-2细胞层）相比，四种模型化学物质在双层培养系统中的ED50值都发生了更高的浓度范围。这导致体内毒性的预测增强，因为在双层培养系统中考虑了体内吸收性并影响了最终毒性。一个有趣的现象是，Caco-2细胞层中的一些化学物质的解毒和主动运输机制被假设（AA…More TEX，已发表）。其次，在使用人肝癌Hep G2细胞而不是TIG-1细胞的类似双层培养系统中，我们重点研究了Caco-2细胞层的解毒和主动运输参与苯并[a]芘的毒性表达机制，苯并[a]芘作为一种模型化学物质，通过细胞色素P450酶的生物活化在人体内表现出强烈的毒性。对苯并[a]芘代谢物（其中一些是致癌物）的定量分析表明，只有不到十分之一的量可以通过Caco-2膜。P450 1A1/2在Hep G2细胞和Caco-2细胞中均有强烈的诱导作用。这些观察结果表明，这种双层培养系统比传统的基于单一群体的细胞毒性试验更有优势，因为它可以很好地模拟人体内发生的非常复杂的毒性表达机制。虽然这种简单的培养系统在体内化学毒性的筛选或排序方面是有效的，但由于它们的细胞密度非常低，因此不适合用于人体毒性表达的定量或动力学分析。因此，我们开发了一个由Caco-2细胞和Hep G2细胞结合生理相关回路组成的灌注培养系统。在一系列实验中，对乙酰氨基酚作为一种模型化学物质，在小肠中被很好地吸收，在肝脏中被生物激活，并在肝脏中表现出特定的毒性。出乎意料的是，在含有Caco-2细胞的系统中观察到的毒性高于不含Caco-2细胞的系统。对人类对乙酰氨基酚毒性负责的细胞色素P450 3A的测量显示，Caco-2细胞中的酶活性非常高。因此，我们得出结论，当对乙酰氨基酚渗透到Caco-2细胞层时，它会转化为毒性更强的代谢物。为了提高培养系统的持续时间，我们正在开发一种新的灌注培养系统，在这种系统中，细胞被不断地摇动，以满足系统中细胞的耗氧量。少

The final goal of this research is to develop a human body simulator that mimics various responses to added chemicals by combining compartments havingorgan-derived cells in a physiologically-relevant manner.First, we developed a simple double-layered culture system consisting of a model small intestine (cultured Caco-2 cell layer) and a model target organs (cultured human diploid fibroblasts, TIG-1 cells). All ED50 values for four model chemicals in the double-layered system changed into higher concentration ranges compared those obtained in a single-layered culture system (without the Caco-2 cell layer), according to the in vitro absorbability of the model chemicals. This resulted in enhancement of predictivity of in vivo toxicity because in vivo absorbability is considered and affects the final toxicity in the double-layered culture system. An interesting phenomenon was that detoxification and active transport mechanisms were postulated for some chemicals in the Caco-2 cell layer (AA … More TEX, published).Second, in the similar double-layered culture system using human hepatoma, Hep G2 cells, instead of the TIG-1 cells, we focused on involvement of detoxification and active transport of the Caco-2 cell layer in the toxicity expression mechanisms of benzo[a]pyrene used as a model chemical that exhibits strong toxicity through bioactivation by cytochrome P450 enzymes in humans. Quantification of benzo[a]pyrene metabolites (some of them are procarcinogens) showed that only less than one-tenth amount could permeate the Caco-2 membrane. P450 1A1/2 was strongly induced not only in the Hep G2 cells but also in the Caco-2 cells. These observations indicate that such a double-layered culture system is advantageous over conventional single-population-based cytotoxicity tests because it can closely mimic very complex toxicity expression mechanism occurring in in vivo humans.Although such simple culture systems are effective in screening or ranking of chemical toxicities in vivo, they are not suitable for quantitative or kinetic analyses of toxicity expressions in humans, because of their very low cell density. Therefore, we developed a perfusion culture system consisting of Caco-2 cells and Hep G2 cells combined with a physiologically-relevant circuit. In a series of experiments using acetaminophen as a model chemical that is well absorbed across the small intestine, biologically-activated in the liver, and expresses specific toxicity in the liver. Unexpectedly, observed toxicity was higher in the Caco-2 cell-containing system than in the Caco-2-cell-free system. Measurement of cytochrome P450 3A that is responsible for acetaminophen toxicity in humans showed the very high enzymatic activity in the Caco-2 cells. We therefore concluded that acetaminophen was transformed into more toxic metabolites when it permeates across the Caco-2 cell layer.To improve duration of culture system, we are developing a new perfusion culture system in which cells are continuously shaken to meet oxygen consumption of the cells in the system. Less