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

开发多室细胞培养系统，用于定量评估化学物质对人类的影响

• 批准号: 11450310
• 负责人: SAKAI Yasuyuki
• 金额: $ 5.76万
• 依托单位: 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: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11450310/
• 关键词: model small intestine; model liver tissue; multi-compartment cell culture system; bioactivation; active transport; static culture; perfusion culture; oral intake; 毒性発現; 小腸上皮; 肝細胞; 吸収; 代謝; アセトアミノフェン

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

本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的细胞在生理相关的方式来模拟各种反应，首先，我们开发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）。根据模型化学品的体外吸收能力，与单层培养系统（无Caco-2细胞层）中获得的ED 50值相比，双层系统中四种模型化学品的所有ED 50值均变为更高的浓度范围。这导致体内毒性的预测性增强，因为考虑了体内可吸收性并影响双层培养系统中的最终毒性。一个有趣的现象是，一些化学物质在Caco-2细胞层（AA）中的解毒和主动转运机制被假定 ...更多信息 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，我们关注了在苯并[a]芘的毒性表达机制中Caco-2细胞层的解毒和主动运输的参与，苯并[a]芘用作通过细胞色素P450酶在人体中的生物活化而表现出强毒性的模型化学品。苯并[a]芘代谢物（其中一些是前致癌物）的定量显示，只有不到十分之一的量可以透过Caco-2膜。P450 1A1/2不仅在Hep G2细胞中被强烈诱导，而且在Caco-2细胞中也被强烈诱导。这些观察结果表明，这种双层培养系统优于传统的基于单群体的细胞毒性试验，因为它可以密切模拟人体内发生的非常复杂的毒性表达机制。尽管这种简单的培养系统在体内筛选或排序化学毒性方面是有效的，但它们不适合用于人体内毒性表达的定量或动力学分析，因为它们的细胞密度非常低。因此，我们开发了一种由Caco-2细胞和Hep G2细胞组成的灌注培养系统，并结合了生理相关的回路。在一系列实验中，使用对乙酰氨基酚作为模型化学品，该化学品在小肠中吸收良好，在肝脏中生物活化，并在肝脏中表达特定毒性。出乎意料的是，在含Caco-2细胞的系统中观察到的毒性高于在无Caco-2细胞的系统中观察到的毒性。对导致人体对乙酰氨基酚毒性的细胞色素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，对乙酰氨基酚在渗透Caco-2细胞层时转化为毒性更大的代谢产物。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，在该系统中，细胞不断振荡以满足系统中细胞的耗氧量。少

项目成果

酒井 康行: "細胞から組織へ"化学工学. 63・4. 214-215 (1999)

酒井康之：“从细胞到组织”化学工程63・4（1999）。

酒井康行: "複合動物細胞培養,動物細胞工学ハンドブック,動物細胞工学会編"朝倉書店(東京). pp.216-217 (2000)

Yasuyuki Sakai：“复合动物细胞培养，动物细胞工程手册，动物细胞工程学会编辑”朝仓书店（东京）第216-217页（2000年）。

Y.Sakai,T.Arai,A.Sakoda and M.Suzuki: "Development of a simple double-layered cell culture system using Caco-2 and TIG-1 cells as a new cytotoxicity test"AATEX. 7(2-3). 47-58 (2001)

Y.Sakai、T.Arai、A.Sakoda 和 M.Suzuki：“使用 Caco-2 和 TIG-1 细胞开发简单的双层细胞培养系统作为新的细胞毒性测试”AATEX。

Y.Sakai, T.Arai, A.Sakoda, and M.Suzuki: "Development of a simple double-layered culture system using Caco-2 and TIG-1 cells as a new cytotoxicity test"AATEX. 7(2-3). 47-58 (2001)

Y.Sakai、T.Arai、A.Sakoda 和 M.Suzuki：“使用 Caco-2 和 TIG-1 细胞开发简单的双层培养系统作为新的细胞毒性测试”AATEX。

酒井康行: "複合動物細胞培養"動物細胞工学ハンドブック. (印刷中). (2000)

Yasuyuki Sakai：“复合动物细胞培养”动物细胞工程手册（印刷中）。