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

基本信息

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

项目摘要

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

这样的最终目标是通过以生理上与生理学的方式组合隔室衍生的细胞来开发人体模拟各种各种各种象征性的化学物质。培养的CACO-2细胞层）和模型靶器官（培养的人二倍体图1细胞）。层，根据模型化学物质的体外吸收性。。通过细胞色素p450酶在人类中的生物活化，表现出强大的thoxics thoxics。培养系统比转化率优于基于ulation的细胞毒性测试，因为它非常模仿体内发生的复杂毒性表达机械疗法。尽管这种简单的培养系统是在体内筛选或排名人类的表达是其非常低的细胞密度。我们开发了一个由可cacaco组成的灌注培养系统。 Wes LL吸收了小肠，在肝脏中被生物学激活，并在肝脏中表达特定的毒a负责人类对乙酰氨基酚的毒性表现出非常高的酶作用，当时Caco-2细胞将theetaminophen转化为更具毒性的代谢物。正在开发一种新的灌注培养系统，其中细胞持续摇动以满足系统中细胞的消耗