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

The final goal of this research is to develop a human body simulator that mimics various responses to added chemicals by combining compartments having organ-derived cells in a physically-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).根据模型化学物质的体外吸收，双层系统中四种模型化学物质的所有ED50值与单层培养系统（无CACO-2细胞层）相比变为较高的浓度范围。这导致了体内毒性预测性的提高，因为考虑了体内吸收并影响双层培养系统的最终毒性。一个有趣的现象是，假设在CACO-2细胞层中的某些化学物质（AA…更多的Tex，发表，发表）。用作模型化学物质，通过在人类中通过细胞色素P450酶生物活化而表现出强毒性。苯并[a] pyrene代谢产物（其中一些是procarcinogens）的定量表明，只有小于十分之一的量可以渗透到Caco-2膜。 P450 1A1/2不仅在HEP G2细胞中，而且在CACO-2细胞中也被强烈诱导。 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 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.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，并结合了与物理相关的电路。在一系列实验中，使用对乙酰氨基酚作为一种模型化学物质，该化学物质在小肠中充分吸收，在肝脏中生物学活化，并表达肝脏中的特异性毒性。出乎意料的是，在含Caco-2细胞的系统中观察到的毒性高于无CACO-2细胞系统。负责人类对乙酰氨基酚毒性的细胞色素P450 3A的测量表明，Caco-2细胞中的酶促活性很高。因此，我们得出的结论是，当对乙酰氨基酚在CACO-2细胞层渗透时，它被转化为更具毒性的代谢产物。为了改善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，细胞持续摇动以满足系统中细胞的氧气消耗。较少的