3D Organotypic Cultures of Human HepaRG Cells: A Tool for In Vitro Toxicity Studies
3D Organotypic Cultures of Human HepaRG Cells: A Tool for In Vitro Toxicity Studies
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DOI：
10.1093/toxsci/kft021
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发表时间：
2013-05-01
影响因子：
3.8
通讯作者：
Noor, Fozia
中科院分区：
文献类型：
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作者：
Gunness, Patrina;Mueller, Daniel;Noor, Fozia
使用目前的体外系统很难预测药物引起的人肝毒性。本研究采用高通量挂滴法制备了人肝癌HepaRG细胞系的长期三维器官型培养物。器官型培养维持3周，并评估(1)肝脏特异性功能，包括I期酶和转运蛋白活性，(2)肝脏特异性蛋白的表达，以及(3)对三种药物（对乙酰氨基酚、曲格列酮和罗格列酮）的反应。我们的结果表明，在3周的培养中，器官型培养物保持了高的肝脏特异性功能。免疫组织化学分析表明，在整个培养过程中，器官型培养物表达肝脏特异性标志物，如白蛋白、CYP3A4、CYP2E1和MRP-2。因此，白蛋白和葡萄糖的产生率以及CYP2E1活性在3D培养中明显高于2D培养。毒性研究表明，与2D培养相比，器官型培养对对乙酰氨基酚和罗格列酮诱导的毒性更敏感，而对曲格列酮诱导的毒性不敏感。此外，对乙酰氨基酚在3D培养物上的EC50值（2.7mM）与体内毒性相似。总之，我们的研究结果表明，3D器官型HepaRG培养是一种有前景的体外工具，可以更准确地评估急性和慢性药物性肝毒性。
Drug-induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long-term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a high-throughput hanging drop method. The organotypic cultures were maintained for 3 weeks and assessed for (1) liver specific functions, including phase I enzyme and transporter activities, (2) expression of liver-specific proteins, and (3) responses to three drugs (acetaminophen, troglitazone, and rosiglitazone). Our results show that the organotypic cultures maintain high liver-specific functionality during 3 weeks of culture. The immunohistochemistry analyses illustrate that the organotypic cultures express liver-specific markers such as albumin, CYP3A4, CYP2E1, and MRP-2 throughout the cultivation period. Accordingly, the production rates of albumin and glucose, as well as CYP2E1 activity, were significantly higher in the 3D versus the 2D cultures. Toxicity studies show that the organotypic cultures are more sensitive to acetaminophen- and rosiglitazone-induced toxicity but less sensitive to troglitazone-induced toxicity than the 2D cultures. Furthermore, the EC50 value (2.7mM) for acetaminophen on the 3D cultures was similar to in vivo toxicity. In summary, the results from our study suggest that the 3D organotypic HepaRG culture is a promising in vitro tool for more accurate assessment of acute and also possibly for chronic drug-induced hepatotoxicity.