Harris Georgina, Hogberg Helena, Hartung Thomas, Smirnova Lena
Center for Alternatives to Animal Testing (CAAT), Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland.
University of Konstanz, Konstanz, Germany.
Curr Protoc Toxicol. 2017 Aug 4;73:11.23.1-11.23.28. doi: 10.1002/cptx.29.
Current neurotoxicity testing and the study of molecular mechanisms in neurodegeneration in vitro usually focuses on acute exposures to compounds. 3D Lund human mesencephalic (LUHMES) cells allow long-term treatment or pulse exposure in combination with compound washout to study delayed neurotoxic effects as well as recovery and neurodegeneration pathways. In this unit we describe 3D LUHMES culture and characterization. Characterization of the model involves immunocytochemistry, flow cytometry, and qPCR measurements. Studying the delayed effects of compounds is more relevant to human exposures and neurodegenerative diseases with a strong genetic or environmental component. Most assays for molecular endpoints have been developed for monolayer cell culture and therefore need to be adapted for 3D models. In this unit, we further describe toxicological assays for molecular endpoints such as ATP levels, mitochondrial viability, and neurite outgrowth, which have been adapted for use in 3D LUHMES cultures. © 2017 by John Wiley & Sons, Inc.
当前，体外神经毒性测试以及神经退行性变分子机制的研究通常聚焦于化合物的急性暴露。3D 隆德人脑中脑（LUHMES）细胞允许进行长期处理或脉冲暴露，并结合化合物洗脱，以研究延迟性神经毒性效应以及恢复和神经退行性变途径。在本单元中，我们描述了 3D LUHMES 细胞培养和特性鉴定。该模型的特性鉴定包括免疫细胞化学、流式细胞术和 qPCR 测量。研究化合物的延迟效应与人类暴露以及具有强大遗传或环境成分的神经退行性疾病更为相关。大多数分子终点检测方法是针对单层细胞培养开发的，因此需要适用于 3D 模型。在本单元中，我们进一步描述了针对分子终点的毒理学检测方法，如 ATP 水平、线粒体活力和神经突生长，这些方法已适用于 3D LUHMES 细胞培养。© 2017 约翰威立国际出版公司
