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{"title":"High-Throughput Assessment of Metabolism-Mediated Neurotoxicity by Co-Culture of Neurospheres and Liver Spheroids","authors":"Pranav Joshi, Soo-Yeon Kang, Prabha Acharya, Manav Goud Vanga, Moo-Yeal Lee","doi":"10.1002/cpz1.70023","DOIUrl":null,"url":null,"abstract":"<p>The liver's role in the biotransformation of chemicals is critical for both augmented toxicity and detoxification. However, there has been a significant lack of effort to integrate biotransformation into in vitro neurotoxicity testing. Traditional in vitro neurotoxicity testing systems are unable to assess the qualitative and quantitative differences between parent chemicals and their metabolites as they would occur in the human body. As a result, traditional in vitro toxicity screening systems cannot incorporate hepatic biotransformation to predict the neurotoxic potential of chemical metabolites. To bridge this gap, a high-throughput, metabolism-mediated neurotoxicity testing system has been developed, which combines metabolically competent HepaRG cell spheroids with a three-dimensional (3D) culture of ReNcell VM human neural progenitor cell line. The article outlines protocols for generating HepaRG cell spheroids using an ultralow attachment (ULA) 384-well plate and for cultivating ReNcell VM in 3D on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds are introduced into the ULA 384-well plate containing HepaRG spheroids and then tested with 3D-cultured ReNcell VM on the 384PillarPlate. This configuration permits the in situ generation of metabolites by HepaRG cells and their subsequent testing on neurospheres. By analyzing cell viability data, researchers can determine the IC<sub>50</sub> values for each compound, thus evaluating metabolism-mediated neurotoxicity. © 2024 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: HepaRG spheroid culture in an ultralow attachment (ULA) 384-well plate and the assessment of drug-metabolizing enzyme (DME) activities</p><p><b>Basic Protocol 2</b>: 3D neural stem cell (NSC) culture on a 384PillarPlate and compound treatment for the assessment of metabolism-mediated neurotoxicity</p><p><b>Basic Protocol 3</b>: Image acquisition, processing, and data analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpz1.70023","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
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Abstract
The liver's role in the biotransformation of chemicals is critical for both augmented toxicity and detoxification. However, there has been a significant lack of effort to integrate biotransformation into in vitro neurotoxicity testing. Traditional in vitro neurotoxicity testing systems are unable to assess the qualitative and quantitative differences between parent chemicals and their metabolites as they would occur in the human body. As a result, traditional in vitro toxicity screening systems cannot incorporate hepatic biotransformation to predict the neurotoxic potential of chemical metabolites. To bridge this gap, a high-throughput, metabolism-mediated neurotoxicity testing system has been developed, which combines metabolically competent HepaRG cell spheroids with a three-dimensional (3D) culture of ReNcell VM human neural progenitor cell line. The article outlines protocols for generating HepaRG cell spheroids using an ultralow attachment (ULA) 384-well plate and for cultivating ReNcell VM in 3D on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds are introduced into the ULA 384-well plate containing HepaRG spheroids and then tested with 3D-cultured ReNcell VM on the 384PillarPlate. This configuration permits the in situ generation of metabolites by HepaRG cells and their subsequent testing on neurospheres. By analyzing cell viability data, researchers can determine the IC50 values for each compound, thus evaluating metabolism-mediated neurotoxicity. © 2024 Wiley Periodicals LLC.
Basic Protocol 1 : HepaRG spheroid culture in an ultralow attachment (ULA) 384-well plate and the assessment of drug-metabolizing enzyme (DME) activities
Basic Protocol 2 : 3D neural stem cell (NSC) culture on a 384PillarPlate and compound treatment for the assessment of metabolism-mediated neurotoxicity
Basic Protocol 3 : Image acquisition, processing, and data analysis
通过神经球和肝脏球体的联合培养高通量评估代谢介导的神经毒性
肝脏在化学品生物转化中的作用对于增强毒性和解毒至关重要。然而，在将生物转化纳入体外神经毒性测试方面一直缺乏努力。传统的体外神经毒性测试系统无法评估母体化学品及其代谢物在人体内发生的质和量的差异。因此，传统的体外毒性筛选系统无法结合肝脏生物转化来预测化学代谢物的神经毒性潜力。为了弥补这一缺陷，我们开发了一种高通量、代谢介导的神经毒性测试系统，它将具有代谢能力的 HepaRG 球形细胞与 ReNcell VM 人类神经祖细胞系的三维（3D）培养相结合。文章概述了使用超低附着力（ULA）384 孔板生成 HepaRG 球形细胞和在带侧壁和缝隙的 384 柱形板（384PillarPlate）上三维培养 ReNcell VM 的方案。将代谢敏感的测试化合物引入含有 HepaRG 球形细胞的 ULA 384 孔板，然后在 384 柱板上用三维培养的 ReNcell VM 进行测试。这种配置允许 HepaRG 细胞原位生成代谢物，然后在神经球上进行测试。通过分析细胞活力数据，研究人员可以确定每种化合物的 IC50 值，从而评估代谢介导的神经毒性。© 2024 Wiley Periodicals LLC.基本方案 1：在超低附着力（ULA）384 孔板中培养 HepaRG 球形细胞并评估药物代谢酶（DME）活性 基本方案 2：在 384 柱板上培养三维神经干细胞（NSC）并进行化合物处理以评估代谢介导的神经毒性 基本方案 3：图像采集、处理和数据分析。
本文章由计算机程序翻译，如有差异，请以英文原文为准。
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